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Anti-Stokes fluorescence spectroscopy and imaging for cutaneous porphyrin detection Tian, Yunxian 2015

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	   	  	  	   	  	  	  	   	  	  	  Anti-­‐Stokes	  Fluorescence	  Spectroscopy	  And	  Imaging	  For	  Cutaneous	  Porphyrin	  Detection	  	   by	  Yunxian	  Tian	  	  	  A	  THESIS	  SUBMITTED	  IN	  PARTIAL	  FULFILLMENT	  OF	  THE	  REQUIREMENTS	  FOR	  THE	  DEGREE	  OF	  	  MASTER	  OF	  SCIENCE	  in	  THE	  FACULTY	  OF	  GRADUATE	  AND	  POSDOCTORAL	  STUDIES	  (Physics)	  	   THE	  UNIVERSITY	  OF	  BRITISH	  COLUMBIA	  (Vancouver)	  	  February	  2015	  	   ©	  Yunxian	  Tian,	  2015	   	  	  	  	  	  	  	  	   ii	  Abstract   Porphyrins produced by Propionibacterium acnes represent the principal fluorophore associated with acne, and appear as orange-red luminescence under the Wood’s lamp. Assessment of acne based on Wood’s lamp (UV) or visible (VIS) light illumination is limited by photon penetration depth and has limited sensitivity for earlier stage lesions. Inducing fluorescence with near infrared (NIR) excitation may provide an alternative way to assess porphyrin-related skin disorders. The objectives of this thesis are (1) to design and develop an optical instrument that perform anti-Stokes fluorescence spectroscopy and imaging measurements under continuous wave CW laser excitation as well as multiphoton fluorescence spectroscopy and imaging under femtosecond (fs) pulsed laser excitation; and (2) using this system for regular (Stokes) fluorescence measurements to evaluate and compare sebum-associated porphyrin fluorescence properties. A NIR multi-modality fluorescence imaging system with fluorescence spectroscopy capability was constructed. The switchable output from a tunable Ti: Sapphire femtosecond (fs) laser (720-950nm) or a (CW) laser (785nm) was scanned over the objective lens. Co-registered confocal imaging and fluorescence (anti-stokes fluorescence & two-photon fluorescence) imaging were acquired in video rate. Under 785 nm CW laser excitation PpIX powder exhibited anti-Stokes fluorescence with an intensity that was linearly dependent on the excitation power and a red spectral emission of 650-720 nm, while the fs laser excited two-photon excitation fluorescence showed a quadratic dependency. Anti-Stokes fluorescence of psoriasis scale image and ex vivo human nasal skin, skin surface smears from the nose, and ex vivo sebum secretions was obtained by the 	   iii	  same system configuration. Regular (Stokes) fluorescence was presented under UV and visible light excitation on ex vivo nasal skin and sebum secretions from non-inflamed acne, but not on skin surface smears from the nose or sebum secretions from inflamed acne.   In conclusion, anti-Stokes fluorescence under NIR CW excitation is more sensitive and specific for porphyrins than UV or visible light-excited regular (Stokes) fluorescence and fs laser-excited multi-photon fluorescence. The anti-Stokes fluorescence of porphyrins within sebum could potentially be applied to detecting and targeting acne lesions for treatment via fluorescence image guidance.  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   iv	  Preface This dissertation is an original intellectual product of the author, Y. Tian. The study reported in Chapters 3 (3.3) was covered by UBC Ethics (certificate #H96-70499).                    	  	   v	  Table Of Contents	  Abstract	  .........................................................................................................................................	  ii	  Preface	  ..........................................................................................................................................	  iv	  Table Of Contents	  .......................................................................................................................	  v	  List Of Tables	  ...........................................................................................................................	  vii	  List Of Figures	  .........................................................................................................................	  viii	  List Of Abbreviations	  ...............................................................................................................	  xi	  Acknowledgements	  ..................................................................................................................	  xii	  Dedication	  .................................................................................................................................	  xiii	  1.	   Introduction	  ..........................................................................................................................	  1	  1.1	   Human skin structure	  ...............................................................................................................	  1	  1.2	   Common skin disorders	  ...........................................................................................................	  2	  1.3	   Fluorescence	  ................................................................................................................................	  5	  1.3.1	   One photon excited fluorescence	  ...................................................................................................	  5	  1.3.2	   Two-photon excited fluorescence	  ..................................................................................................	  9	  1.3.3	   Anti-Stokes fluorescence	  ...............................................................................................................	  11	  1.4	   Overview of optical imaging technology for skin disorders	  ...........................................	  14	  1.4.1	   Wood’s lamp	  .....................................................................................................................................	  14	  1.4.2	   Dermoscopy	  .......................................................................................................................................	  14	  1.4.3	   Confocal Laser scanning microscopy	  .......................................................................................	  15	  1.4.4	   Two-photon fluorescence microscopy	  ......................................................................................	  17	  1.4.5	   Fluorescence spectroscopy	  ...........................................................................................................	  19	  1.5	   Objectives	  ..................................................................................................................................	  20	  2.	   System Development	  ........................................................................................................	  21	  2.1	   Versatile multi-modality imaging and spectroscopy system for ex vivo sample study…	  .................................................................................................................................................	  21	  2.1.1	   Introduction	  ........................................................................................................................................	  21	  2.1.2	   System construction	  ........................................................................................................................	  23	  2.1.3	   Testing extracted biological samples	  .........................................................................................	  30	  2.2	   Video rate in vivo multi-modality imaging system	  ...........................................................	  31	  3.	   Anti-Stokes Fluorescence Imaging Of Porphyrin	  .....................................................	  34	  3.1	   Sample preparation	  .................................................................................................................	  35	  3.2	   Methodology	  .............................................................................................................................	  37	  3.3	   Results	  ........................................................................................................................................	  40	  3.3.1	   Anti-Stokes fluorescence spectra and power dependence of PpIX powder	  .................	  40	  3.3.2	   Anti-Stokes fluorescence imaging of PpIX powder	  .............................................................	  45	  3.3.3	   Anti-Stokes fluorescence image of porphyrin in psoriasis plaque scales	  .....................	  46	  3.3.4	   Anti-Stokes fluorescence image of porphyrin in skin surface smears from the nose	  49	  3.3.5	   Anti-Stokes fluorescence image of sebum secretion from non-inflamed acne	  ...........	  49	  3.3.6	   Anti-Stokes fluorescence image of sebum secretion from inflamed acne	  ....................	  51	  	   vi	  3.3.7	   Anti-Stokes fluorescence image of porphyirn in human ex vivo nasal skin	  .................	  53	  3.3.8	   Wood’s lamp examination of psoriasis plaque scale	  ...........................................................	  58	  3.3.9	   Wood’s lamp examination of sebum secretion from non-inflamed acne	  ......................	  59	  3.3.10	   Wood’s lamp examination of human ex vivo nasal skin	  ..................................................	  60	  3.3.11	   EEM spectra of sebum secretion from non-inflamed acne	  ..............................................	  61	  3.3.12	   EEM Spectra of human ex vivo nasal skin	  ............................................................................	  63	  3.4	   Discussions	  ................................................................................................................................	  63	  3.4.1	   PpIX powder anti-Stokes fluorescence has first order power dependency	  ...................	  63	  3.4.2	   High sensitivity of anti-Stokes fluorescence imaging	  .........................................................	  65	  3.4.3	   Anti-Stokes fluorescence image contrast and image resolution	  .......................................	  67	  3.4.4	   Depth-resolved anti-Stokes fluorescence image	  ....................................................................	  68	  3.4.5	   Anti-stokes fluorescence of inflamed acne lesion and non-inflamed acne lesion	  ......	  68	  3.5	   Conclusions and future work	  ................................................................................................	  69	  3.6	   Potential clinical applications	  ...............................................................................................	  70	  Bibliography	  .............................................................................................................................	  72	                              	  	   vii	  List Of Tables  Table 1 Wavelength calibration comparison table. C - calibration wavelength number [nm]. S - standard wavelength number [nm].	  ..........................................................................	  26	  Table	  2	  Summarizes the performance of Wood’s lamp, EEM, and anti-stoke fluorescence imaging for porphyrin detection. In conclusion, anti-Stokes fluorescence may provide an alternative and superior way to assess acne condition in the presence of low porphyirn content.	  ....................................................................................................................	  66	                                      	   viii	  List Of Figures   Figure 1. 1 Diagram of human skin. Skin cells of different type are noted on the left side. Reproduced with permission from [2].	  ........................................................................................	  2	  Figure	  1.	  2	  Scheme of Stokes shift in one-photon fluorescence. The purple line represents the absorption spectrum, the green line represents the emission fluorescence spectra. The fluorescence is red-shifted.	  .....................................................................................................	  6	  Figure 1. 3 Jablonski diagram of the fluorescence process	  ............................................................	  7	  Figure 1. 