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Investigations into inkjet cell printing hydrodynamics through microscopy imaging techniques Cheng, Eric 2015

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Investigations into Inkjet Cell Printing Hydrodynamics through Microscopy Imaging Techniques by  Eric Cheng  B.ASc., The University of British Columbia, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (BIOMEDICAL ENGINEERING)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2015  © Eric Cheng, 2015 ii  Abstract Inkjet bioprinting technology aims to accurately and precisely dispense biological materials in a spatially predefined pattern within a three-dimensional space. The technology has a multitude of applications in the biomedical field such as in drug discovery and tissue or organ engineering. However, there are known limitations in an inkjet nozzle's capabilities in dispensing cells as the cell ejection rate does not follow any predictable distributions. In this work, the cell behaviors within a piezoelectric nozzle due to droplet ejection were classified through high speed brightfield imaging. With each ejected droplet, one of three cell behaviors was observed to occur: cell travel, cell ejection, or cell reflection. Cell reflection is an undesirable phenomenon which may adversely affect an inkjet's capability to reliably dispense cells. To further study how the hydrodynamics within a nozzle can influence the cell's behavior, µPIV was performed to identify the flow field evolution during droplet ejection. Through the study of cell motion, it was observed that the viscosity of the media in the cell suspension plays an important role in influencing the cell behavior. This was experimentally studied with the tracking of cells within the inkjet nozzle in a higher viscosity 10% w/v Ficoll PM400 cell suspension. As hypothesized, the addition of Ficoll PM400 was effective in preventing the occurrence of cell reflection which promises to increase the reliability in inkjet bioprinting systems.  iii  Preface Chapter Two is based on work conducted at the BioMEMS lab by Dr. Karen C. Cheung, Dr. Ali Ahmadi and Eric Cheng. I was involved on all aspects of the project including the designing of the experiments, conducting the experiments, data processing, analysis, and writing of the results. Figure 1 was created with the assistance of Horace Yu. A portion of this chapter has been published:   Eric Cheng, Ali Ahmadi, Karen C. Cheung, "Imaging Method Developed for the Tracking of Single Cell Behaviours in Inkjet Printing Systems", Proceedings of the CMBEC37 Conference, Vancouver, BC, Canada, May 21-23, 2014.  Eric Cheng, Ali Ahmadi and Karen C. Cheung, "Investigation of the Hydrodynamics of Suspended Cells for Reliable Inkjet Cell Printing", Proceedings of the ASME 2014 12th International Conference on Nanochannels, Microchannels and Minichannels, Chicago, Illinois, USA, August 3–7, 2014, Paper No. ICNMM2014-21583, pp. V001T03A010; 8 pages, doi:10.1115/ICNMM2014-21583 Chapter Three is based on work conducted at the BioMEMS lab by Dr. Karen C. Cheung, Dr. Ali Ahmadi and Eric Cheng. I was involved in all aspects of the project including designing of the experiments, conducting the experiments, data processing, analysis and writing of the results. A version of this chapter is under review for publication in a journal . Chapter Four is based on work conducted at the BioMEMS lab by Dr. Karen C. Cheung, Dr. Ali Ahmadi, Haoran Yu and Eric Cheng. I was involved on all aspects of the project including the designing of the experiments, conducting the experiments, data processing, analysis, and writing of the results.  Material from Chapters 2 and 4 will be submitted to for journal publication. iv  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Figures ............................................................................................................................... vi List of Symbols ........................................................................................................................... viii List of Abbreviations ................................................................................................................... ix Acknowledgements ........................................................................................................................x Dedication ..................................................................................................................................... xi Chapter 1: Introduction ................................................................................................................1 Chapter 2: Characterization of Cell Behaviors in PBS ..............................................................7 2.1 Materials and Methods .................................................................................................... 7 2.1.1 Piezoelectric Inkjet Nozzle ......................................................................................... 7 2.1.2 Cell Suspension Preparation ....................................................................................... 7 2.1.3 Imaging Setup ............................................................................................................. 8 2.1.4 Droplet Formation ....................................................................................................... 9 2.1.5 Cell Tracking Algorithm ........................................................................................... 11 2.1.6 Cell Mapping ............................................................................................................ 12 2.2 Results ........................................................................................................................... 12 2.2.1 Cell Printing in 115 pL Droplets ............................................................................... 13 2.2.2 Cell Printing in 310 pL Droplets ............................................................................... 19 Chapter 3: µPIV in a Cylindrical Inkjet Nozzle .......................................................................29 v  3.1 µPIV Materials and Methods ........................................................................................ 29 3.1.1 Inkjet System ............................................................................................................ 29 3.1.2 PDMS Nozzle Holder ............................................................................................... 29 3.1.3 µPIV Imaging Setup ................................................................................................. 32 3.1.4 PIV Analysis ............................................................................................................. 36 3.2 Results ........................................................................................................................... 37 Chapter 4: Cell Printing in a High Viscosity Fluid ...................................................................49 4.1 Materials and Methods .................................................................................................. 50 4.2 Results ........................................................................................................................... 51 Chapter 5: Conclusion and Future Work ..................................................................................55 Bibliography .................................................................................................................................60   vi  List of Figures  Figure 1: Diagram of the imaging setup ......................................................................................... 8 Figure 2: A droplet ejection event of the 115 pL droplet ............................................................. 10 Figure 3: The droplet ejection phases of the 310 pL droplet ........................................................ 10 Figure 4: The process of the image segmentation routine ........................................................... 11 Figure 5: Selected frames of a cell exhibiting cell travel .............................................................. 13 Figure 6: The vertical cell tracking results of a cell experiencing cell travel. .............................. 14 Figure 7: Selected frames of a cell exhibiting cell ejection. ......................................................... 15 Figure 8: The vertical cell tracking results of a cell experiencing cell ejection. .......................... 16 Figure 9: Selected frames of a cell exhibiting cell reflection ....................................................... 16 Figure 10: The vertical cell tracking results of a cell experiencing cell reflection. ...................... 17 Figure 11: Cell mapping with 115 pL dispensed droplet volume. ................................................ 18 Figure 12: Select frames of cell travel. ......................................................................................... 19 Figure 13: Cell tracking demonstrating cell travel. ...................................................................... 20 Figure 14: Select frames of cell ejection....................................................................................... 20 Figure 15: Cell tracking results for cell ejection. .......................................................................... 21 Figure 16: Selected frames of a cell demonstrating cell reflection ............................................... 22 Figure 17: Cell tracking results of cell reflection ........................................................................ 23 Figure 18: The vertical cell position and velocity of a cell exhibiting reflection and travel ........ 24 Figure 19: The radial motions of a cell normalized over the cross-sectional radius .................... 26 Figure 20: Cell mapping ............................................................................................................... 27 Figure 21: Image of the inkjet nozzle in the PDMS nozzle holder ............................................... 30 vii  Figure 22: Schematic of the nozzle holder fabrication process .................................................... 31 Figure 23: Schematic of the µPIV experimental setup. ................................................................ 34 Figure 24: Brightfield image of the inkjet nozzle ......................................................................... 34 Figure 25: A double frame fluorescence image pair used for PIV analysis ................................. 36 Figure 26: PIV vectors .................................................................................................................. 39 Figure 27: Measured centerline velocity at 50 µm away from the nozzle orifice. ....................... 41 Figure 28: Diagram depicting a cross-section of the liquid-glass interface of the inkjet nozzle .. 42 Figure 29: The degrees of deviation in the radial direction due to the liquid-glass interface ....... 44 Figure 30: Centerline velocity for selected time points ................................................................ 45 Figure 31: Axial velocity plotted across the inkjet nozzle radial cross-section ............................ 46 Figure 32: Consecutive frames of a droplet dispensing the Ficoll PM400 cell suspension ......... 51 Figure 33: Cell tracking of a droplet ejection event of cell travel in the 10% Ficoll PM400. ...... 52 Figure 34: Cell mapping for printing a cell solution suspended in Ficoll PM400 ........................ 53  viii  List of Symbols Θ Angle of light beam to tangential plane Bo Bond number D Cell diameter ρp Density of particle d Distance of particle from center µ Dynamic viscosity e Error ρf Fluid density tf Fluid flow timescale g Gravitational acceleration  r Nozzle radius Pa Pascal n Refractive index v Sedimentation velocity St Stokes Number σ Surface Tension ∆t Time between frames τ Time Constant φ Volume fraction ix  List of Abbreviations  BSA Bovine Serum Albumin DOC Depth of correlation DoD Drop on demand FPS Frames per second µPIV Micro-Particle Image Velocimetry PBS Phosphate buffered saline          x  Acknowledgements I would like to acknowledge Dr. Cheung and Dr. Ahmadi for their guidance and mentorship throughout my graduate studies. xi  Dedication I dedicate my work to my parents, my brother and my significant other.  1  Chapter 1: Introduction Bioprinting is an additive manufacturing technology with the goal of producing complex biological constructs through the selective patterning of live cells, biomolecules, extracellular matrices or engineered biomaterials [1–6]. The technology allows for a one step process [7–10] of three-dimensional bottom up fabrication enabled by the layer-by-layer stacking of scaffolding material [11–15]. Approaches towards biofabrication can fall into one of or a hybrid of two main material deposition categories: extrusion based and jetting based. Extrusion based printing continuously dispenses filaments of materials [7, 16–19] whereas inkjet printing ejects droplets onto a substrate [20– 27]. Extrusion based printing can be further sub-categorized into pneumatic and mechanical filament extrusion and jetting based droplet deposition can be sub-categorized into laser, thermal or piezoelectric actuation. Each method has their own unique advantages and limitations. Extrusion based printing offers short fabrication time, low sensitivity to the viscosity of the liquid and simultaneous deposition of multiple cell lines or materials. However, the method is limited by low printing resolution (>100 µm) and high shear stresses resulting in reduced cell viability [28–32]. Inkjet based deposition methods typically exhibit high throughput, high resolution (20-100 µm), and high reported cell viability rates [33–35]. However, this technique is limited by a strong dependence on ink viscosity and low dispensed volumes [36–38]. For the presented work, cell deposition by piezoelectric inkjet nozzles was investigated.  The first experiment demonstrating the feasibility of cell dispensing from inkjet printers was performed by Wilson and Boland using a modified commercial thermal inkjet printer [39–40]. Drop-on-demand (DoD) inkjet printing technology have been widely adapted for biofabrication applications which aims to generate complex 2D or 3D biological constructs through the selective patterning of cells, extracellular matrix or scaffolding material [2]. The 2  controlled deposition of cells through inkjet nozzles has been utilized in many applications for tissue engineering [36, 41–43], cell assays [35], stem cell research [44] or therapeutics [45–46]. Piezoelectric printheads generate droplets through the deformation of the element causing a pressure wave to propagate through the inkjet channel. As the pressure wave reaches the nozzle orifice, the pressure gradient across the liquid-air interface causes the liquid to be extruded and subsequently ejected [47]. Although the pressure waves generated by the piezoelectric element can potentially damage the cell membrane [48] high dispensed cell viability rates of greater than 85% have been studied and reported under normal printing conditions [36, 49–51]. Similarly, high cell viability rates were also observed in thermal inkjet printers [48, 52–53].  However, it was found that for cells suspended in culture media or saline solutions, the rate of cells dispensed from inkjet systems did not follow the Poisson or any other statistical distributions [54–57]. This was determined experimentally by dispensing discrete droplets on a substrate and counting the frequency of the number of encapsulated cells per droplet. This rate was then compared with a theoretical expected Poisson distribution. If the rate of dispensed cells followed the expected Poisson distribution, it would indicate that the cells suspended in the printhead were randomly distributed in the media and that the droplet dispensing process does not have any effect on a cell's likelihood to be dispensed. Therefore, if the rate of dispensed cells did not follow the Poisson distribution, it would indicate that other forces are affecting the cell's ability to be dispensed with the ejected droplet. Previously, this has been mainly attributed to the cells settling within the inkjet reservoir as the cells are typically denser than their surrounding medium resulting in a decrease in the number of cells reaching the nozzle to be ejected over time [1, 54]. This has led to research investigating methods to counteract the effects of cell settling including gentle agitation of the inkjet reservoir [58] or the use of a neutrally buoyant media [59, 3  60]. It was discovered that gentle periodic agitation through the use of a magnetic stirrer placed in the ink reservoir was not effective in resuspending the cells and in fact promoted cell aggregation. However, through rheological manipulation of the suspension, improvements in the cell deposition rates was observed in both studies.  The curved air-glass and glass-liquid interface present within a tapered cylindrical inkjet nozzle presents challenges in imaging within the nozzle due to light refraction at the refractive index mismatched material boundaries. In a study conducted with Yamaguchi et al. [61] the tapered cylindrical geometry was flattened to facilitate imaging within the nozzle. In that study, regions with a high probability of cell encapsulation were identified. Another similar study was conducted by Gross et al. [62] where a flat microfabricated inkjet-like cell manipulator was developed. As the geometry of the nozzle is planar, it does not experience any significant optical distortion during imaging within the nozzle. Both studies conducted by Yamaguchi et al. and Gross et al. are similar in that a region within the nozzle near the nozzle orifice was identified where cells experience a high probability of being encapsulated in the ensuing droplet. As a result, the nozzle volume was divided into two regions of ejection and non-ejection. In both studies, a camera equipped with a long working, large depth of field (~200 µm) objective lens was used for autonomously identifying the presence of a single cell within the ejection region within the nozzle and selectively dispensing that droplet while disposing of all other remaining droplets. While high single cell encapsulation rates of 80-98% were achieved using this visual feedback approach, the developed system would be limited by the processing time necessary to identify a cell within the ejection region and the high number of disposed droplets of empty droplets or droplets with more than one cell.  4  While visual feedback cell printing systems can overcome the inhomogeneous cell dispensed rates by selectively depositing droplets with single cells, there has been no previous research conducted on the cell's motions within the inkjet nozzle during droplet actuation.  In order to further understand the hydrodynamics behind inkjet cell dispensing, micro-particle image velocimetry (µPIV) can be performed on the inkjet nozzle to experimentally resolve the flow field evolution during droplet dispensing. µPIV provides a non-intrusive method for measuring the velocity fields present within a flow [63, 64].µPIV is a microscopy based flow visualization technique used to resolve the instantaneous velocity fields within a fluid which is achieved by seeding the fluid with small fluorescent tracer particles. The tracer particles are assumed to faithfully follow the fluid's motions. The entire volume within the microchannel is illuminated by a pulsed laser excitation source. The excited fluorescent tracer particles emits light at a longer wavelength which is then captured by a camera mounted on the microscope. The objective lens is focused on a plane of interest where the tracer particles’ motions within the depth of correlation are used to calculate the velocity vector. An image pair acquired at a pre-determined time separation is used to determine the present velocity field. Each image is subdivided into interrogation windows. Each interrogation window is cross-correlated to determine a corresponding velocity vector for that specific region. The cross-correlation function is a sliding dot product mathematical operation of two images where the peak of the calculated cross-correlation array would indicate the displacement of the particles within the interrogation window between the two frames. Using the time between the acquisition of the two images, and the cross-correlation peak, the velocity vector can be determined. The cross-correlation of all the interrogation windows within the image would produce a velocity vector field for that instance in time. As a result, the interrogation window size determines the spatial resolution of the µPIV 5  system. To increase the density of velocity vectors within an image, the interrogation windows can be subdivided to overlap. As long as the interrogation window size remains the same, the spatial resolution would not be altered; only the density of the velocity vectors would change.  Fluorescence µPIV was first performed on a planar custom inkjet cartridge [65] to measure the instantaneous velocity across droplet actuation and the shape of the meniscus. However, the geometry of a planar inkjet cartridge differs from that of a tapered cylindrical nozzle and may result in different velocity fields during the droplet ejection process. From that work, it was observed that a recirculation region begins to develop at the periphery of the inkjet nozzle before droplet break-off. After droplet break-off, complete flow reversal was observed to occur. Castrejon-Pita et al.