4. Broad PpIX absorption spectrum together with characteristic PpIX fluorescence emission with a dominant peak at 635 nm. Reproduced with permission from  [22].	  .............................................................................................................................................	  8	  Figure	  1.	  5	  Scheme of (a) one-photon excitation fluorescence and (b) two-photon excitation fluorescence. In one-photon excitation, photon with energy hν excites the molecule from ground state to the excited state. In two-photon excitation, one photon with energy hνA excites the molecule to a virtual intermediate state, and another photon with energy hνB excites the molecule from virtual state to the excited state.	  10	  Figure	  1.	  7	  Scheme of anti-Stokes fluorescence in three-level energy state system. Reproduced with permission from [32].	  ...................................................................................	  12	  Figure 1. 8 Schematic diagram of the principle of RCM. Reproduced with permission from [48].	  ............................................................................................................................................	  16	  Figure 1. 9 Scheme diagram of multiphoton microscope.	  ............................................................	  18	  	  Figure 2. 1 Schematic diagram of the versatile simplified multi-modality imaging and spectroscopy system for ex vivo sample study. APD: avalanche photodiode, PMT: photomultipliers.	  ...............................................................................................................................	  22	  Figure 2. 2 Simplified configuration of the section for fluorescence imaging and fluorescence spectroscopy.	  ............................................................................................................	  24	  Figure 2. 3 Hg lamp spectra measured by the fluorescence spectroscopy subunit. The major wavelength numbers were marked on the chart.	  .......................................................	  26	  Figure	  2.	  4	  Examples of spectral response of a typical holographic diffraction grating (green curve) and a back-thinned CCD camera (black curve).	  .........................................	  27	  Figure 2. 5 Halogen lamp spectrum measured by the fluorescence spectroscopy subunit.	  ................................................................................................................................................................	  28	  Figure 2. 6 Intensity calibration spectra of the system. The lower the value in y-axis, the lower the sensitivity of the spectrometer at the corresponding wavelength.	  ................	  28	  Figure 2. 7	  Schematic configuration of the subunit for confocal imaging. L: lens; BP: bandpass filter; WP: waveplate; M: mirror; PBS: polarization beamsplitter; PMT: photomultiplier tube; LP: long pass filter; SP: short pass dichroic.	  ................................	  30	  Figure	  2.	  8	  (a)	  Reflectance confocal image, (b) integrated two-photon fluorescence and second harmonic generation image, (c) emission spectrum showing two-photon fluorescence and second harmonic generation signals of collagen type I sample.	  .....	  31	  	   ix	  Figure	  2.	  9	  In vivo video rate RCM/MPM system setup. The SHG/TPF dichroic and filters preceding PMTs were changed or removed according to the desired imaging modalities [58].	  ..................................................................................................................................	  33	  	  Figure 3. 1 Anti-Stokes emission spectrum of PpIX powder at different excitation laser intensities from 3 to 13 mW, λ excitation = 785 nm, CW laser excitation.	  ..........................	  41	  Figure 3. 2 Two-photon emission spectrum of PpIX powder at different excitation laser intensities from 3 to 13 mW,  λ excitation = 785 nm, fs laser excitation.	  .............................	  42	  Figure 3. 3 Plot of integrated Anti-Stokes fluorescence intensity versus excitation laser power. The dashed line represents first-order fit. R2= 0.986. The sample was PpIX powder, 785 nm CW laser excitation. The power range was from 3 to 13 mW.	  ........	  43	  Figure	  3.	  4	  Plot of Log (integrated Anti-Stokes fluorescence) intensity versus excitation laser power. The dashed line represents first-order fit. R2= 0.992. The sample was PpIX powder, 785 nm CW laser excitation. The power range was from 3 to 13  mW.	  ................................................................................................................................................................	  44	  Figure 3. 5 Plot of integrated two-photon fluorescence versus excitation laser power. The dashed line represents second-order polynomial fit. R2= 0.999. The sample was PpIX powder, 785 nm fs laser excitation. The power range was from 3 to 13 mW.	  ............	  44	  Figure 3. 6 Plot of logarithm (integrated two-photon fluorescence) intensity versus logarithm excitation laser power. Dashed line represents first-order fit. R2= 0.997. Sample was PpIX powder, 785 nm fs laser excitation. Power range was from 3 to 13 mW.	  .......................................................................................................................................................	  45	  Figure	  3.	  7	  Multimodality	  images	  of	  PpIX	  power	  sample:	  (a)	  Reflectance	  confocal	  image	  (785	  nm	  CW	  laser	  illumination),	  (b)	  multi-­‐photon	  fluorescence	  image,	  785	  nm	  fs	  laser	  excitation,	  (c)	  anti-­‐Stokes	  fluorescence	  image,	  785nm	  CW	  laser	  excitation.	  All	  are	  single	  frame	  images	  extracted	  from	  video	  form	  image	  data	  (frame	  rate	  14	  fps).	  ........................................................................................................................	  46	  Figure	  3.	  8	  (a) anti-Stokes fluorescence image of psoriasis plaque scale, CW laser excitation at 785nm. (b) confocal reflectance image (c) Overlay image (red channel: confocal reflectance image, green channel: anti-stokes fluorescence image). Single frame extracted from data in video type. FOV: 50 μm×50μm (frame rate 14fps)	  .....	  47	  Figure	  3.	  9	  (a) anti-Stokes fluorescence, (b) two-photon fluorescence, (c) overlay image (red channel: confocal reflectance image, green channel: anti-stokes fluorescence image). Single frame extracted from data in video. FOV: 100μm ×100μm (frame rate 14fps)	  ............................................................................................................................................	  48	  Figure 3. 10 (a) Fluorescence image of gold-coated glass slide (Control), (b) Anti-Stokes fluorescence image of nasal skin surface smears.  FOV: 300μm× 300μm	  ...................	  49	  Figure	  3.	  11	  (a) CW laser excited Anti-Stokes fluorescence and (b) CW laser reflectance confocal image of sebum secretion from non-inflamed acne. FOV= 100μm.	  .............	  50	  Figure 3. 12 (a) fs laser excited two photon fluorescence and (b) fs laser reflectance confocal image of sebum secretion from non-inflamed acne. FOV= 100μm.	  .............	  51	  Figure 3. 13 (a) CW laser excited Anti-Stokes fluorescence and (b) CW laser reflectance confocal image of sebum secretion from inflamed acne. FOV= 100μm.	  ......................	  52	  Figure	  3.	  14	  (a) fs laser excited two photon fluorescence and (b) fs laser reflectance confocal image of sebum secretion from inflamed acne. FOV= 100μm.	  ......................	  52	  	   x	  Figure 3. 15 (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 1.	  ............................................................	  54	  Figure	  3.	  16	  (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image. FOV= 200μm, depth 2.	  ............................................................	  55	  Figure	  3.	  17	  (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 3.	  ............................................................	  56	  Figure 3. 18 (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 4.	  ............................................................	  57	  Figure 3. 19 (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 5.	  ............................................................	  58	  Figure 3. 20 Red fluorescence under wood’s lamp on psoriatic skin.	  ......................................	  59	  Figure 3. 21 Wood’s lamp examination of sebum secretion. Upper left was overnight sebum secretion, lower right was freshly extracted sebum secretion. Fresh secretion appeared more in orange pink tone than over night secretion.	  ..........................................	  60	  Figure 3. 22 Wood’s lamp examination of nasal skin.	  ...................................................................	  61	  Figure 3. 23 The measured EEM spectrum of sebum secretion from non-inflamed acne.	  62	  Figure 3. 24 The measured EEM spectrum of Pp IX powder.	  ....................................................	  62	  Figure 3. 25 The measured EEM spectrum of nasal skin.	  ............................................................	  63	                     	   xi	  List Of Abbreviations  APD Avalanche photodiode BCC Basal cell carcinoma  BS Beamsplitter CCD Charge coupled device CSLM Confocal scanning laser microscopy CW Continuous wave DAQ Data acquisition FOV Field of view fps Frames per second fs Femtosecond LP Long pass MPM Multiphoton microscopy N.A. Numerical aperture NAD NAD(P)H Reduced nicotinamide adenine dinucleotide (phosphate)  NIR Near infrared PBS Polarization beamsplitter PMT Photomultiplier tube RCM Reflectance confocal microscopy SB Stratum basale SC Stratum corneum SCC Squamous cell carcinoma  SG Stratum granulosum SHG Second harmonic generation SNR Signal-to-noise ratio SS Stratum spinosum TPF Two-photon fluorescence  UV Ultraviolet 	   xii	  Acknowledgements I would like to express my deepest gratitude to my supervisor Dr. Haishan Zeng, Distinguished Scientist in the Integrative Oncology Department – Imaging Unit, BC Cancer Agency. I thank Dr. Zeng for providing guidance and support through my graduate study, both academically and financially. His wide knowledge in the field has encouraged and inspired my interest in Biophotonics research.  Secondly, I would like to say thank you to Dr. Harvey Lui and Dr. David McLean for their helpful suggestions on my work. You two taught me not only how to be a good student, but also how to be a good researcher, a good mentor.  I would like to thank the Canadian Institutes of Health Research (CIHR), the Canadian Dermatology Foundation,  and the CIHR- Skin Research Training Centre for funding this project.  I wish to express my particular thanks to Dr. Jianhua Zhao for enlarging my vision of science and providing coherent answers to my endless questions. His logical way of thinking has been of great value for me. His understanding, encouraging and personal guidance have provided a good basis of this thesis. I warmly thank Dr. Michael A. Short and Dr. Mladen Korbelik for their constructive comments and support throughout my Master’s study.  I offer my enduring gratitude to the faculty, staff and my fellow students at UBC who have inspired me to continue my work in this field.  I wish to thank my co-workers in the BC Cancer Agency.   I appreciate all the group members from Dr. Haishan Zeng’s lab and from the Photomedicine Institute in the Department of Dermatology and Skin Science at University of British Columbia. Special thanks to Dr. Gerald Li, Dr. Tracy Wang, Dr. Wenbo Wang, Dr. Shangyuan Feng, Dr. Yimei Huang, Dr. Guannan Chen, Ms. Hanna Pawluk, Mr. Zhenguo Wu, and Mr. Kam Chow, Ms. Jessie Fu for their valuable suggestions to my dissertation. Thank you all!            	   xiii	          Dedication To my parents                             	   1	  1. Introduction  1.1   Human skin structure   Human skin is the largest organ of our body, which plays an important role in protecting us from environmental influence. Figure. 1.1 shows the basic structure of human skin. Skin consists of epidermis and dermis. The epidermis is a thin layer at around 0.1 mm thickness. Epidermis is composed of 5 layers including stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS), stratum basale (SB).  