[66] performed a hydrodynamics study by ultra-high speed shadowgraph imaging of the flow inside a tapered cylindrical glass inkjet nozzle. Using frame rates of 500,000 fps, a time between frames of 2 µs was achieved. However, as a limitation to high speed imaging, acquiring at the higher frame rates results in a reduction in the captured image resolution. The camera utilized in the study was only capable of capturing at 310 × 260 pixels. The reduced image resolution results in a reduced field of view and a decrease in the number of velocity vectors which could be defined. From their study, recirculation zones at the periphery of the nozzle were observed to occur before droplet break-off with complete flow reversal after droplet break-off. IAlso in that work, the authors used shadowgraph images and a setup that gave a depth of field of approximately 200 μm in an inkjet nozzle with an 80 μm diameter orifice. As a result, their velocity vectors were integrated across the entire depth of field. However, with the presence of a non-uniform flow field, as indicated by the recirculation zone, errors would be present within the measurement due to the large depth of field. In addition, due to optical aberration from the glass curvature and radial magnification due to refractive index 6  mismatch, the nozzle appeared approximately 50% wider in the captured images. As a result of refraction of the light at the curved air-glass and glass-fluid interface, only the velocity vectors calculated along the centerline could be accurate as all other measured velocity vectors would be influenced by radial optical distortion.  The goal of the work presented here is to improve our understanding of the phenomena underlying current inkjet biofabrication technologies, specifically with the behaviors of cells within the inkjet nozzle during printing. This is achieved through 1) classification of the cell behaviors within the inkjet nozzle during printing through the study of the cell motions, 2) performing µPIV on the inkjet nozzle to understand the flow field evolution during droplet ejection and 3) improving the reliability of cell printing through the understanding of the cell's motions and hydrodynamics within the inkjet nozzle.  7  Chapter 2: Characterization of Cell Behaviors in PBS A study of cell motion within a piezoelectric inkjet nozzle was carried out using a high speed imaging system designed to produce images with a low depth of field and minimal distortion. The study aims to characterize the cell's behaviors in response to a droplet ejection under conditions typical to current inkjet printing setups.  2.1 Materials and Methods 2.1.1 Piezoelectric Inkjet Nozzle A piezoelectric inkjet nozzle with an 80 μm diameter orifice (MicroFab MJ-ABP-01) was utilized in this study. The inkjet consisted of a 21.7 mm long glass cylindrical channel with an inner diameter of 0.475 mm which tapered at one end to form the head of the inkjet nozzle. An annular piezoelectric element positioned at the middle of the glass channel actuates the system.  For printing, the cell suspension was supplied to the nozzle through a 0.79 mm inner diameter flexible tubing (Tygon AAC00001) from an external reservoir by gravitational forces and capillary action. The back pressure at the nozzle inlet was measured continuously throughout the experiment by a differential pressure sensor (Omega PX139-0.3D4V) and read by a microcontroller (Arduino Uno). This inkjet nozzle was utilized in all of the presented experiments. The back pressure during the printing was fixed at -500 Pa during the printing of cells suspended in PBS by adjusting the relative height of the external reservoir to the inkjet nozzle.  2.1.2 Cell Suspension Preparation MCF-7 breast cancer cells with an average diameter of 12 μm and density of 1.068 g/ml were cultured in an incubator (37 oC, 5% CO2) until confluency in RPMI 1640 culture media [67]. During cell preparation for printing, the cells were separated from the flask with 0.25% trypsin-8  EDTA and treated with bovine serum albumin (BSA). BSA is a protein used to prevent non-specific binding of the cells which may prevents clogging of the nozzle during printing. To increase the contrast of the cells during brightfield imaging, the cells were stained with 0.1% Toluidine Blue which stains the nucleic acid of the inherently transparent cells to dark blue in color [68]. After staining, the excess Toluidine Blue was washed away and the cells were re-suspended in PBS to a final concentration of 1,500,000 cells/ml as verified by a hemocytometer. The surface tension of the PBS solution was measured to be 65.36 mN/m using a pendant-drop tensiometer (Attension Theta) and the viscosity was 1.07 mPa·s as measured using a rheometer (Physica MCR 301). 2.1.3 Imaging Setup  Figure 1: Diagram of the imaging setup designed for imaging cells within the inkjet nozzle. The system consists of an inkjet nozzle fixed horizontally across a microscope stage. The inkjet nozzle was driven by an amplified actuation signal produced by an arbitrary function generator. An image of the inkjet nozzle in the PDMS holder is shown in the circular inset. High speed images were acquired with a high speed camera attached to the viewport of the microscope which allows for the imaging of the cell's behaviors within the 9  inkjet nozzle during droplet actuation. A grayscale image captured by the high speed camera with the presented imaging setup is shown in the rectangular inset. Figure was produced with the assistance of Horace Yu.  An inverted microscope (Nikon Eclipse TE2000-U) and a high speed camera (Phantom Miro 4) was used to capture brightfield images of the inkjet nozzle during printing (Figure 1). A Nikon CFI Plan Fluor 10×, 0.3 NA objective lens was used for this study and adjusted to be focused on the middle plane of the inkjet nozzle. As the inkjet nozzle's body is 3.75 mm in diameter, a super long working objective lens was required for overcoming the thickness of the inkjet printhead and the PDMS holder. The utilized objective lens had a working distance of 16 mm. The objective lens has a depth of field of approximately 15 μm which was comparable to the diameter of the cells in the inkjet nozzle. This ensured that only cells within a 15 μm thick volume around the middle plane of the nozzle would be focused and captured through high speed imaging. The high speed camera was capable of capturing at a rate of 23,121 frames per second (fps) and output an 8-bit greyscale video with a resolution of 120 x 160 pixels. Due to the low resolution of the high speed camera, the magnification of the objective lens utilized was limited in order to capture a sufficiently large field of view to study the cell's motions.  2.1.4 Droplet Formation The pressure wave necessary to generate an ejected droplet was driven by an arbitrary waveform generator (Agilent 33220A) and a 50× amplifier (Trek Model 603) in series. Droplet ejection was performed at 60 Hz using a bipolar waveform with varying voltage amplitudes for printing droplets of different volumes [69]. As there is a direct relation between the amplitude of the actuation waveform and the ejected droplet volume, droplets of different sizes can be easily studied. Two different droplets were produced, a ±16.5 peak-to-peak voltage (Vpk-pk) (Figure 2) 10  and a ±22.5 Vpk-pk (Figure 3) actuation waveform was used which produced a 115 pL and 310 pL droplet respectively. The two actuation wavefom amplitudes were identified by iteratively increasing the actuation waveform amplitude by increments of 0.5 Vpk-pk and imaging of the resulting droplet. Through this two actuation amplitudes were identified which created consistent droplets of different volumes and did not produce any trailing satellite droplets. The droplet volume was determined by fitting an image of a droplet to a circle and modeling the droplet as a sphere.   Figure 2: A droplet ejection event as captured by the high speed imaging of the 115 pL droplet. The three phases of any droplet ejection was: a) The initial liquid column extrusion phase, b) the droplet break-off and c) the damping oscillating meniscus. The scale bar represents 100 µm.   Figure 3: The droplet ejection phases of the 310 pL droplet: a) liquid extrusion, b) droplet break-off and c) damping meniscus. Scale bar represent 100 µm.  Aside from the differences in droplet volume, the two droplets break-off at different points from the extruded liquid. The smaller droplet breaks-off outside the inkjet nozzle, leaving a portion of the initially extruded liquid to retract back into the nozzle printhead. The larger 11  droplets breaks-off within the nozzle orifice resulting in the liquid meniscus moving outwards of the printhead after the droplet has been ejected. The two droplets were both tested to observe for any differences in cell dispensing performance due to the droplet break-off process.  2.1.5 Cell Tracking Algorithm The presence of a cell within the inkjet nozzle was identified through an image processing and cell segmentation algorithm using Matlab® (MathWorks). For each frame in the captured high speed video, an image segmentation routine was implemented to highlight the features of the cell present within the nozzle. The process of the cell tracking algorithm is displayed in Figure 4.  Figure 4: The process of the image segmentation routine. a) A frame captured from the high speed camera of a cell within the inkjet nozzle. b) The resulting image from part (a) after the spatial bandpass filter. c) The center of the cell as identified by the bandpass filter plotted over a blue star marker (*) over the original image. d) A surface plot of the cell in the original image highlighted by the blue square. e) The surface plot of the filtered image (b) of the region outlined in red. The gradient between the cell and background was increased through background subtraction and enhancing the features of the cell.    12  The grayscale image was first filtered with a Gaussian low pass filter to remove any high frequency noise from the high speed camera. The image was then inverted and filtered with a spatial bandpass filter [70]. The filter performs background subtraction and highlights the spherical cell within the image which would appear as a bright spot on a dark background. The cell was then identified as the local maximum value within the filtered image. The vertical and horizontal position of the cell was recorded and the process is repeated for the next frame in the video.   2.1.6 Cell Mapping As a cell advances through the inkjet nozzle, for each droplet dispensed, the vertical and horizontal position of a cell within the inkjet nozzle before a droplet ejection was plotted on an image of the inkjet nozzle. Each point was categorized according to the behavior exhibited by the cell on the subsequent droplet ejection. The process was repeated for every captured high speed video until the map was fully populated. The resulting map allows for the identification of localized regions within the nozzle which will exhibit a high probability of a certain cell behavior. The small depth of field in our imaging system allows for the extrapolation of the mapped behaviors to be representative of the entire 3D volume of the inkjet nozzle due to the axis-symmetric geometry of the nozzle and assuming a radial symmetry in the observed cell behaviors.  2.2 Results Through the analysis of the high speed imaging and cell tracking results, the cells were observed to exhibit three possible behaviors with each droplet ejection event. Each behavior was classified by the cell's longitudinal motions and were termed: cell travel, cell ejection and cell reflection. 13  Only longitudinal motions were considered as the radial component of the fluid flow are relatively small with respect to the longitudinal component and related to the tapering geometry of the inkjet nozzle. Additionally, optical aberration in the longitudinal direction are negligible within the inkjet nozzle geometry [66, 71]. 2.2.1 Cell Printing in 115 pL Droplets Cell travel (Figure 5) was observed to occur to cells and characterized according to the vertical cell position before and after a droplet ejection event (Figure 6). This behavior is defined as the net longitudinal displacement of the cell towards the nozzle orifice in response to a droplet ejection.   Figure 5: Selected frames captured from the high speed camera of a cell exhibiting cell travel. The initial and final position of the cell within the inkjet nozzle is highlighted by the red arrow. The scale bar represent 100 µm.  Cell tracking results demonstrated that during cell travel, the cell initially displaces towards the inkjet nozzle with the extruded liquid column then oscillates with the damping meniscus before settling at a final position closer to the inkjet orifice than before the droplet ejection event. On all cell tracking plots, the inkjet nozzle orifice is defined as y = 0 and plotted over one droplet ejection event. 14   Figure 6: The vertical cell tracking results of a cell experiencing cell travel.   Cell ejection (Figure 7) was defined as the cell exiting the inkjet nozzle with the ejected droplet. As the droplet was produced, the cell displaces out of the inkjet nozzle with the extruded liquid column. The cell can no longer be observed within the inkjet nozzle and is assumed to be encapsulated within the ejected droplet.  15   Figure 7: Selected frames captured from the high speed camera of a cell exhibiting cell ejection. The scale bar represent 100 µm.  From the cell tracking results (Figure 8), as the extruded liquid column forms during the first phase of the droplet ejection event, the cell was initially observed to displace downwards towards the nozzle orifice then disappears from view inside the liquid column. After the droplet breaks-off and the meniscus fully retracts back into the nozzle, the cell disappeared from the field of view and is assumed to have exited the nozzle with the ejected droplet. 16   Figure 8: The vertical cell tracking results of a cell experiencing cell ejection.  Lastly, cell reflection (Figure 9) was observed in the smaller 115 pL droplets for cells suspended in PBS.   Figure 9: Selected frames captured from the high speed camera of a cell exhibiting cell reflection. The scale bar represent 100 µm.  17  During cell reflection (Figure 10), the cell was initially observed to displace towards the nozzle orifice with the extruded liquid column. However, after droplet break-off, as the meniscus begins to retract, the cell reappears inside the nozzle and was displaced to a vertical position further upstream than before the droplet was produced. The observation of the occurrence of cell reflection is significant because it demonstrated that the droplet production process can have a negative effect on a cell's likelihood to be dispensed. This, in addition to the effects of cell sedimentation and aggregation, could explain the observed inhomogeneous cell deposition rates found in current inkjet cell printing systems.   Figure 10: The vertical cell tracking results of a cell experiencing cell reflection.  18  Through cell mapping (Figure 11), a cell ejection region was observed near the nozzle orifice. Surrounding the ejection region was a region of cell travel. Overlapping the cell travel region and surrounding the cell ejection region was a reflection region where a cell has a chance of exhibiting cell reflection.   Figure 11: Cell mapping performed for cells suspended in PBS with 115 pL dispensed droplet volume. The blue '+' marker represents a cell's initial position before it was ejected. The yellow 'o' marker represents the initial position of cells that experienced cell travel, and the red '' represents the initial position of cells that experienced cell reflection. In total, 130 cells were studied to populate this map with 66 instances of cell travel, 30 instances of cell reflection and 34 instances of cell ejection.  19  2.2.2 Cell Printing in 310 pL Droplets Cell travel (Figure 12) was defined as a net displacement of the cell towards the nozzle orifice after a droplet ejection event. During the process of cell travel, the cell remains within the nozzle throughout the droplet ejection event. Through the vertical cell tracking in Figure 13, as the initial liquid column was extruded from the inkjet nozzle, the cell experiences a displacement towards the nozzle orifice. After droplet break-off, the cell was observed to oscillate with the damping meniscus before settling to a final position closer to the nozzle orifice than the initial longitudinal position before the droplet production.   Figure 12: Select frames captured with high speed imaging of cell travel. Scale bar represent 100 µm.  20   Figure 13: Cell tracking demonstrating the vertical cell position over a droplet ejection event demonstrating cell travel. Cell ejection (Figure 14 and 15) was observed to occur for cells near the inkjet nozzle orifice.  Figure 14: Select frames captured by high speed imaging of cell ejection. Scale bar represent 100 µm.  21   Figure 15: Cell tracking results for cell ejection. The cell disappears from the field of view as soon as the liquid column forms and is ejected with the droplet.   Cell reflection (Figure 16) was defined as the net displacement of the cell away from the nozzle orifice after a droplet ejection event. Similar to the case of cell travel, (as shown in Figure 13) during the initial liquid extrusion, the cell displaces towards the nozzle. However, as the droplet breaks-off, as shown in the cell tracking results (Figure 17), the cell was projected away from the nozzle orifice. As the liquid meniscus oscillates, the cell follows the damping motions of the meniscus but in addition, continues to displace away from the nozzle orifice. After the droplet ejection event, the cell settles at a position further away from the initial position than before the droplet ejection. The occurrence of cell reflection will be detrimental to the ability to 22  reliably dispense cells from inkjet systems. This is the first time which the cell reflection behavior has been observed and it is  hypothesize that its effect plays an important role in the reliability of printing cells.  Figure 16: Selected frames during a droplet ejection event of a cell demonstrating cell reflection. Scale bar represents 100 µm.  23   Figure 17: Cell tracking results of cell reflection. The break in the graph represents the frames in which the cell momentarily leaves the field of view of the system.   Plotting the cell motions and velocity with the phases of the droplet ejection showed that cell reflection occurs during droplet break-off (Figure 18). During cell reflection, the cell experiences a large velocity (1.85 m/s) into the inkjet nozzle while a cell experiencing travel experiences a much lower velocity (0.34 m/s). The high velocity which was imparted on the cell allows for it to be displaced further upstream from its initial position before the droplet ejection. At all other phases of the droplet ejection (droplet formation, exiting meniscus and retracting meniscus), the cell's motions were similar. During the droplet formation for both cases, the cells were observed to be displaced towards the nozzle orifice (note: in this case of cell reflection, the 24  cell was initially displaced to a position outside of the field of view but was assumed to be inside the extruded liquid column and thus displaced towards the nozzle orifice). After droplet break-off, the cells were observed to oscillate with the exiting and retracting meniscus.   Figure 18: The vertical cell position and velocity of a cell exhibiting reflection and travel. The background of the plots are color coded to reflect the phase of droplet ejection. Selected images from each phase of the droplet ejection event are displayed. 25   To show that the radial motion of the cell was mostly influenced by the tapering of the inkjet nozzle, the radial position of the cell (r) was normalized by the cross-sectional radius of the inkjet nozzle (R), where R depends linearly on the axial position, and plotted over a droplet ejection of a cell experiencing cell travel (Figure 19). If the cell's radial motion is due to the tapered nozzle geometry, the normalized radial position should be equal to a constant value. During the initial phases of the droplet ejection, the cell's motions are influenced by the hydrodynamics of the droplet break-off and can experience radial motion. However, during the oscillating meniscus, the cell's normalized radial position was observed to be nearly consistent around 0.5. Therefore, in characterizing the observed cell behaviors, the radial motions of the cells were neglected as the radial motions are mainly attributed to the geometry of the inkjet nozzle. 26    Figure 19: The radial motions (r) of a cell normalized over the cross-sectional radius (R) plotted across a droplet ejection. The background of the plot was color coded to reflect the phases of the droplet ejection event.   