Stratum corneum mainly consists of dead skin cells. Keratin from the SC is the major fluorescence signal. Living cells are located beneath SC, which are originated from keratinocytes. These cells keep migrating up at a cycle of 28-30 days and eventually become dead cells. The morphologies of the cells in different layers are different. The dermis can be divided into two sub-layers: papillary dermis (PD) and reticular dermis (RD), both of which consist of collagen fibers and elastin fibers.   The main functions of skin include thermal regulation, protection and sensation. In the process of thermal regulation, body temperature at 37° is maintained by blood flow, sweat production and heat insulation by dermis. Melanin plays a key role in protecting our skin from ultraviolet (UV) light radiation. Collagen and elastin fibers in the dermis protect our skin by forming as a physical barrier between the inner organ and the environment. In addition, the sebaceous glands excreting oil help to lubricate and waterproof the skin. Langerhans cells which are spread out in the basal layer are active in capturing, uptaking 	   2	  and processing of antigens. All of these protect our skin against the environment. Skin sensation is even a more complicated process.   	  Figure 1. 1 Diagram of human skin. Skin cells of different type are noted on the left side. Reproduced with permission from [2].   1.2  Common skin disorders  	  Among the numerous abnormal skin conditions, there are some skin disorders seriously threatening our life, such as skin cancer. The main skin cancers are divided by two categories: melanoma skin cancer and non-melanoma skin cancer. Melanoma is a malignant tumor of melanocytes originated from basal layer. Although malignant melanoma accounts for less than 5% of skin cancers, it is the most severe skin cancer causing highest mortality rate in North America [3]. Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the major non-melanoma skin cancers, which are mainly caused by sun exposure. Neither BCC nor SCC are easily metastatic. In addition 	   3	  to skin cancers, other common skin disorders involve acne vulgaris (acne), psoriasis and seborrheic keratosis (SK).   Acne, characterized as inflammatory disease of the pilosebaceous duct occurs most commonly during adolescence [4]. Sebum production, ductal cornification and bacterial colonization are the main three factors of acne [5]. Acne may have long lasting psychosocial effects including depression and suicide. It typically begins from production of sebum and proliferation of follicular epidermis [6]. The early stage of non-inflammatory acne is named as micro-comedones. It then developed into clinically evident comedones [7, 8].  The late and severe inflammatory lesions are papules, pustules, nodules and cysts. The clinical examination includes grading and lesion counting. Grading is subjective, and lesion counting is done by successively recording the number of acne lesions[9]. The treatment effectiveness is closely related to the early detection of acne. The later the acne is detected, the less effective the treatment will be [10]. However, the early stage acne referred as “the invisible time bombs” are usually unseen by naked eyes [11]. Non-invasive high-resolution acne imaging has potential to detect early stage acne so that to optimize the treatment.   Acne treatment aims to improve the disfiguring, psychological distress, and to prevent scarring [5] . Drugs to cure acne are retinoids and oral isotretinoin. Despite these drugs, there is a growing demand of light therapy. It has been found that laser therapy can be effective and safe for treating mild and moderate inflammatory acnes [12]. The current drug and laser treatment evaluation is mainly based on extracting the region of interest 	   4	  and analyzing the color of acne compared to adjacent areas [13]. This imaging has limitations in early stage acne and non-inflammatory acne detection. The alternative way is to quantify the single photon fluorescence signal generated by porphyrin inside acne. However, porphyrin fluorescence is undetectable when the density of P. acnes is low such as inflammatory lesions and post treatment lesions[14]. A more sensitive high-resolution imaging technique will provide more information in evaluating acne therapy outcomes.   Psoriasis is a common chronic immune mediated skin disease, which affects about 2% of the population. [15] It can be characterized as red, scaly patches, papules, and plaques. Among all types of psoriasis, plaque is the most common manifestation affecting 80% to 90% of psoriasis patients [16]. Psoriasis has a profound impact on patients who experiencing scaling, itching and skin redness [17]. To treat psoriasis, light therapy named photodynamic therapy (PDT) has been proved to be an effective methodology [18].  PDT treatment involves a reaction between light of a specific wavelength and a drug called a photosensitizer or photosensitizing agent. Singlet oxygen is produced during the reaction, and it further kills the nearby cells. For psoriasis treatment, 5-aminolevulinic acid (ALA) is one of the drugs (photosensitizers). Topically applied ALA produces protoporphytin (Pp IX) inside the tissue, and it acts as photosensitizer under red light exposure.    Beyond skin disorders, photo-aging, an important condition which affects people’s lives, is caused by chronic sun exposure in middle aged and elderly adults [19]. Some of the skin cancers and disorders discussed above might be developed by Cutaneous sun 	   5	  exposure. Therefore, understanding the relationship between skin and sun light radiation is crucial to early skin cancer diagnosis [19].   The current gold standard diagnosis of skin diseases is obtained from biopsy and histology. It is time consuming and costly. Moreover, a biopsy increases patient inconvenience and pain by leaving a permanent scar on the skin. Biopsy sites are usually selected following clinical diagnosis, i.e. by a physician’s visual examination of the skin or by measurements of more advanced optical devices. Therefore, improving the accuracy of non-invasive clinical diagnosis is of great interest. Recently numerous optical imaging technologies have been developed and applied to clinical diagnosis to reduce the number of false positive biopsies and also improve disease detection sensitivity. These will be briefly introduced in the next two sections. 	  	  1.3 Fluorescence 1.3.1 One photon excited fluorescence 	  The fluorescence Stokes shift was discovered by George Gabriel Stokes in 1852 [20]. As shown in Figure 1.2, it is the process of the re-emission of photons of lower energy after a molecule absorbs the energy from incident photons of higher energy. The Jablonski diagram (as shown in Figure. 1.3) illustrates the transitions of electronic states of a molecule between excited and ground states during this process. In general, the emitted fluorescence light has a longer wavelength than that of the absorbed light. Thus regular one photon excited fluorescence is called Stokes fluorescence.   	   6	  	  Figure	  1.	  2	  Scheme of Stokes shift in one-photon fluorescence. The purple line represents the absorption spectrum, the green line represents the emission fluorescence spectra. The fluorescence is red-shifted.   (Source:http://www.public.asu.edu/~laserweb/woodbury/classes/chm467/bioanalytical/spectroscopy/absflr.html)  	  	  	  	  	  	  	   7	  	  Figure 1. 3 Jablonski diagram of the fluorescence process  (Source: http://en.wikipedia.org/wiki/Jablonski_diagram#mediaviewer/File:JablonskiSimple.png)   Previous studies concluded that native psoriatic plaques can exhibit red auto-fluorescence that is due to elevated levels of protoporphyrin IX (Pp IX) within scales[21].	  Porphyrin produced by Propionibacterium acnes (P. acnes) is the main fluorophore in acne, which shows an orange-red luminescence under the Wood’s lamp. An example of porphyirn one-photon fluorescence is shown in Figure. 1.4.  	   8	  	  Figure 1. 4. Broad PpIX absorption spectrum together with characteristic PpIX fluorescence emission with a dominant peak at 635 nm. Reproduced with permission from  [22].   Porphyrin in skin surface smears from the nose Skin surface smears from the nose provide an oily, greasy substance secreted from sebaceous follicles on our facial area. The content of skin surface smears from the nose can be analyzed for bacteria, lipids and porphyrins [23, 24]. The fluorescence of skin surface smears from the nose has rarely been studied systemically. This may be due to the  extremely low content of prophyrin in smears.  Porphyrin in Sebum secretion from inflamed and non-inflamed acne lesions Sebum secreted from sebaceous glands contains mostly squalene, wax esters, triglycerides, as well as porphyin [25]. Porphyrin secreted from sebaceous glands to the skin pore 	   9	  generates strong fluorescence [5]. Studies reported the orange-red fluorescence excited by wood’s lamp has strong positive correlation with the presence of non-inflammatory acne lesions and high sebum amount, whereas a negative relationship was found with increasing clinical grade of acne. The late stage inflammatory acnes show less or no fluorescence compared to what is found during the early stage [5].  Currently, no fluorescence study on inflammatory acne has been published.  Porphyrin in nasal skin tissue The distribution of porphyrin on nose and chin were first discovered by fluorescence [26]. It was found that the red fluorescence due to Pp IX was generated in the sebaceous glands [27].  	  1.3.2 Two-photon excited fluorescence 	  	  The theory of two-photon excitation was mentioned in Maria Goeppert-Mayer’s  PhD dissertation in 1931 [28]. Two-photon excitation is a non-linear process. A diagram comparing one-photon excitation and two-photon excitation is shown as Figure. 1.5. The total energy of two photons in two-photon excitation is the same as the energy of a single photon in corresponding one-photon excitation. The excitation results a similar emission compared with one-photon excitation. Several factors influence fluorescence intensity such as wavelengths of excitation and detection, intensity of excitation light, quantum efficiency of fluorophores, resonant energy transfer partners and oxidation in the medium. There are endogenous fluorophores inside human skin that can generate two-photon 	   10	  fluorescence signals, such as porphyrin, keratin, melanin, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), flavin, and elastin [29]. Two-photon excited fluorescence has been largely applied to biological system by the development of multi-photon microscopy (MPM) which will be discussed in detail in section 1.4.4 [30].   	  Figure	   1.	   5	   Scheme of (a) one-photon excitation fluorescence and (b) two-photon excitation fluorescence. In one-photon excitation, photon with energy hν excites the molecule from ground state to the excited state. In two-photon excitation, one photon with energy hνA excites the molecule to a virtual intermediate state, and another photon with energy hνB excites the molecule from virtual state to the excited state. 	  	  	  	  	   11	  1.3.3 Anti-Stokes fluorescence 	  	  Anti-Stokes fluorescence, also called anti-Stokes photoluminescence, is a phenomenon different from traditional fluorescence. Fluorescence emitted by the material has shorter wavelength than the excitation light. On the contrary from traditional Stokes fluorescence shown in Figure. 1.2, anti-Stokes fluorescence is blue-shifted. This up-conversion process is closely connected to the thermal interactions with the excited molecules[31]. To generate anti-Stokes fluorescence, a system consisting of manifold ground state and manifold excited state well separated from one another is a prerequisite (Figure. 1.6) [1]. Similar to a cooling cycle mechanism, anti-Stokes starts the process by optical pumping of the thermally populated high-lying levels of the manifold ground state to low-lying states of the manifold excited state [31]. In going from the excited state back to the ground state the molecule undergoes radiative relaxation in which the energy is emitted as fluorescence.    	  Figure 1. 6 Scheme of anti-Stokes fluorescence. Reproduced with permission from [1]. 	   12	  	   In a three-level system (Figure. 