Cell mapping (Figure 20) revealed three regions where each of the three observed behaviors were localized similar to the previous map (Figure 11). Surrounding the nozzle orifice is a cell ejection region. Outside of the cell ejection region is a region exhibiting a high probability of cell travel. However, overlapping parts of the travel region and surrounding the ejection region is a region of cell reflection.  27   Figure 20: Cell mapping demonstrating the regions within the inkjet nozzle experiencing cell travel (yellow Ο markers), cell reflection (red  markers) or cell ejection (blue + markers). In total, 134 cells were characterized: 78 experiencing cell travel, 18 experiencing cell reflection and 37 experiencing cell ejection.   The cell travel region was observed to be the largest of the three regions in area and localized upstream of the inkjet nozzle. It was by this phenomenon which the cells displace towards the nozzle orifice to be dispensed. If the cell travels into the ejection region, the cell would be dispensed in the ensuing droplet. However, if the cell was carried to the reflection region, the cell can be given an initial momentum during the droplet break-off and reflected to a position further upstream from its initial position before the droplet ejection actuation.  In the first cell map of the smaller ejected droplet (Figure 11), the cell ejection region appears smaller and may be due to the lower volume of the ejected droplet. All three possible 28  cell behaviors previously observed in printing with PBS with the smaller droplets were again observed in the larger volume droplets. Therefore, the cell's motions are minimally affected with the volume of the ejected droplet. 29  Chapter 3: µPIV in a Cylindrical Inkjet Nozzle In order to further understand the hydrodynamic conditions present within the inkjet nozzle during cell printing, micro-Particle Image Velocimetry was performed on the tapered cylindrical inkjet nozzle during printing. Imaging through cylindrical geometries presents difficulties in that optical aberrations at the air-glass interface critically reduce the fluorescent signals received from the seeded flow tracing particles such that no useable images can be captured for µPIV measurements. This problem was overcome with a specially designed PDMS nozzle holder which allows for imaging of fluorescent particles within a cylindrical inkjet nozzle with good signal quality. Using the custom holder and a microsecond resolution image capture triggering system, the flow field evolution during droplet ejection was captured. 3.1 µPIV Materials and Methods 3.1.1  Inkjet System We used a MicroFab MJ-ABP-01 (MicroFab, USA) piezoelectrically actuated tapered cylindrical inkjet nozzle as previously described in Chapter 2. Actuation of the piezoelectric element is achieved by a ±11.25V, 60 Hz bipolar waveform generated by an arbitrary function generator (Agilent 33220A) and a 50x amplifier (TRek Model 603) placed in series.  3.1.2 PDMS Nozzle Holder One of the challenges for μPIV in cylindrical channels, such as the nozzle used in this study, is refraction of the excitation laser source at the curved air-glass interface, such that insufficient light would reach the seeded fluorescent particles to produce a detectable image. To facilitate fluorescence imaging for µPIV, the inkjet nozzle is fixed across the stage of an inverted microscope (Nikon Eclipse TE 2000-U) by a PDMS holder as shown in Figure 21. The PDMS holder serves two major functions: (1) it affixes that nozzle horizontally above the objective lens 30  of the microscope; (2) it provides a refractive index matching material of n = 1.4 [72] to the glass nozzle of n = 1.5 for imaging with minimal distortion and attenuation of the excitation beam and emitted fluorescence signal at the PDMS-glass interface. The PDMS holder effectively transforms the outside of the tapered cylindrical geometry of the nozzle to a flat rectangular prism. To achieve these goals, the PDMS holder is fabricated to closely fit the inkjet nozzle with the glass nozzle, thus effectively altering the outside of the nozzle's cylindrical geometry to a rectangular prism without impeding the nozzle's function.   Figure 21: Image of the inkjet nozzle in the PDMS nozzle holder. The region highlighted with the dashed box provides a magnified view of the inkjet nozzle within the PDMS holder acquired with a stereo microscope under 4x optical zoom. For clarity, the features of the inkjet nozzle within the PDMS are outlined.    The PDMS from the holder fully surrounds the glass nozzle, transforming the tapered cylindrical geometry to a trapezoidal geometry, thus effectively removing the effect of light refraction at the curved air-glass interface.   31   Figure 22: Schematic of the nozzle holder fabrication process, showing the cross section across the middle plane of the inkjet nozzle. a) An identical inkjet nozzle is fixed horizontally across a container used as a mould for the PDMS holder. b) A 3D printed polymeric part is fitted to the front face of the nozzle orifice in order to prevent PDMS from filling the nozzle and impeding the droplet ejection process. c) PDMS is mixed and prepared for casting. d) PDMS is cast on the inkjet nozzle and 3D printed part. PDMS fills all open cavities including the space between the inkjet nozzle and the 3D printed part, while the inkjet nozzle cavity remains free of PDMS. The PDMS is then cured. e) The 3D printed part is removed leaving the front face of the inkjet nozzle exposed. The inkjet nozzle is then removed. f) The completed PDMS inkjet nozzle holder. It allows the tapered cylindrical inkjet nozzle to be encased in PDMS, giving optical access with minimal distortion while leaving the inkjet orifice free to eject droplets. The fabricated PDMS holder is then bonded onto a microscope slide.   The nozzle holder is fabricated by casting PDMS around an identical inkjet print-head and a three-dimensional (3D) printed negative mold (Figure 22). The 3D printed mould, composed of an opaque photopolymer (VeroWhitePlus), is designed using CAD software (SolidWorks®, Dassault Systemes) and printed by a 3D printer (Objet24, Stratasys). It is designed to fit in front of the inkjet nozzle orifice during PDMS casting in order to prevent PDMS from entering inside the nozzle mould. To fabricate the PDMS holder, an inkjet nozzle which is identical to the nozzle that will be used in the µPIV work is placed horizontally over a container. To eliminate any tilting of the inkjet nozzle within the PDMS holder, the PDMS was cast on a leveled surface. The 3D printed part is then carefully placed in contact with the orifice 32  face of the inkjet nozzle. PDMS base and hardener is mixed in a 10:1 ratio and degassed before being cast around the nozzle and 3D printed part in the container. In order to minimize disturbance of the sensitive setup of the inkjet nozzle and the attached negative mold, the PDMS is allowed to cure at room temperature for 6 hours. Once cured, the negative mold is peeled away from the PDMS revealing a cavity and exposing the front face of the inkjet nozzle. The inkjet nozzle is then removed leaving a PDMS prism with a hollow core with the features of the glass inkjet nozzle tip. The PDMS is then bonded to a 1 mm thick glass microscope slide using oxygen plasma to structurally reinforce the PDMS holder.  Once fabricated, the PDMS fully surrounds the periphery of the glass nozzle while leaving the front face and nozzle orifice exposed, permitting unimpeded droplet formation and ejection.  3.1.3 µPIV Imaging Setup The flow tracing particles used for this experiment are fluorescent polystyrene particles which are 1 µm in diameter (Thermo Scientific R0100).  The particles have a peak excitation wavelength of 542 nm and a peak emission wavelength of 612 nm. The bead solution is suspended in phosphate buffered saline at a concentration of 0.03% w/v in preparation for printing [73]. The viscosity of the bead solution was measured to be 0.00112 Pas at 20.4 °C using a rheometer (Physica MCR 301, Anton Paar) and surface tension was 67.61 mN/m as measured using a tensiometer (Attension Theta, KSV Instruments). The volume fraction (φ) of our bead solution was φ = 2.8×10-4; it can be assumed to be a Newtonian fluid as shear-thinning behavior was not observed for low volume fraction (φ  < 0.25) suspensions of spherical particles [74]. The particle solution is supplied into the inkjet device by gravitational forces and the back pressure in the system, as measured by a differential pressure sensor (Omega PX139) connected near the nozzle's inlet, is maintained at -500 Pa.  33  Volume illuminated µPIV is achieved through an inverted microscope setup with a Nd:YAG laser (New Wave Research Solo PIV) which is capable of producing a 5 ns pulsed laser at 532 nm for an illumination source. The average laser power per double pulsed cycle is measured to be 350 µW (ThorLabs PM100). A schematic of the complete imaging setup is displayed in Figure 23. A 20x, 0.35 NA super long working distance objective lens (Nikon CFI L PLAN EPI SLWD) is focused across the middle plane of the inkjet nozzle. Due to the tapered geometry of the inkjet nozzle, the middle plane was determined as the plane in which the nozzle cross-section appears the widest. The depth of correlation (DOC) of the objective lens is calculated to be approximately 25 µm [75]. The DOC was determined with a threshold value (ε) of ε = 0.01. Therefore, fluorescence intensity of particles outside of the DOC is sufficiently low that it does not significantly influence the velocity vector calculation. Double frame images for PIV analysis were captured by a LaVision sCMOS camera which captures two consecutive 16 bit greyscale images spaced ∆t apart at 2160 x 2576 pixels with a resolution of 0.654 µm/pixel. The time between frames were acquired with a ∆t of 2 or 5 µs delay depending on the expected flow velocity at each measured time point. A brightfield image of the middle plane of the inkjet nozzle acquired with the described imaging setup shows the features of the inkjet nozzle (Figure 24). 34   Figure 23: Schematic of the µPIV experimental setup. At every droplet ejection event, the arbitrary function generator produces an actuation waveform while simultaneously sending a trigger output which is received by the microcontroller. The microcontroller then implements a short delay and sends an image capture trigger which is received as a cyclic trigger by the PIV camera. Image capture is then achieved at every least common multiple between the droplet ejection frequency and the Nd: YAG laser's maximum pulsing frequency.   Figure 24: Brightfield image of the inkjet nozzle, using a mercury arc lamp as the light source. In µPIV a pulsed laser is used to acquire the image; other components of the setup remain the same. 35   The timing of the PIV frame capture is synchronized with the inkjet droplet actuation by an external cyclic trigger system.  At the beginning of each actuation cycle, the arbitrary function generator produces a +3.3 V TTL trigger output, the rising edge of which is received by a microcontroller (Arduino Uno) as an external hardware interrupt. The interrupt service routine on the microcontroller implements a delay (~900 µs) that allows the transient propagation of the pressure wave from the piezoelectric element to the nozzle orifice as well as a controllable microsecond resolution delay which permits imaging of the different phases of the periodic droplet ejection events. At the end of this delay, the microcontroller outputs a +5 V TTL signal to the PIV camera where the rising edge initiates a double pulse from the excitation laser spaced with ∆t spacing, synchronized with the double frame image capture which would be used to produce the PIV velocity vectors (Figure 25). As the droplet ejection rate of 60 Hz is greater than the maximum double pulsing frequency of the laser excitation source of 10 Hz, the cyclic trigger initiates a phase-locked image capture at every least common multiple between the two periods, which corresponds to a PIV image at every 6th ejected droplet. By tuning the delay in the microcontroller, the triggering system permits control over the phase in which the periodic droplet ejection event is captured. 36   Figure 25: A double frame fluorescence image pair used for PIV analysis. Two consecutive images acquired in close temporal proximity permits the motion and thus velocity of the seeded particles to be measured. The camera initiated the capture of the first frame of the image pair at t = 90 µs and a ∆t of 5 µs was utilized to capture the second frame at t = 95 µs. The acquired greyscale image pair is pseudo-colored blue. The onset of droplet formation is normalized to t=0 µs as determined by the time point before fluid motion was observed.   3.1.4 PIV Analysis Before PIV analysis, the image is preprocessed using a power filter of two by multiplying each pixel value in an image by itself. This improves the gradient between the fluorescent signals and background, and has been demonstrated to decrease the DOC by a factor of two which in this case would reduce the volume integrated for PIV analysis to a thickness of only 12.5 µm  [76]. Cross-correlation for PIV analysis was performed using Davis 8 software (LaVision, Germany) 37  with an interrogation window of 46 x 46 pixels acquired at 50% overlap. This corresponds to a 15 µm x 15 µm interrogation window and a spatial resolution of 15 µm with velocity vectors calculated every 7.5 µm. Therefore, each velocity vector represents the average velocity within the interrogation window, vertically integrated around a 12.5 µm thick slice across the middle plane of the inkjet nozzle. Due to the axis-symmetry of the cylindrical nozzle, the observed velocity fields within the middle plane of the nozzle can be extrapolated to be representative of the entire volume by assuming radial symmetry in the velocity fields. The orientation of the PIV velocity vectors is oriented in such a way that the lateral flow is positive out of the inkjet nozzle away from the nozzle orifice.  3.2 Results Through the use of a refractive index matching PDMS holder, fluorescent µPIV was performed on a cylindrical inkjet nozzle. The system utilizes an inverted microscope with the nozzle affixed horizontally above the objective lens. The long working objective lens allows for high resolution, low depth of correlation imaging of the center plane of the inkjet nozzle. Due to the axisymmetric geometry, the 2D images are representative of the entire 3D volume of the nozzle cavity and the low DOC allows us to assume that out-of-plane particle motion in the acquired imaging volume is negligibly small. In this setup, the surface tension forces dominate over the gravitational forces, allowing droplet ejection when the inkjet nozzle is placed horizontally over the inverted microscope stage [77]: the Bond number              (1) where ρf is the fluid density, g is the gravitational acceleration, R is the nozzle radius and σ is the surface tension, in the inkjet nozzle is Bo ≈ 0.0002. Due to the density differences between 38  polystyrene and PBS, bead sedimentation is expected to occur over time. The sedimentation velocity for the suspended particles can be calculated by:                    (2) where v is the sedimentation velocity, ρp is the particle density, ρf is the fluid density, g is the gravitational acceleration, D is the particle diameter and µ is the dynamic viscosity. With the 1 µm particles, the sedimentation velocity was 0.028 µm/s. Therefore, the effects of particle sedimentation of the seeded fluorescence particles are negligible due to the small density differences, small bead diameter and small time scales [78]. µPIV was performed on the first 210 µs of the droplet ejection cycle with the onset of droplet formation normalized to t = 0 µs as determined by the time point before fluid motion was observed.  PIV double frame images were acquired 2 to 5 µs apart. Using the experimental setup, the velocity field inside the nozzle can be measured at different times. As seen in Figure 26, the overall velocity trend agrees with previously modeled flow fields with an oscillating pressure boundary condition [79]. Both modeled results from Suh and Son and our measured instantaneous µPIV velocity fields show that the flow was uniform in direction for each sampled time point and no recirculation flows occur. During flow reversal, the velocity vectors gradually decrease in magnitude then completely reverse in direction. Along the centerline, the velocity vectors are entirely composed of the longitudinal component as radial flow along the centerline of the inkjet nozzle is zero. Away from the centerline, radial components of the velocity vector can be observed which can be attributed to the tapering in the nozzle geometry.  39   Figure 26: PIV vectors at a) t = 12 µs, b) t = 32 µs, c) t = 50 µs and d) t = 65 µs. Reference velocity vector found on the bottom left corner of each velocity field represents 2 m/s.   To get a better understanding of the changes in the velocity, the centerline axial velocity at 50 µm away from the orifice was plotted over time with each point averaged across 3 identical samples as plotted in Figure 27. For expected velocity fields greater than ±1 m/s, a time between frame (∆t) of 2 µs was chosen; this included time points at t = 12, 14, 16, 32, 34, 36, 50 and 65 µs. A ∆t of 2 µs permits more accurate measurement of higher velocities; as an example, a measured velocity of 2.3 m/s correlated to an average particle displacement of 15 pixels or 33.3% of the interrogation window. All the other data points were acquired at ∆t = 5 µs. At the 40  onset of droplet formation, the velocity field was observed to be positive as the flow was directed outwards of the nozzle. A peak positive centerline velocity of 2.29 m/s was observed at t = 14 µs. After the peak positive centerline velocity, the velocity field gradually decreases in magnitude until flow reversal occurs between t = 20-25 µs. The flow field then reverses until a peak negative velocity of -2.20 m/s at t = 32 µs. The flow field continues to oscillate for another cycle reaching a secondary positive peak of 1.52 m/s at t = 50 µs and a secondary peak negative value of -1.37 m/s at t = 65 µs. After this, the droplet breakup was observed to occur at t = 85 µs. Before droplet break-off, the net velocity flow was positive as the peak positive velocities were greater than the peak negative velocities and the general flow direction was towards the inkjet orifice. After the droplet break-off, the flow produces a peak negative flow velocity of -1.07 m/s at t = 98 µs and a peak positive velocity of 0.85 m/s at t = 120 µs. For the first time, the peak negative velocity was greater than the subsequent peak positive velocity. This is due to the retracting meniscus which occurs after droplet break-off imparting a negative overall flow on the system.  41   Figure 27: Measured centerline velocity at 50 µm away from the nozzle orifice, averaged over three trials. The onset of droplet formation is normalized to t = 0 µs. The error bars represent the maximum and minimum range of velocities measured at the specific time point.  The observed oscillating velocity fields are a result of the propagating actuation pressure wave as it is reflected at the boundaries of the inkjet channel. Due to the low viscosity of the ink used, the pressure wave experiences low acoustic damping. The oscillating flow fields would be mainly produced by the actuation pressure wave, however other forces such as the capillary action of the extruded liquid would also influence the measured velocity fields within the system.  While the PDMS holder provides a refractive index match to that of the cylindrical glass nozzle, reducing distortion at the air-glass interface, a liquid-glass interface still remains in the system. The distortion observed from this interface is mainly in the radial direction within a tapered cylindrical geometry [66, 71].  The distortion in the axial direction is negligible due to the linear tapering of the nozzle in the axial direction causing a constant refraction angle of the 42  emitted fluorescence signal at the liquid-glass interface. As the velocity vectors are measured by the relative displacement of the particles between the captured double frames images, a constant shift in the particle coordinates would not affect the magnitude of the axial velocity vectors measured. As a result, measurements along the centerline of the inkjet nozzle such as those shown in Figure 26 and Figure 27 are free of any optical distortion in both the position and magnitude of the velocity vectors calculated. The optical distortion in the measurement in the radial direction can be theoretically calculated through ray tracing in equation (6) and is in agreement with previous work [74–75]. The distortion in the radial measurements is dependent on the distance of the particle away from the center point (d) and the cross-sectional radius (R).  Using a ray tracing method, the distortion within the inkjet nozzle can be characterized as shown in Figure 28:  Figure 28: Diagram depicting a cross-section of the liquid-glass interface of the inkjet nozzle with a cross-sectional radius of R. Inset shows the emitted and refracted light path and the subsequent radial measurement error (e) which occurs.  