1.7), anti-Stokes fluorescence occurs under the following conditions: a molecule is excited from ground state to the excited state 1, during the transitions between ground state and excited state 1 there is thermal population from excited state 1 to excited state 2, transition from excited state 2 to ground state generates radiative energy. The similar mechanism has been mentioned in reference [32].  	  Figure	   1.	   7	   Scheme of anti-Stokes fluorescence in three-level energy state system. Reproduced with permission from [32]. 	  	  	   The simplified physical background of anti-Stokes fluorescence can be as follows [33]: PA = σAIPν            (1) Pν =𝑒?∆??𝐵?                (2) PA is the probability of one-photon excitation, σA is the cross-section of one photon excitation, I is the photon flux in the focus. The thermal population Pν is derived from 	   13	  equation (2). KB is the Boltzmann constant, T is the thermal dynamic temperature. Anti-Stokes fluorescence has been observed in various materials. Semiconductor macrocrystals such as undoped crystals of CdS, chromium-doped ZnSe and CdS have provided a solid evidence of anti-Stokes fluorescence generation [34-36]. This phenomenon has also been reported in structure materials. Anti-Stokes fluorescence can be used for laser cooling of matter. The first report of cooling of a solid was done by Richard I. Epstein in 1995 [37]. Recently, anti-Stokes fluorescence drew great attention with the development of new technologies such as multi-color display and biomedical imaging [38, 39]. Most recently, it is proved to be applicable in chloroplasts and maize [40] by continuous wave laser excited anti-Stokes fluorescence.    Anti-Stokes fluorescence has several advantages for biomedical imaging. Firstly, it can be excited by near-infrared wavelength laser light, which is beneficial to deep penetration of biological tissue. Secondly, as opposed to two-photon fluorescence which is created by ultra short pulses, femtosecond laser, anti-Stokes fluorescence can be generated by a lower cost, continuous-wave laser which produces a continuous output beam, source. Anti-Stokes fluorescence allows us to focus on specific molecules since most other molecules fail to fulfill the excitation conditions.  	  	  	  	  	  	   14	  1.4   Overview of optical imaging technology for skin disorders 1.4.1 Wood’s lamp 	  	  Wood's lamp is a technology that uses ultraviolet (UV) light to inspect the skin. In 1925, the wood’s lamp was first reported to be used in dermatology for detection of fungal infection of hair [41]. The excitation wavelength is centered at 365nm with power output less than 1mW/cm2 [42]. When exposed to a wood’s lamp, skin tissue absorbs the light energy and fluorescence occurs, which is visible to clinicians. Wood’s lamp can be used to examine the localization of melanin pigmentation in the skin [43]. Acne, caused by disorders of porphyrin metabolism can also be evaluated by using Wood’s lamp because it shows orange-red fluorescence. Wood’s lamp is also useful in detection of hypopigmentation and depigmentation, and infection disease [42]. In brief, until now Wood’s lamp has been largely used in dermatology due to its convenience and utility. This technology is less reliable in darker skin because the high level of melanin absorption.  	  1.4.2 Dermoscopy 	  	  Dermoscopy is a non-invasive optical technology for evaluation of colors and distributions of microstructures of skin visible to the naked eye. For decades, the clinical diagnosis of dermatology had been mostly dependent on observation. The accuracy of melanoma diagnosis is only slightly higher than 50-75% [44]. Dermoscopy increases the accuracy by providing more detailed inspection of pigmented skin lesions and by increasing the detection depth [45]. Currently, dermoscopy has opened a new dimension in screening pigmented skin lesions. Similar to Wood’s lamp, one limitation of 	   15	  dermoscopy is low optical resolution. The lack of capability of sectioning reduces the accuracy of diagnosis for  pigmented skin lesions [46].  	  1.4.3 Confocal Laser scanning microscopy  Confocal scanning laser microscopy (CSLM) is a valuable imaging tool for investigating living skin in vivo and fixed specimens in biological and medical laboratories. Compared to previous optical techniques, confocal microscopy provides optical sectioning capacity of tissues up to a depth of 200 micron. It has recently gained popularity in skin disease detection. In general, CSLM includes reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM). In this section, we will mainly discuss the RCM modality since confocal imaging in our system (section 2) is developed based on the RCM principle. The concept was first introduced by Marvin Minsky in 1957 [47]. The image contrast of RCM comes from the refractive index variations within the sample.  Figure. 1.8 shows the basic RCM working scheme. There are 5 major components in CSLM: light source, microscopic objective lens, 50/50 beam splitter, pinhole and detector. A focused laser beam scans the specimen, and reflectance signal is focused to the photodetector. A pinhole in front of the detector rejects the out-of-focus light.    	   16	  	  Figure 1. 8 Schematic diagram of the principle of RCM. Reproduced with permission from [48].  Confocal microscopy is a viable technique for non-invasive assessment of human skins in vivo [14]. This technology has been applied recently on patients with acne to provide microscopic images of the dermal-epidermal junction in small pustules and epidermis images for small comedones [49]. A similar study was done to demonstrate a strongly undulated epidermal-dermal junction around facial pores using RCM [50]. So far, studies of investigating porphyrin in acne is scarce. RCM is useful in morphological and structural analysis, however it is less reliable for selective chemical mapping such as porphyrin imaging.   	  	  	   17	  1.4.4 Two-photon fluorescence microscopy 	   Multiphoton microscopy (MPM) techniques such as two-photon fluorescence (TPF) microscopy recently gained popularity in dermatology [2]. By introducing nonlinear effects to achieve image contrast, cellular metabolism and biological structures can be imaged [3]. The simplified diagram is shown in Figure. 1.9. A Titanium-sapphire laser beam is scanned over the back aperture of the objective lens. The TPF is collected by the same objective and detected by photomultiplier (PMT). In the next section, anti-Stokes fluorescence and two-photon fluorescence imaging are developed based on this principle. MPM has several advantages including self-sectioning capability and minimizing of photodamage and photo-bleaching effects.    	  	   18	  	  Figure 1. 9 Scheme diagram of multiphoton microscope.  (source:http://biophotonics.illinois.edu/technology/mpm/) 	   MPM in skin detection provides both morphological and biochemical information abour skin tissue, and it has potential for skin treatment as well [51, 52]. There are numerous studies about two-photon absorption of porphyrin for photodynamic therapy (PDT) [53]. However, very few studies studied porphyrin in acne by two-photon fluorescence microscopy. One drawback of this technique is that the two-photon absorption bands for many fluorophores are similar. For label free non-invasive detection, two-photon fluorescence microscopy is incapable of mapping any single chemical substance without interference from other fluorophores.  	  	   19	  1.4.5 Fluorescence spectroscopy 	   Fluorescence spectroscopy also named as fluorometers or fluorimeters. It is a type of electromagnetic spectroscopy, which records fluorescence intensity as a function of wavelength from a sample. There are three major optical components in the system: laser source, diffraction grating monochromator and detector. In general, the fluorescence signal excited by a laser beam passes through a grating. The fluorescence signal is diffracted during the process and is directed to a photodetector such as a charge-coupled device (CCD). Fluorescence spectroscopy plays an important role in discriminating chemical ingredients by analyzing spectrum shape and peak values because each chemical substance yields to a unique characteristic spectrum. In principle, fluorescence spectroscopy can be defined as one-photon or two-photon fluorescence spectroscopy by adopting different excitation laser sources. Both spectroscopies have been studied massively on skin.     Excitation-emission matrix (EEM) fluorescence spectroscopy is a type of spectroscopy that requires a scanning excitation source. Emission spectrum from each excitation wavelength is recorded, and then the excitation wavelength, emission wavelength and intensity are plotted as 2-D or 3-D contour map. In a biological sample, EEM might be used to identify the fluorophores and their relative proportions. EEM spectroscopy has been studied on skin [54]. Since current commercial EEM uses UV to visible excitation light, the penetration depth is restricted. More detailed properties will be discussed in section 3 when comparing EEM to anti-Stokes fluorescence. 	   20	  1.5  Objectives   The first objective is to generate porphyrin anti-Stokes fluorescence signal by designing and constructing an anti-Stokes fluorescence spectroscopy system with 785nm CW laser excitation. The generation of anti-Stokes fluorescence signal will be demonstrated as fluorescence emission spectra in Section 3.3.1. The second objective is to further prove anti-Stokes fluorescence imaging is capable of differentiating poprhyrin from other fluorophores in a biological sample known to contain porphyrin. A multi-modality optical instrument was designed and constructed (please refer to section 2) aiming to obtain the microscopic image of porphyirn distribution in psoriasis scales, which will be illustrated in section 3.3.3.  The third objective is to push the limits of our optical system for porphyrin detection in facial sebum.  Our ultimate goal is to apply this technology in early acne diagnosis. The rest of Section 3 will discuss the potential of anti-Stokes porphyrin fluorescence imaging for acne management.     	  	  	  	  	  	  	  	  	  	  	  	   	  	   21	  2. System Development 	  	  This thesis used two multimodality systems. One was an existing video rate in vivo multimodality imaging system. The other was a versatile multimodality imaging and spectroscopy system for ex vivo sample studies developed specifically in this thesis work.    In both systems, confocal reflectance microscopy was used as imaging guidance, providing basic morphology information to localize the measurement region in a sample.  	  2.1  Versatile multi-modality imaging and spectroscopy system for ex vivo sample study 	  A version of Sections 2.1 has been presented as: Wang, W., Tian, Y., Zhao, J., Lui, H., Zeng. H.: A LabVIEW based graphic user interface for multimodality confocal and multi-photon microscopy imaging and spectroscopy measurement, Canadian Medical and Biological Engineering Society, Vancouver. Wenbo Wang was responsible for LabVIEW programming for the system. 2.1.1 Introduction 	  Our group at BC Cancer Research Centre (BCCRC) has successfully developed a multimodality imaging microscope capable of simultaneous RCM and MPM imaging. Considering the complexity and the ever-expanding measurement capability of the multimodality microscope system, a software platform that meets all the stringent needs for multiple channel imaging and optical measurements is highly desirable. Using National Instrument’s graphic programming language LabVIEW and data acquisition 	   22	  (DAQ) device, we have developed a software platform that enables multi-channel imaging and spectroscopy measurement within one single optical system.   This section is mainly focused on the optical and electronics configuration, whereas the software programming is omitted. The simplified integrated system setup is shown in Figure. 2.1. Confocal imaging, fluorescence imaging (anti-Stokes fluorescence & two-photon fluorescence) and fluorescence spectroscopy will be introduced respectively.  	  Figure 2. 1 Schematic diagram of the versatile simplified multi-modality imaging and spectroscopy system for ex vivo sample study. APD: avalanche photodiode, PMT: photomultipliers. 	   	   