43  The error due to distortion from the liquid-glass interface is calculated by projecting the refracted light ray back to the imaging plane and finding the difference of the projected position relative to the original particle position (Figure 28). This is first done with Snell's law                       (3)                          (4) The angle which the refracted light is deviated from the undistorted path (θd) is then identified by             (5)                         (6)                                (7) Once θd was identified, the radial error (e) can be easily calculated from simple trigonometric identities as depicted by the inset in Figure 28.  e=                                        (8) The radial error (e) and distance of the particle (d) can then be normalized by the total cross-sectional radius (R) to produce a profile of the expected radial error measurements along R. As expected the error is zero during the case of d = 0 and d = R with the error generally increasing with d (Figure 29). 44   Figure 29: The degrees of deviation in the radial direction due to the liquid-glass interface at the boundary of the inkjet nozzle as a function of the distance along the nozzle radius normalized to the cross-sectional radius of the inkjet nozzle. Due to the close refractive indices, the degree of deviation is small relative to the distortion present in a cylindrical air-glass interface.  Figure 30 shows the centerline velocity for selected time points (14 µs, 32 µs, 50 µs, 65 µs and 85 µs) across the axial direction of the inkjet nozzle. As expected, due to the tapered geometry of the nozzle, the centerline velocity decreases (in magnitude) as the distance from the nozzle orifice increases.   45   Figure 30: Centerline velocity for selected time points (14 µs, 32 µs, 50 µs, 65 µs and 85 µs) across the axial direction of the inkjet nozzle. The nozzle orifice is normalized to x=0, with x decreasing with distance into the nozzle.   Due to the nozzle geometry, the magnitude of the centerline velocity increases with distance towards the nozzle orifice as the tapered cross-section constricting the flow.   Due to the liquid-glass interface in the inkjet nozzle, the position of the acquired velocity vectors are radially distorted. This can be easily corrected by shifting each velocity vector position (d/R) by the factor (e/R) according to Figure 29.Although the magnitude of the radial distortion is small, this shifting was performed to correct the measured velocity values at different radial positions (as shown in Figure 31).   46   Figure 31: Axial velocity plotted across the inkjet nozzle radial cross-section at 50 µm, 75 µm and 105 µm away from the nozzle orifice. The selected time points plotted were at a) t = 14 µs, b) t = 32 µs, c) t = 50 µs and d) t = 65 µs. The radial position of each velocity point plotted is corrected based on the expected degree of distortion as shown in Figure 29. No correction to the measured velocity is necessary as the error is only found in the radial direction of the inkjet nozzle. The work presented here is the first direct measurement of the velocity fields within a cylindrical inkjet nozzle. The µPIV results shows an oscillating velocity field within the inkjet nozzle during the droplet ejection cycle. This corresponds with previous work on numerical simulation and measurements of pressure waves within the inkjet nozzle. Wijshoff developed a system named Piezo-Acoustic sensing of INk channels in the Time domain (Paint) to directly measure the pressure waves within the inkjet channel [82]. The system utilized the piezoelectric element which actuates the inkjet nozzle to both produce and record the reflected pressure waves at the piezoelectric element which is placed at the center of the inkjet channel. For this study, a uni-polar actuation waveform was used to generate the droplet. After the actuation signal was 47  produced, the electronics switched the piezoelectric element from actuation mode to receive mode. This allowed the same piezoelectric element to be used (1) to generate the actuation pressure wave, and (2) to measure that same pressure wave as it propagates and reflects across the inkjet channel. For this system, there would be a delay in the beginning of the measured signal as the electronics switch, resulting in a loss of information during the initial actuation of the droplet. Nevertheless, this work provided the first direct measurement of the pressure wave within the inkjet nozzle. The measured Paint signal shows a damped oscillating pressure wave propagating within the inkjet channel.  Additionally, numerical simulations of the flow fields within an inkjet nozzle with an oscillating pressure wave as an input calculated the occurrence of uniform flow oscillations during the droplet formation process, which is also in good agreement with our measured results [79].  In this work, Suh and Son modeled the flow field evolution across a droplet ejection. The major difference from their work compared to other numerical simulations or droplet production was their actuation calculation of the pressure wave input into the system. Using an equivalent circuit model where the physical properties of the inkjet nozzle were converted to equivalent electrical components, it was calculated that an oscillatory flow field was present within the inkjet nozzle. This is analogous to the results observed by Wijshoff's measured Paint signal. Through the use of an oscillatory pressure wave input, Suh and Son demonstrated a uniform flow field to occur during droplet production. In addition, complete flow reversal occurred without the presence of a recirculation region. That numerical result is in good agreement with the presented experimental results obtained from the µPIV study (Fig. 27).  For the first time, fluorescence µPIV was performed on a tapered cylindrical inkjet nozzle. Overcoming refraction of the excitation and emitted light during epi-fluorescence 48  imaging, a refractive index matching holder was developed reducing imaging distortion while the objective lens gave a low depth of correlation, permitting for direct measurement of the flow field. Oscillating flow fields were observed to develop during the droplet formation process as well as after droplet ejection. A peak positive velocity of 2.29 m/s and a peak negative velocity of -2.2 m/s was observed to occur before droplet break-off. The direct measurements of the oscillating flow field confirm previous work on flow field modeling and previous measurements of the pressure waves which implied an oscillatory flow.  49  Chapter 4: Cell Printing in a High Viscosity Fluid With the observation of cell reflection occurring in cells suspended in PBS through cell tracking and an understanding of the flow field present within the inkjet nozzle through µPIV, it is hypothesized  that rheological manipulation of the cell suspension can improve the cell printing outcome by counteracting the effects of cell sedimentation and cell reflection. A high viscosity, biocompatible medium was utilized through the addition of 10% w/v Ficoll PM400 into PBS to experimentally test the hypothesis in an effort to prevent the occurrence of cell reflection and improve the reliability of cell encapsulation in inkjet cell printing.  Cell reflection may be caused by a detachment of the particle's motion from the surrounding fluid flow. This can be analyzed further by considering the Stokes number (St) which relates a particle’s response in a fluid flow and can be expressed as             (9) where τ is the time constant of a particle under acceleration and tf is the fluid flow timescale. The time constant can be expressed as               (10) where ρp is the particle's density, D is the particle's diameter and µ is the dynamic viscosity of the liquid in which the particle is suspended [58, 77]. From the µPIV study performed on the inkjet nozzle, the fluid flow time scale was measured to be tf ≈ 40 µs. The particle time constant was calculated to be τ  = 8.4 µs with ρp = 1050 kg/m3, D = 12×10-6 m, and µ = 0.001 Pa∙s. The Stokes number is then St = 0.21, as the St > 0.1 [84], the cell does not follow the fluid's motions faithfully. Therefore, if the cell experiences an initial momentum away from the nozzle orifice 50  due to the droplet break-off, the cell would be allowed to be displaced further into the inkjet nozzle than before the droplet ejection event. The sensitivity of the response time to the cell's diameter could also explain why the cell reflection region overlaps the cell travel region as cells of different sizes would behave differently within the inkjet nozzle.  In an effort to mitigate the effects of cell reflection and to test our hypothesis on the hydrodynamics behind the phenomenon, it would be theoretically possible to prevent cell reflection through rheological manipulation by decreasing the Stokes number of the system. This can be achieved by decreasing the time constant. One variable from the time constant calculation that is not a property of the cell is the dynamic viscosity of the liquid. The time constant can be increased with a higher viscosity fluid. One approach to increasing the viscosity of the liquid could be the addition of Ficoll PM400. The addition of Ficoll PM400 exhibits no detrimental effects on cell viability [85] with the benefit of allowing for a means of creating a neutrally buoyant suspension to the seeded cells.   4.1 Materials and Methods The addition of Ficoll PM400 (GE Healthcare) was used to create a high viscosity and neutrally buoyant cell suspension. 10% w/v Ficoll PM400 dissolved in phosphate buffered saline (PBS) solution created a solution with a density of 1.050 g/ml and a viscosity of 4 mPa∙s. The Ficoll in PBS solution is optically transparent with a surface tension and viscosity at 22 °C were 72.19 mN/m and 4.86 mPa·s, measured by a pendant-drop tensiometer and rheometer, respectively. The presence of Ficoll PM400 was previously demonstrated to have no adverse effects on cell viability [85]. 51  In preparation for printing, cells were prepared in the same way as described in Chapter 2, then suspended at a final concentration of 1,500,000 cells/ml in the Ficoll solution. To overcome the higher viscosity fluid an actuation waveform of ±30 Vpk-pk was required to produce a droplet. During printing of the Ficoll PM400 solution, the back pressure at the inkjet nozzle inlet was maintained at 25 Pa. Under these conditions, a droplet of 380 pL was produced with no trailing satellite droplets.  4.2 Results Printing with a 10% Ficoll PM400 solution (as shown in Figure 32), which has a viscosity of 4.86 mPa∙s at a shear rate of 50 s-1, would decrease the time constant to τ = 2.1 µs resulting in a Stokes number of St = 0.053. Cell tracking (Figure 33) and cell mapping (Figure 34) was performed on a suspension of cells in 10% Ficoll PM400 in order to study the effect of increased viscosity solutions on the occurrence of cell reflection.   Figure 32: Consecutive frames of a droplet dispensing the Ficoll PM400 cell suspension. Scale bar represent 100 µm.  52   Figure 33: Cell tracking of a droplet ejection event of cell travel in the 10% Ficoll PM400 suspension.   From performing cell mapping on the cells suspended in the 10% Ficoll PM400 solution, it was observed that only either cell travel or cell ejection occurred during a droplet ejection event. In the higher viscosity solution, the cells more closely follow the fluid flow field as the cells were not allowed to reflect further upstream of the nozzle from the initial position during droplet ejection.  53   Figure 34: Cell mapping for printing a cell solution suspended in Ficoll PM400. Cell travel was represented with (yellow o markers) and cell ejection with (blue + markers). No instances of cell reflection were observed. In total, 138 cells were studied to populate this map with 102 cases of cell travel and 36 cases of cell ejection.   Cell reflection was attributed to the cell slipping within the fluid's streamlines within the nozzle during the production of the droplet. Therefore, a higher viscosity solution would increase the drag force on the cell preventing the detachment of the cell's and fluid's motions. Through the manipulation of the rheological properties of the inkjet ink, it is evident from the resulting cell map, that as hypothesized the cell printing outcome was improved with the elimination of the effects of cell reflection. From printing in Ficoll PM400, upstream of the inkjet nozzle, all cells experienced cell travel and were observed to be displaced towards the nozzle orifice with every 54  ejected droplet. As the cell displaces closer to the nozzle orifice, it will enter the cell ejection region and be dispensed in the subsequent droplet.  55  Chapter 5: Conclusion and Future Work Although there are known limitations, inkjet cell printing is a rapidly progressing field of interest for researchers and industry due to the potential impact it can have on applications such as drug discovery and tissue engineering. Due to the tapered cylindrical geometries typically utilized in piezoelectric inkjet printheads, imaging within the nozzle is difficult due to the optical aberrations at the curved air-glass interface. However, this work overcomes the optical distortions through the use of a refractive index matching PDMS nozzle holder. Using the PDMS holder, the first in-depth analysis of cell behavior within the inkjet nozzle during printing was observed. Additionally, the flow fields within an inkjet nozzle have been experimentally studied in order to provide insight to the hydrodynamics behind the observed cell motions.  This research provides novel insight into the behaviors of cells within a nozzle during printing operations and identified potential sources behind the unpredictability observed in current cell printing systems. This was achieved through the development of a robust imaging method allowing for low depth of field imaging with minimal distortion. Through the tracking of the cell's behaviors and the study of the flow field evolution, it was hypothesized that the viscosity of the fluid in the cell suspension would be a driving factor behind the occurrence of cell reflection. This hypothesis was tested experimentally thorough use of a higher viscosity medium with the addition of Ficoll PM400 into the buffer saline solution. With the use of the high viscosity medium, the occurrence of cell reflection was effectively eliminated.  The presented work would contribute to achieving reliable cell dispensing rates in highlighting important parameters to consider when dispensing cells such as fluid viscosity and cell size. Through the careful selection of cell dispensing parameters, it can lead to the development of an efficient and reliable cell dispensing system.  56  Future directions of the work could aim to further understand the hydrodynamics of a cell within a nozzle by performing µPIV with seeded fluorescent tracer particles and cells or cell sized beads to visualize the flow developing around a cell during printing operations. To date, conducting such a study has faced several technical limitations which must be addressed before a comprehensive study can be conducted. Firstly, visualizing the flow around a cell or a cell-sized particle would require high resolution imaging achieved through a higher magnification objective lens. As the current spatial resolution of the system (15 µm) is on the same order  of magnitude as a cell or a cell sized particle (~12 µm). This limitation would reduce the number of velocity vectors the system would be able to determine around the cell or cell sized particle. However, this limitation cannot be resolved through the selection of a higher magnification objective lens alone. Other considerations would have to be taken into account as the system is limited by the thickness of the glass sidewalls of the inkjet nozzle which would require the employment of a large magnification and long working distance objective lens. However, the working distance of an objective lens decreases with magnification and numerical aperture; therefore the ideal objective lens may not be available. Moreover, it is not possible to physically control where a cell would be spatially within the inkjet nozzle at a given time. As the imaging rate of the PIV camera used is less than the rate of droplet ejection, it would not be possible to study the flow around an individual cell. Under ideal circumstances, a single cell at a fixed location would be imaged at high speed of 2-5 µs between frames with good image resolution across the entire droplet ejection event. However, as the PIV camera utilized in the presented experiment is only capable of capturing double frame images at a maximum frequency of 10 Hz, this would not be possible. This greatly limits the ability to study the cell printing conditions under controlled parameters. In addition, if a single image pair was captured of a cell within the 57  inkjet nozzle, it would be difficult to determine the behavior exhibited by the cell (travel, ejection or reflection) during the printing process given only those two images acquired only 2-5 µs apart. As it is known that there is an oscillatory flow within the nozzle channel, if a cell is observed to be displaced upstream of the nozzle, it cannot be determined with certainty if the cell was undergoing reflection or travel as transient upstream displacement of the cell is observed in both cases. To overcome this, it could be technically feasible to simultaneously acquire high speed videos and double frame images by splitting the fluorescent signal from the tracer particles to two separate viewports and cameras. The double frame images would be used for µPIV analysis to determine the flow around a particle and the high speed video can be used to determine the exhibited cell behaviors. For this, the current hardware available to the microscope housing the µPIV setup would need to be modified with a beam splitter to support the two cameras. In addition, special considerations would have to be made with the amount of signal which would reach the two cameras. Due to the short exposure times of both cameras (~1-2 µs), the resulting image quality would be degraded as the amount of photons reaching the sensors are halved. Therefore, a higher energy pulsed laser would be required if any meaningful data is to be acquired. While the current microscope has two camera ports, the hardware is incapable of splitting the light path to facilitate simultaneous imaging with both view ports. While the technology currently exists in one form or another to properly conduct the proposed study, with the described limitations, the currently available experimental setup utilized for µPIVdoes not yet permit us to effectively study the flow field around a particle within a nozzle.  In addition, future directions of the work can investigate printing with higher cell concentrations to improve on the rate of cell dispensing. Although the rate of cells dispensed can be improved with the addition of Ficoll PM400, under the current printing conditions and cell 58  concentrations in the suspension, the optimal rate of cell deposition is only 5.8 droplets/cell and 2.2 droplets/cell for the 115 pL and 310 pL droplet respectively. Future studies can investigate the effects of higher cell concentrations to achieve higher cell deposition rates to be practical for bioprinting applications. Lastly, a benchmark of the efficacy of the addition of Ficoll PM400 into the media to improving cell deposition rates would be to print a number of droplets and fitting the number of cells per droplet to the Poisson distribution. If the rate of cell deposition follows the Poisson distribution, it would indicate that the cells are randomly distributed in the suspension and that the printhead is randomly encapsulating a cell within an ejected droplet. From the study conducted by Ferris et al.[60] through the use of a density matching microgel solution, the group discovered that the rate of cell deposition followed the expected Poisson distribution. Moreover, to decouple the effects of density and viscosity, the addition of sodium polytungstate to a PBS suspending medium would give minimal changes to the fluid's viscosity [86]. However, while sodium polytungstate is non-toxic, the biocompatibility of the solution would have to be studied before being used in any bioprinting applications. Nonetheless, printing with cells suspended in sodium polytungstate should be performed to study the effects of fluid density on cell printing without the viscous forces induced while printing the Ficoll solution.   In conclusion, this work highlights the importance of the considerations of the rheology of the liquid medium used in the cell suspension. If a low viscosity medium is used when dispensing cells or micrometer scale particles with inkjet printheads, three cell behaviors were observed to occur. Reflection of the cells/particles was observed to occur which can diminish the performance of the bioprinting system. This work contributes towards achieving reliable cell 59  deposition rates for biofabrication applications through the understanding of the interactions of cells in inkjet printing systems.  60  Bibliography [1] A. B. Dababneh and I. T. Ozbolat, “Bioprinting Technology: A Current State-of-the-Art Review,” J. Manuf. Sci. Eng., vol. 136, no. 6, pp. 061016–061016, Oct. 2014. [2] V. Mironov, T. Trusk, V. Kasyanov, S. Little, R. Swaja, and R. Markwald, “Biofabrication: a 21st century manufacturing paradigm,” Biofabrication, vol. 1, no. 2, p. 022001, Jun. 2009. [3] Y.-J. Seol, H.-W. 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