23	  	  2.1.2 System construction 	   Anti-Stokes fluorescence imaging and two-photon fluorescence imaging The system setup is shown schematically in Figure. 2.2. The switchable output from a tunable femtosecond Ti: Sapphire laser (720-950nm) or continuous wave (CW) laser (785nm) was scanned over the rear aperture of a 60× water immersion objective lens. The fs laser at wavelength 785nm was the light source for two-photon fluorescence excitation, whereas a CW laser (StarBright 785XM), with a central wavelength of 785nm was the light source for anti-Stokes fluorescence excitation. Both the CW laser beam and fs laser beam were aligned to follow the same beam path directed to the imaging head. In the imaging head, both beams were expanded to the same spot size to fill the rear aperture of the 60×-water immersion objective (LUMPLFLN60X/W, Olympus Canada, Markham, Ontario). Two galvanometer-scanners were mounted as close as possible together to raster scan the laser beam. A data acquisition (DAQ) device (USB-6353, National Instruments, Austin, TX, USA) was used to control timing and synchronization of different components. Adjusting the scanning angles of the galvanometer scanners permitted a variable field of view (FOV) from 10μ m×10μ m to 300μm×300μm. A 710 nm long pass dichroic (F710Di02, 25×36, Semrock, Rochester, NY, USA) was used behind the objective to reflect the epi-directed multiphoton fluorescence signals or anti-Stokes fluorescence signals towards the detection arm. In the detection arm, a 750nm short pass filter was inserted to remove the rest of the excitation light at 785nm.  A photomultiplier tube (PMT) was used to detect epi-directed fluorescence signals.   	   24	   	  Figure 2. 2 Simplified configuration of the section for fluorescence imaging and fluorescence spectroscopy.  Fluorescence spectroscopy The configuration difference between fluorescence spectroscopy and fluorescence imaging is in the detection arm. The angle of the 705 nm long pass dichroic beam splitter can be tuned 90 degrees as shown in Figure. 2.2. In consequences, fluorescence signal reflected by dichroic passes a 750 nm short pass filter and is coupled into a customized fiber bundle (Fiberguide Industries) and is finally collected by the spectrometer (SpectraPro-150, Roper Scientific). The detailed description of fiber bundle has been mentioned in [55]. In brief, the customized fiber bundle is to increase the collection efficiency and the signal-to-noise ratio (SNR) during spectral acquisition. From Figure. 	   25	  2.3, entrance and exit patterns are magnified and shown.  The fiber bundle has 90 fibres with diameter of 100 μm (NA=0.12) arranged in a hexagon pattern at the input end to provide a collection diameter of  900 μm. The output end is arranged as 2 straight lines with 45 fibres in each line so that most of the collected light can be coupled into the narrow entrance slit of the spectrometer. The width of this line-shape bundle of fibers is 200 μm. Precise alignment has been carried out. Wavelength calibration and intensity calibration were performed as shown in Figure. 2.3 and Figure. 2.4.   Wavelength calibration The purpose of wavelength calibration is to readjust the position of the CCD pixels to spectral wavelengths. The most commonly used light source for wavelength calibration is Hg lamp (Ocean optics, HG-1). The calibration procedure can be found in [56]. Calibration was done in a dark environment. Figure. 2.3 is the spectrum measured by the spectrometer. The measured and standard numbers were listed as comparison in Table. 1. The standard deviation is less than 0.1%.   	   26	  	  Figure 2. 3 Hg lamp spectra measured by the fluorescence spectroscopy subunit. The major wavelength numbers were marked on the chart.	  	  	  Table 1 Wavelength calibration comparison table. C - calibration wavelength number [nm]. S - standard wavelength number [nm]. 	   Intensity calibration The intensity displayed in the computer is modified by the optical elements by their varying sensitivities at different wavelengths. Figure. 2.4 demonstrates two examples of spectral response: a holographic diffraction grating (a) and a back-thinned CCD camera (b). The Y-axis represents the measured intensity, and the curve demonstrates the C 404.67 435.98 546.22 578.96 696.63 750.33 763.23 772.44 794.93 S 404.66 435.84 546.08 579.07 696.54 750.39 763.51 772.40 794.82 	   27	  variation of intensity with wavelength. The technique of intensity calibration has been well studied [56, 57]. During the calibration, halogen lamp light through an integrating sphere was collected by an objective lens and eventually detected by the spectrometer CCD. The spectrum of the measured halogen lamp is presented in Figure. 2.5. The intensity calibration spectrum (Figure. 2.6) was derived after dividing the standard halogen lamp spectrum.   	  Figure	   2.	   4	   Examples of spectral response of a typical holographic diffraction grating (green curve) and a back-thinned CCD camera (black curve).  (Source:	  http://www.princetoninstruments.com)   	   28	  	  Figure 2. 5 Halogen lamp spectrum measured by the fluorescence spectroscopy subunit. 	   	  Figure 2. 6 Intensity calibration spectra of the system. The lower the value in y-axis, the lower the sensitivity of the spectrometer at the corresponding wavelength.    0	  500	  1000	  1500	  2000	  2500	  300	   350	   400	   450	   500	   550	   600	   650	   700	   750	   800	  Intensity	  [arb.]	  wavelength	  [nm]	  0	  0.005	  0.01	  0.015	  0.02	  0.025	  0.03	  450	   500	   550	   600	   650	   700	  Intensity	  [arb.]	  wavelength	  [nm]	  	   29	  Confocal imaging  Confocal imaging provides the reflectance property of the sample. The configuration of the optical setup can be found in Figure. 2.7. The reflectance signals from the sample are descanned (in descaned geometry, the emitted light returns along the same path as the excitation light, the beam remains non-scanned after passing scanning mirror in the returning path). For 785nm CW laser excited confocal imaging, the signal is reflected by a 750nm short pass filter and a Polarizing beam splitter (PBS) and half wave plate and finally is coupled through a 10μm pinhole to an avalanche photodiode (APD) module (C10508, Hamamatsu Corp., Bridgewater, NJ). The long pass (LP) filter is used to clean up residual laser noise lower than 700 nm. The short pass (SP) dichroic filter reflects light of wavelength longer than 750 nm and allows short wavelengths (shorter than 750 nm) to pass through. For fs laser excited confocal imaging, the signal is directed to the second APD with a similar configuration except for skipping the 750 nm short pass filter. During the system testing, both APD and PMT were used for signal detector. PMT is known to have higher sensitivity than APD, however, PMT has the defect of a higher noise level as well. Noise intensity from room light can be much higher than signal intensity. Therefore dark conditions are required when PMT is used. On the contrary, confocal reflectance images acquired by APD are less sensitive to room light, which allows us to do the measurement without interference with room light.    	   30	  	  Figure 2. 7	   Schematic configuration of the subunit for confocal imaging. L: lens; BP: bandpass filter; WP: waveplate; M: mirror; PBS: polarization beamsplitter; PMT: photomultiplier tube; LP: long pass filter; SP: short pass dichroic.  	   	  2.1.3 Testing extracted biological samples  	  	  Type I Collagen sample was obtained from Sigma-Aldrich. The sample was placed on a glass slide and covered by a cover slip. Figure 2.8 shows the RCM image (a), integrated TPF/second harmonic generation (SHG) image (b), and the emission spectrum containing both TPF and SHG signals (c) of the collagen sample at the xy plane. Both images were 	   31	  acquired simultaneously and co-registered. In Figure. 2.8 (c), the SHG signal is shown as a strong narrow peak located at half of the excitation wavelength (785/2=392.5 nm).  	  Figure	  2.	  8	   (a)	  Reflectance confocal image, (b) integrated two-photon fluorescence and second harmonic generation image, (c) emission spectrum showing two-photon fluorescence and second harmonic generation signals of collagen type I sample. 	  	  In conclusion, we have successfully developed a custom-built multimodality imaging and spectroscopy system that is capable of co-registered RCM imaging and TPF imaging or anti-Stokes fluorescence imaging, as well as performing TPF and anti-Stokes fluorescence spectral measurements. The anti-Stokes fluorescence spectra and images acquired from this system will be presented and discussed in section 3.	  	  	  2.2  Video rate in vivo multi-modality imaging system  	  	  The video rate in vivo multi-modality imaging system has been published elsewhere [58] [59]. The system setup (shown in Figure. 2.9) has many features in common with the 	   32	  system shown in Figure. 2.2, but used a resonant scanner for fast image acquisition and is less versatile for ex vivo sample study. The resonant scanner oscillates at a fixed frequency of 8kHz, and it enables fast scanning speed. In brief, a tunable fs Ti:Sapphire laser is scanned over the back aperture of a 60× water-immersion objective lens. Images are acquired by using an 8 kHz resonant scanner on the fast axis and a galvanometer scanner on the slow axis. The minimum FOV achievable is only 20μm×20μm. The MPM signal collection is realized by epi-detection using a 665nm long pass dichroic mirror. TPF and SHG signals are separated into two channels. RCM signal is collected by an APD module. As reference [58] emphasized, RCM, TPF, and SHG images are perfectly co-registered because all the imaging modalities share the same laser, scanners, and objective ensuring with complete certainty that the images are comparable. To conduct the experiment of porphyrin anti-Stokes fluorescence, the system has been modified and improved. First of all, the CW laser (StarBright 785XM) has been adopted as the light source for anti-Stokes fluorescence imaging. Secondly, the resolution of the RCM imaging has been improved by reducing the pinhole size from 50 μm to 20 μm. Thirdly, collection efficiency of fluorescence signal was optimized by selecting different lenses based on optical modeling using Zemax software. Finally, fluorescence spectroscopy has been integrated into the system.  	   33	  	  Figure	   2.	   9	   In vivo video rate RCM/MPM system setup. The SHG/TPF dichroic and filters preceding PMTs were changed or removed according to the desired imaging modalities [58]. 	    	   34	  3. Anti-Stokes Fluorescence Imaging Of Porphyrin 	   Porphyrin single photon absorption and excitation is well understood [60]. It exhibits fluorescence peaka at around 630 nm and 670 nm under the UV and visible light excitation. Several technologies such as Wood’s lamp and digital photography have successfully demonstrated this orange-red fluorescence [61]. With the development of multiphoton microscopy, two-photon fluorescence has been mainly applied to imaging biological samples at the cellular level. This technology can be realized by femtosecond  laser at near infrared wavelength excitation. Compared to UV-visible one-photon fluorescence imaging, near-infrared excitation wavelength of two-photon fluorescence is beneficial for deeper penetration of biological tissues. However, a previous study demonstrated that two-photon fluorescence of PpIX was not successful [33]. A two-photon anti-Stokes fluorescence imaging was proposed [38] [33]. Endogenous PpIX was visualized in skin tumors by anti-Stokes fluorescence imaging with femtosecond laser excited multi-photon microscopy. The drawback of femtosecond laser excited anti-Stokes fluorescence imaging is that the laser triggers two-photon fluorescence of other endogenous fluorophores in the skin, such as collagen, elastin, NAD(P)H, and keratin. The anti-Stokes fluorescence of porphyrin overlaps with the two-photon fluorescence of these fluorophores. Extra filtering is needed for separating endogenous porphyrin and other fluorophores in order to provide good tumor contrast.   We discovered that anti-Stokes fluorescence of porphyrin could be generated by continuous wave (CW) laser excitation.  It was found that CW laser excited anti-Stokes 	   35	  fluorescence is only sensitive to porphyrin with no signal from other fluorophores. To further prove anti-Stokes fluorescence imaging is capable of differentiating porphyrin from other fluorophores in biological tissues, we measured the porphyrin distribution in psoriasis plaque scales in section. Our ultimate goal is to apply this technology to a complex biological system - facial sebum.  	  	  3.1  Sample preparation  PpIX Powder  PpIX (A3785) was purchased from Sigma-Aldrich (St. Louis, MO, United States of America) which was in the form of a dry powder. The container with PpIX powder was kept in a dark environment to avoid potential photo-bleaching effects. Before the experiment, a small amount of PpIX power was spread on a microscope slide and then covered by a coverslip for imaging.  Psoriasis plaque scale Psoriasis scales examined in this study were collected from patients who were a subset of the participants from a major clinical trial at the Skin Care Center of the Vancouver General Hospital. The experiment was approved by the local ethics committee. The scales were located in patients’ buttock and calf. These psoriasis scales were examined under a Wood’s lamp (UVP, Upland CA, Model B 100AP) in a dark environment, and those exhibited an orange-pink fluorescence (as shown in Figure. 3.15 later on) were collected for further measurement. Care was taken to minimize exposure of the scales to light. Prior 	   36	  to the measurements, scales were placed on a glass slide, and covered by a transparent thin film with a thickness of 2 µm.  Skin surface smears from the nose Skin surface smears from the nose were extracted from a healthy male volunteer by pressing a gold coated glass slide on to the skin nasal area for a few seconds. The slide was then covered by a transparent thin film and placed under the objective lens for measurements. The thickness of the smear was less than 5μm. It was worth noting that the gold-coated glass slide prevented endogenous fluorescence of the glass material without interfering with the signal from porphyrin. 	  Sebum	  secretion	  from	  non-­‐inflamed	  acne	  and	  inflamed	  acne	  Acne lesions were tested under Wood’s lamp excitation in the dark environment. The non-fluorescence lesions were classified as inflamed acne, whereas the fluorescence lesions were classified as non-inflamed acne. The extrusion method was applied in this experiment to collect sebum sample. The gold-coated glass slide was pressed under pressure on the wing of nose for few seconds. The extracted sebum was placed on the gold-coated glass side. Only water was added between sebum and objective lens. Care was taken to avoid contamination.  Human ex vivo nasal skin Human nasal skin was obtained from the Skin Care Center, Vancouver General Hospital. The study of the ex vivo tissue samples has been approved by the University of British 	   37	  Columbia Research Ethics Board (certificate #H96-70499). The nasal skin was adjacent to a BCC lesion, which had been removed by surgery. The fresh tissue was submerged in PBS solution and kept in a fridge at 4ºC degrees for 2-3 hours before measurement.	  	  3.2 Methodology 	  Wood’s lamp examination Wood’s lamp (UVP, upland CA, Model B 100AP) was turned on and warmed up for 5 minutes. Psoriasis scales were placed on a blackened foil film horizontally. They were examined with Wood’s lamp in a dark environment. Skin surface smears from the nose, sebum secretion and human nasal skin were placed on gold-coated slides and an aluminum foil respectively for examination. The examination process for each sample was finished within 5 seconds. Samples with orange-red fluorescence were selected and photographed by a digital camera. For ex vivo human nasal skin, one red fluorescence spot was picked as region of interest for further microscopy study.   Excitation Emission Matrix measurement A commercial EEM system (FluoroLog3, Horiba Jobin Yvon, Edison, NJ, USA) was used for this study, consisting of light source, double grating excitation spectrometer, sample compartment, double-grating emission spectrometer and detector. The detailed system setup has been published elsewhere [62]. In brief, a broadband 450W Xeon lamp is directed onto the two gratings to reject stray light and side bands. The monochromatic light beam then enters the sample compartment, where the sample was placed at the 	   38	  center. The fluorescence emission is collected by a fiber bundle (containing 62, 250μm core-diameter fibers) and is delivered to another double-grating emission monochrometer. Finally, fluorescence signal was detected by a PMT (R928, Hamamatsu, Bridgewater, NJ, USA) connected to the emission monochrometer. Intensity calibration has been performed for both the excitation subunit and the emission subunit.   The measured EEM has excitation wavelengths ranging from 300 nm to 450 nm and emission wavelengths ranging from 300 nm to 700 nm. The fiber probe was mounted vertically. Skin surface smears from the nose, sebum secretion and human nasal skin were placed underneath the fiber probe for measurements. The distance between sample and the entrance of fiber probe was restricted to less than 5 mm. Measurement was done in a dark environment.   Multi-modality microscopy imaging and spectroscopy Prior to the measurement, the multi-modality microscopy imaging and spectroscopy system was optimized and calibrated as discussed in section 2.1.  Fluorescence spectra and confocal reflectance images were co-registered. A background spectrum was captured with exposure time of 1 second in the dark environment and subtracted from the signal. PpIX powder was spread uniformly on a glass slide with a size of 5 by 5 mm2 and covered by a cover slip. Configuration blank slide without PpIX powder was prepared as control. Water was added between the cover slip and the objective lens for refractive index matching. Both the PpIX samples and the blank slides (as control) were measured by CW laser excited fluorescence spectroscopy and confocal reflectance imaging. PpIX powder 	   39	  was visualized in confocal image mode, FOV= 20 μm×20 μm. In the meantime, fluorescence emission spectra were recorded with the same exposure time. For the control group, the measurement was targeted on a layer of air gap between the cover slip and the glass slide. The experimental parameters were kept the same as with the PpIX powder measurement. All the measurements were done in a dark environment.  Video rate in vivo multi-modality microscopy Prior to the measurement, the in vivo multi-modality microscopy system was optimized and calibrated, as discussed in section 2.2. Femtosecond laser excited two-photon fluorescence imaging and CW laser excited anti-Stokes fluorescence imaging could be implemented by simply switching a flip mirror. Two-photon fluorescence imaging, anti-Stokes fluorescence imaging and confocal reflectance imaging had been co-registered. It is noted that there was a chance that the sample might be oxidized (photobleaching or photo oxidization) when excited at high power level. Therefore, the minimum exposure power was maintained during the measurement. During human ex vivo nasal skin measurements, one sebaceous gland within the region of interest was measured by three imaging modes. When recording depth-resolved images with nasal skin, the microscopy stage moved downward with constant speed. Video rate data were recorded with the sample in movement.  	  Image processing Image processing involved only frame extraction using software ImageJ (Wayne Rasband, National Institute of Health, USA). No further color editing or frame processing was 	   40	  performed. Overlay images of different imaging mode were synthesized by Matlab programming.  	  3.3  Results 	  3.3.1 Anti-Stokes fluorescence spectra and power dependence of PpIX powder 	  	  Control experiment To prove that the signal was not from laser leakage or other environmental factors, controlled samples (mirror without porphyrin sample) were studied first. The noise intensity level ranged from -10 counts to +10 counts. No visible fluorescence signal was obtained. The result of this study eliminated the possibility of artificial signals generated by the laser, the imaging system or other environmental factors.   Porphyrin anti-Stokes fluorescence spectra The anti-Stokes fluorescence spectra of porphyrin powder were obtained under the excitation of the 785 nm CW laser. Figure. 3.1 presents the anti-Stokes fluorescence emission spectra of porphyrin powder under different incident powers from 3 mW to 13 mW. The fluorescence signals were bracketed by the long-pass dichroic mirror (705 nm) and the short-pass filter (750nm) in the microscopy. After system intensity calibration, the effective detection wavelength range was extended to 720 nm. The spectral shapes were similar for all the excitation powers. For comparison, the two-photon fluorescence spectra are shown in Figure.3.2. 	   41	  Comparison study on other chemical samples Further tests were performed by measuring several chemical samples including elastin, collagen, tryptophan, hemoglobin, and carotene under the same incident power. Under the same power level, no blue-shifted fluorescence signal was generated. One can speculate that this special optical property of anti-Stokes fluorescence has potential to selectively image the porphyrin distribution in biology samples.  	  Figure 3. 1 Anti-Stokes emission spectrum of PpIX powder at different excitation laser intensities from 3 to 13 mW, λ excitation = 785 nm, CW laser excitation.  	  0	  50	  100	  150	  200	  250	  300	  350	  400	  450	  650	   660	   670	   680	   690	   700	   710	   720	  Fluorescence	  intensity	  [arb.]	  wavelength	  [nm]	  3	  5	  7	  9	  11	  13	  control	  	   42	  	  Figure 3. 2 Two-photon emission spectrum of PpIX powder at different excitation laser intensities from 3 to 13 mW,  λ excitation = 785 nm, fs laser excitation.   Power dependency measurement The integrated Pp IX fluorescence intensity with different excitation 785 nm CW laser power is shown in Figure. 3.3. The number on Y-axis represents the fluorescence emission intensity summation of each spectrum shown in Figure. 3.1. As seen on Figure. 3.3, a linear trend line approximation was found: y=4472x was derived from excel trend line calculation. The R2 value of 0.986 indicates that almost 99% of the variance in fluorescence was determined by the laser power. Furthermore, we performed logarithm operation on the fluorescence emission intensity summation and incident power respectively as shown as Y- axis and X- axis in Figure. 3.4. Identical to the above, linear trend line approximation: y=1.009x + 4.6434 was derived, R² = 0.992. The fluorescence therefore has first order dependence on the excitation light power, which implies a one-0	  500	  1000	  1500	  2000	  2500	  3000	  600	   610	   620	   630	   640	   650	   660	   670	   680	  Fluorescence	  intensity	  [arb.]	  Wavelength	  [nm]	  3	  5	  7	  9	  11	  13	  15	  	   43	  photon excitation process. Note that the emission wavelengths were shorter than the excitation wavelength of 785 nm. These results, therefore, confirm that we observed anti-Stokes fluorescence from PpIX. For comparison, 785nm fs laser excited two-photon fluorescence power dependencies were acquired and are shown in Figure. 3.5 and Figure. 3.6.  From the logarithm operation shown in Figure. 3.6, the trend line approximation: y=2.131x + 2.453 indicates that fs laser excitation depends quadratically on laser power..   	  Figure 3. 3 Plot of integrated Anti-Stokes fluorescence intensity versus excitation laser power. The dashed line represents first-order fit. R2= 0.986. The sample was PpIX powder, 785 nm CW laser excitation. The power range was from 3 to 13 mW. 	  y	  =	  44742x	  R²	  =	  0.986	  0	  100	  200	  300	  400	  500	  600	  0	   2	   4	   6	   8	   10	   12	   14	  Fluorescence	  [arb.]	  Laser	  Power	  [mW]	  	   44	  	   	  Figure	  3.	  4	  Plot of Log (integrated Anti-Stokes fluorescence) intensity versus excitation laser power. The dashed line represents first-order fit. R2= 0.992. The sample was PpIX powder, 785 nm CW laser excitation. The power range was from 3 to 13  mW.  	  Figure 3. 5 Plot of integrated two-photon fluorescence versus excitation laser power. The dashed line represents second-order polynomial fit. R2= 0.999. The sample was PpIX powder, 785 nm fs laser excitation. The power range was from 3 to 13 mW.     y	  =	  1.0098x	  +	  1.5647	  R²	  =	  0.99182	  2	  2.1	  2.2	  2.3	  2.4	  2.5	  2.6	  2.7	  0.5	   0.6	   0.7	   0.8	   0.9	   1	   1.1	  Log	  (?luorescence)	  [arb.]	  Log	  (laser	  power)	  [mW]	  y	  =	  1003.9x2	  -­‐	  4339.2x	  +	  113413	  R²	  =	  0.99959	  10	  12	  14	  16	  18	  20	  22	  0	   2	   4	   6	   8	   10	   12	   14	  Fluorescence	  [arb.]	  Laser	  Power	  [mW]	  	   45	   	  Figure 3. 6 Plot of logarithm (integrated two-photon fluorescence) intensity versus logarithm excitation laser power. Dashed line represents first-order fit. R2= 0.997. Sample was PpIX powder, 785 nm fs laser excitation. Power range was from 3 to 13 mW.     3.3.2 Anti-Stokes fluorescence imaging of PpIX powder 	  	  Figure. 3.7 shows (a) a confocal image, (b) a two-photon fluorescence image and (c) a anti-Stokes fluorescence image of PpIX powder. Without inserting a pinhole in the system setup of fluorescence imaging mode, the resolution of anti-Stokes fluorescence image was lower than the that of the two-photon fluorescence image and confocal image, but the distribution of PpIX was still discernable (Figure. 3.7).         y	  =	  2.131x	  +	  2.453	  R²	  =	  0.99704	  3.5	  3.7	  3.9	  4.1	  4.3	  4.5	  4.7	  4.9	  0.5	   0.6	   0.7	   0.8	   0.9	   1	   1.1	  Fluorescence	  [arb.]	  Log	  (laser	  power)	  [mW]	  	   46	  	  Figure	   3.	   7	   Multimodality	   images	   of	   PpIX	   power	   sample:	   (a)	   Reflectance	   confocal	  image	  (785	  nm	  CW	  laser	  illumination),	  (b)	  multi-­‐photon	  fluorescence	  image,	  785	  nm	  fs	  laser	  excitation,	  (c)	  anti-­‐Stokes	  fluorescence	  image,	  785nm	  CW	  laser	  excitation.	  All	  are	  single	  frame	  images	  extracted	  from	  video	  form	  image	  data	  (frame	  rate	  14	  fps).	  	  	  3.3.3 Anti-Stokes fluorescence image of porphyrin in psoriasis plaque scales 	  	  Anti-Stokes fluorescence image vs confocal image   The anti-Stokes fluorescence image (Figure. 3.8(b)) and correspondence confocal image (Figure. 3.8(a)) were captured with FOV 50 μm×50μm. Figure. 3.8 (c) is the overlay images of Figure. 3.8 (a) and Figure. 3.8 (b). The red image is the confocal image; the green image is the anti-Stokes fluorescence image. As guidance of anti-Stokes fluorescence images, confocal images revealed the scales’ optical property as moderately irregular refractive structures corresponding to hyperkeratosis. This image is similar to those acquired in previous study of psoriasis scale confocal images in reference [63]. Figure. 3.8 (c) reveals the information of porphyrin distribution, which will be discussed in detail in section 3.4.2. It might be questioned why the most of the porphyrin signals 	   47	  were detected in the lower part of the image. One possible explanation is that the confocal imaging has sub-optimal sectioning ability compared to anti-Stokes fluorescence imaging. It is likely that the axial resolution of the confocal image is lower than that of anti-Stokes fluorescence image. The supporting evidence is that the image resolution is better in the lower part of the image compared to the image resolution in the top left part of the image (the lower region is less blurry).   	  Figure	   3.	   8	   (a) anti-Stokes fluorescence image of psoriasis plaque scale, CW laser excitation at 785nm. (b) confocal reflectance image (c) Overlay image (red channel: confocal reflectance image, green channel: anti-stokes fluorescence image). Single frame extracted from data in video type. FOV: 50 μm×50μm (frame rate 14fps) 	  Anti-Stokes fluorescence image vs two-photon fluorescence image   Figure. 3.9 compares anti-Stokes fluorescence (a) images and two-photon fluorescence (b) with FOV=100 μm×100μm.  The overlay images are shown in Figure. 3.9 (c). The red image is from two-photon fluorescence; the green image is from anti-Stokes fluorescence.  In Figure. 3.9 (a), all the anti-stoke fluorescence signals were scattered spots in high 	   48	  intensity. In Figure. 3.9 (b), homogenous fluorescence signals were shown within the whole scale region. Beyond those homogenous signals, there were several spots with higher intensity with sizes ranging from 1μm to 3μm scattered in the region as well. In Figure. 3.9 (c), nearly 80% of the porphyrin anti-Stokes signal coincided with the bright spots shown in Figure. 3.9 (b). Non-coincident signal in the anti-Stokes fluorescence image was small or had moderate signal intensity. Those porphyrin signals of smaller size are hard to discern in the two-photon fluorescence image. This leads us to conclude that anti-Stokes fluorescence image has higher porphyrin image contrast than two-photon fluorescence image.  	  Figure	  3.	  9	  (a) anti-Stokes fluorescence, (b) two-photon fluorescence, (c) overlay image (red channel: confocal reflectance image, green channel: anti-stokes fluorescence image). Single frame extracted from data in video. FOV: 100μm ×100μm (frame rate 14fps)   	   49	  3.3.4 Anti-Stokes fluorescence image of porphyrin in skin surface smears from the nose  	  Figure. 3.10 (b) shows anti-Stokes fluorescence from nasal skin surface smears. The control image is shown in Figure. 3.10 (a). Most of the porphyrin signals appeared in the  form of bright individual spots. Some lateral distortion occurred during the laser scanning due to system artifacts. Compared to the control image, anti-Stokes fluorescence in nasal skin surface smears has been verified.  	  Figure 3. 10 (a) Fluorescence image of gold-coated glass slide (Control), (b) Anti-Stokes fluorescence image of nasal skin surface smears.  FOV: 300μm× 300μm 	  	  3.3.5 Anti-Stokes fluorescence image of sebum secretion from non-inflamed acne 	  	  The 785nm CW laser excited anti-Stokes fluorescence is shown is Figure. 3.11 (a). Similar to Figure. 3.10 (b) some lateral distortion occurred during the laser scanning due 	   50	  to the porphyrin fluorescence life time. The fs laser excited two-photon fluorescence image is shown in Figure. 3.12 (a). The anti-Stokes fluorescence and two-photon fluorescence image were co-registered. These data shared similar patterns with those from psoriasis scales (Figure.3.8). The anti-Stokes fluorescence had the advantage over two-photon fluorescence in terms of porphyrin specificity again.   	  Figure	  3.	  11	  (a) CW laser excited Anti-Stokes fluorescence and (b) CW laser reflectance confocal image of sebum secretion from non-inflamed acne. FOV= 100μm.    	   51	  	  Figure 3. 12 (a) fs laser excited two photon fluorescence and (b) fs laser reflectance confocal image of sebum secretion from non-inflamed acne. FOV= 100μm.  3.3.6 Anti-Stokes fluorescence image of sebum secretion from inflamed acne 	  Figure. 3.13 (a) demonstrates 785nm CW laser excited Anti-Stokes fluorescence image of porphyrin from inflamed acne lesion. Figure. 3.13 (b) shows the co-registered reflectance confocal image. Anti-Stokes signals in inflamed acne were similar to those from non-inflamed acne. Figure. 3.14(a) shows the two-photon fluorescence image of inflamed acne lesion and (b) co-registered reflectance confocal image.   	  	   52	  	  Figure 3. 13 (a) CW laser excited Anti-Stokes fluorescence and (b) CW laser reflectance confocal image of sebum secretion from inflamed acne. FOV= 100μm. 	  	   	  Figure	  3.	  14	  (a) fs laser excited two photon fluorescence and (b) fs laser reflectance confocal image of sebum secretion from inflamed acne. FOV= 100μm.        	   53	  3.3.7 Anti-Stokes fluorescence image of porphyirn in human ex vivo nasal skin 	  	  Figure.3.15-Figure.3.19 show images of one sebaceous gland at different depths. Figure.3.15 corresponds to the sample surface, while Figure.3.19 is at the deepest penetration. In each depth, CW laser excited anti-Stokes fluorescence image (a), CW laser confocal image (b), fs laser excited two-photon fluorescence image (c), and fs laser confocal image (d) were obtained. The sebaceous gland appears as a straight tubular structure in the confocal image. Firstly, anti-Stokes fluorescence signal was comparable with previous results shown in Figure. 3.10 (b) and Figure. 3.9 (a) by presenting as bright scattered spots of size less than 3μm. Secondly, anti-Stokes fluorescence signals were successfully shown in the depth-resolved images at five different penetration depths. As the scanning section plane moved downward, the signals shifted from top left in Figure. 3.15 (a) to the bottom right in Figure. 3.19 (a). Thus anti-Stokes fluorescence microscopy successfully demonstrated sectioning ability when the cross section image plane moved vertically downwards. It could be substantiated by comparison with two-photon fluorescence. For the fs laser excited two-photon fluorescence images, the maximum fluorescence was found in the center in Figure. 3.15 (c) and shifted to the bottom right with decreasing depth in Figure. 3.19 (c). Based on the above explanation, anti-Stokes fluorescence successfully showed depth-resolved images in nasal skin. 	  	   54	  	  	  	  	  	  Figure 3. 15 (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 1. 	   55	  	  	  Figure	   3.	   16	   (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image. FOV= 200μm, depth 2. 	  	   56	  	  Figure	   3.	   17	   (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 3. 	  	   57	   Figure 3. 18 (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 4.  	   58	  	  Figure 3. 19 (a) CW laser excited Anti-Stokes fluorescence, (b) CW laser reflectance confocal image, (c) fs laser excited two-photon fluorescence, and (d) fs laser reflectance confocal image, FOV= 200μm, depth 5. 	  	  3.3.8 Wood’s lamp examination of psoriasis plaque scale 	  	  	  A Woods lamp image was taken of a psoriasis scale as shown in Figure. 3.20. The normal skin area was darker than the plaque scale in the center. The pink-red color represents fluorescence from PpIX in the scales [14].,. 	   59	  	  Figure 3. 20 Red fluorescence under wood’s lamp on psoriatic skin. 	  	  3.3.9 Wood’s lamp examination of sebum secretion from non-inflamed acne 	  	  Wood’s lamp provided evidence of prophyrin presence as shown in Figure. 3.21. An interesting phenomenon was that if sebum placed onto the glass slide was left overnight, the fluorescence appeared less orange-red color than that of freshly extracted sebum. The explanation is not certain. One possible reason is that porphyrin fluorescence is quenched by the photo-bleaching process when sebum is exposed in the ambient light for a few hours. However, this explanation hasn’t been completely verified. To avoid reducing the fluorescence, minimum light exposure was performed and the time period of the experiment was shortened to within 30 minutes.  	   60	  	  Figure 3. 21 Wood’s lamp examination of sebum secretion. Upper left was overnight sebum secretion, lower right was freshly extracted sebum secretion. Fresh secretion appeared more in orange pink tone than over night secretion. 	  	  3.3.10 Wood’s lamp examination of human ex vivo nasal skin 	  	  Woods lamp image was taken as shown in Figure. 3.22. The scattered orange-red fluorescence spots were from porphyrin related substances. One red fluorescence spot on the edge of the tissue was marked for further microscopy fluorescence measurement.  	   61	  	  Figure 3. 22 Wood’s lamp examination of nasal skin. 	  Also the inflamed acne lesion and skin surface smears from the nose were examined under Wood’s lamp light. No fluorescence signal was found. The results will be discussed further in Section 3.4.2.   3.3.11 EEM spectra of sebum secretion from non-inflamed acne 	  	  	  Figure. 3.23 Shows the EEM spectra of sebum secretion from non-inflamed acne. Figure. 3.24 shows the EEM spectra of PpIX power. The fluorescence peak could be discerned and compared with previous porphyrin spectra [56][57][58]. The PpIX fluorescence spectrum is usually characterized with a peak at around 630nm [64]. Some other studies reported another peak at 675 nm, which is due to a photoproduct transformed from PpIX, named as photoportoporphyrin (PPp) [65]. From the EEM, both 630nm and 675nm presented as peaks. The other two peaks at 588nm and 595nm may be contributions from metalloporphyrin and zinc porphyrin [66]. In addition, there are indications that between 600 nm and 630 nm, coproporphyrin might induce fluorescence [66]. All of these data showed solid evidence of porphyrin fluorescence in the sebum.   	   62	  	  Figure 3. 23 The measured EEM spectrum of sebum secretion from non-inflamed acne. 	  	  	  Figure 3. 24 The measured EEM spectrum of Pp IX powder. 	  	  	   63	  3.3.12 EEM Spectra of human ex vivo nasal skin 	  	  	  Figure. 3.25 Shows EEM spectrum of ex vivo human nasal skin. At excitation wavelength 416nm, there were three weak emission peaks at the region with emission wavelength longer than 580nm: 595nm, 640nm, and 650nm. 	  	  Figure 3. 25 The measured EEM spectrum of nasal skin.   	  3.4   Discussions 3.4.1 PpIX powder anti-Stokes fluorescence has first order power dependency 	  	  It is known that the relationship between incident laser power and corresponding fluorescence intensity can give information on the mechanism of photon absorption [67, 68]. For example, with two-photon excitation or two-stage excitation of electrons from 	   64	  the valence to the conduction band, a quadratic fluorescence dependence should be observed [68]. A previous study (ref- 68?) investigated the dependence of the PpIX fluorescence signal with respect to the fs laser power.  It was found that for shorter wavelengths approaching 700 nm there was a gradual transition of non-linearity of the excitation process into a linear process [4]. For anti-Stokes fluorescence, a pure linear dependency is expected.   Data in section 3.3.1 investigated the behavior of the power dependency of the porphyrin powder fluorescence (Figure. 3.3 & 3.4). We assumed a complex system mixing first order power dependency with second, third and n order power dependency shown as in formula (3) below. F is the fluorescence intensity, p is the excitation light power. In formula (4), we performed logarithm operation. F=ap1+bp2+cp3…+xpn                                                               (3) From the linear curve fit y=ax (a=44742) in Figure. 3.3, we can infer the dependency was highly close to first order. Furthermore, in the log plot of Figure. 3.4, the slop k=1.009 rejected the interpolation of second order and other higher order contributions. Thus the fluorescence signal dependency on excitation light power proved the generation of pure anti-Stokes fluorescence.  	  	  	  	  	   65	  3.4.2 High sensitivity of anti-Stokes fluorescence imaging  	  	  	  Section	  3.3.7,	  3.3.8	  and	  3.3.9	  showed	  Wood’s	   lamp	  examination	  of	  psoriasis	  plaque	  scale,	   sebum	   secretion	   and	   human	   ex	   vivo	   nasal	   skin.	   Regular	   fluorescence	   (i.e.	  Stokes)	  was	  present	  under	  woods	  lamp	  on	  ex	  vivo	  nasal	  skin	  and	  sebum	  from	  non-­‐inflamed	   acne,	   but	   not	   present	   on	   nose	   surface	   smears	   and	   inflamed	   acne.	   	   No	  similar	  work	  with	  UV	   facial	   smear	   imaging	   has	   been	   reported	   from	   the	   literature.	  Interestingly,	  the	  porphyrin distribution in skin surface smears from the nose and sebum secretion from anti-Stokes fluorescence images revealed similar concentration by calculating the total number of porphyrin signals in the same FOV. During sample preparation, only one layer of skin surface smears from the nose (less than 5μm) was collected during the touch-on process, however, the volume of sebum secretion located in hair follicle was thousands of times higher than the volume of smear. Therefore, the detection failure by Wood’s lamp may be due to its low sensitivity in the condition of low porphyrin content. In order to discern the porphyrin fluorescence, the density of P. acnes had to be more than 1000 organisms since the intensity of fluorescence was proportional to the density of the P. acnes population [26]. Previously, porphyrin in horny casts had been tested by digital fluorescence photography, and the system failed to present a reduction in the degree of P. acnes colonization with treatment [69]. Porphyrin fluorescence was undetectable when the density of P. acnes became less than 1000 organisms [26]. Neither the digital photography nor Wood’s lamp techniques have been considered by clinicians to be reliabile for low porphyrin concentration detection. Another explanation relies upon on the reflectance of illumination. When passing through the skin 	   66	  surface smears from the nose, the residual UV excitation light can be reflected or scattered by the glass slide. This strong scattered UV lights hinders the fluorescence observation. Therefore, a special filter fixed onto the camera is necessary.  	  Section 3.3.17, 3.3.18 and 3.3.19 showed EEM measurements of sebum secretion, PpIX powder and human ex vivo nasal skin, but it was not capable of providing any porphyrin information for nasal skin surface smears. Similar to the above reasoning, EEM sensitivity may drop considerably when imaging low concentration porphyirn contents. Comparing EEM of sebum secretion and nasal skin absolute values of porphyrin fluorescence intensities, the latter one was much lower. The scattered porphyrin signal in sebaceous gland or hair follicle, though visible under Wood’s lamp examination and anti-Stokes fluorescence imaging, can be low when averaged by  EEM measurement. In addition, the penetration depth might limit the EEM assessment. It is possible that porphyrin located in deeper tissue failed to be excited by EEM measurements.   Wood’s lamp EEM Anti-Stokes Fluorescence Imaging Skin surface smears from the nose No No Yes Sebum secretion from inflammatory acne No No Yes Sebum secretion from non-inflammatory acne Yes Yes Yes Nasal skin Yes Yes Yes Table	  2	  Summarizes the performance of Wood’s lamp, EEM, and anti-stoke fluorescence imaging for porphyrin detection. In conclusion, anti-Stokes fluorescence may provide an 	   67	  alternative and superior way to assess acne condition in the presence of low porphyirn content.   3.4.3 Anti-Stokes fluorescence image contrast and image resolution 	  	  Two-photon fluorescence images from the different samples were different. The two-photon fluorescence image from psoriasis scales was mostly homogenous, which might come from keratin due to hyperkeratosis. However, the porphyrin signals in smaller size regions were hard to discern in the two-photon fluorescence image. In sebum secretions, the fluorophors besides porphyrin mostly came from keratin in dead skin cell fragments and lipids. Different from psoriasis scales, the two-photon fluorescence signals formed as several flakes. The size of each flake was close to the size of a single cell. In addition, lipids might emit weak fluorescence helping to form a nebulous picture. Thus lipid and dead skin cell fragments overwhelmed the fluorescence signals in sebum secretions. The situation was similar in nasal skin; fs laser excited all the endogenous fluorophore in the nasal skin including the transparent cover film. In general, while the two-photon fluorescence images provided high resolution of structural information by exciting all the endogenous fluorophores, they had less selectivity in porphyrin imaging. Porphyrin signals were almost indiscernible in sebum secretion and nasal skin.   On the contrary, porphyrin anti-Stokes fluorescence signals were identical in biological samples: psoriasis scale, skin surface smears from the nose, sebum secretion and nasal skin. The size of the signal sources was consistently between 1μm and 3μm. This distinct property dramatically improved the image contrast and specificity of porphyrin and more 	   68	  importantly it precluded interference of other skin fluorophores. Compared to two-photon fluorescence imaging, anti-Stokes fluorescence imaging has great potential to generate high specificity, high contrast, and high-resolution images of porphyrins. 	  	  3.4.4 Depth-resolved anti-Stokes fluorescence image 	  	  Microscopy with near-infrared excitation increased penetration depth compared to UV and short wavelength visible light excitation. Confocal imaging and two-photon fluorescence imaging have sectioning ability. The anti-Stokes fluorescence signals were depth sensitive as has been shown in nasal skin. Anti-Stokes fluorescence has great potential to generate depth-resolved, image of porphyrins.  	  	  3.4.5 Anti-stokes fluorescence of inflamed acne lesion and non-inflamed acne lesion 	   Fluorescence detection has been applied mainly to non-inflamed acne lesion because of the strong correlation between the signal and porphyrin produced by P. acne. However, the fluorescence signal of inflamed acne lesions is largely suppressed. For severe inflamed acne lesions, the fluorescence signal is almost negligible. The possible reason is that sebum and bacteria escape from rupturing infundibulum, and P.acne are possibly destroyed [8]. It is well known that inflamed acne lesions fluoresce much less than non-inflamed acne lesion under wood’s lamp examination. Moreover, effectively treated acne also presents as low fluorescence intensity, which increases the evaluation’s difficulty. Anti-stokes fluorescence images of both non-inflamed and inflamed acne showed high-	   69	  resolution porphyrin fluorescence signals in microscopic scale. Although regular two-photon fluorescence images were discernable, it lacked specificity for porphyrin content. No fluorescence signal from inflamed acne was detected under Wood’s lamp and EEM. Therefore, Anti-Stokes fluorescence imaging has potential to track and analyze porphyrin distribution in inflamed acne lesions.   	  3.5 Conclusions and future work 	  	  In conclusion, we discovered that PpIX powder has anti-Stokes fluorescence under 785 nm CW laser excitation. It had been validated in psoriasis plaque scale measurement, skin surface smears from the nose, sebum secretion, and nasal skin. Depth-resolved anti-Stokes fluorescence images had also been generated successfully in an ex vivo nasal skin sample. The anti-Stokes fluorescence technology has higher sensitivity for pophyrin detection than Wood’s lamp. In addition, anti-Stokes fluorescence imaging makes porphyin signal more distinct (high detection specificity) without interference from other fluorophores. The Anti-Stoke fluorescence technique might also open the field of investigating the porphyrin features of inflamed acne. All of those characteristics point to great potentials for its application in porphyrin related disease diagnosis such as early stage acne imaging.  The principle of anti-Stokes fluorescence should be explored in depth. The relationship between temperature and anti-Stokes fluorescence was reported in previous studies as 	   70	  positive correlation from the Boltzmann equation (Pν=e?∆?™   ? ), which had impact on fluorescence intensity. Therefore future work should involve investigating relationship between temperature changing and porphyrin anti-Stokes fluorescence intensity. In addition, the studies based on PpIX did not provide the whole picture. EEM spectra of sebum secretion and nasal skin provided the possibility of the existence of several other tetrapyrrole molecules. Thus coproporphyrin III, zinc porphyirn and photoprotoporphyrin should be investigated to examine whether anti-Stokes fluorescence has selectivity between them. Moreover, the preliminary data suggests that the biological sample can be easily oxidized. Several studies suggested that tissue or molecule oxidation caused by thermal effects may enhance fluorescence [70]. Thus the oxidation effect of porphyrin by CW laser excitation should be studied in detail.  	  3.6 Potential clinical applications   In psoriasis scale and sebum, anti-Stokes fluorescence has shown high image contrast and high sensitivity and specificity in low porphyrin content imaging. Currently, our lab is developing a hand-held video rate in vivo multi-modality imaging system. It can be applied to the measurement of acne patients in vivo. Furthermore, anti-Stokes fluorescence images had high image contrast without a complicated imaging processing procedure. Combining with imaging tracking software such as OpenCV, we will be able to calculate the porphyirn signal during the in vivo measurement. 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