KEYWORDS: Atomic force microscopy, Modeling, Polystyrene, Mechanics, Equipment, Composites, Beam analyzers, Analog electronics, 3D modeling, 3D metrology
This study explores Atomic Force Microscopy's (AFM) applicability for 3D analysis by determining its "mechanical focal plane" in soft composites. The investigators determined the AFM's ability to measure mechanics from a distance when target components are immersed in a secondary medium. Using the Kelvin-Voigt model under quasi-static and dynamic conditions, a sample material with polystyrene beads embedded in agarose gel is analyzed at varying scanning parameters. The results include a model of the effective depth and the effect that a secondary medium has on the ability to measure an embedded component's properties.
Articular cartilage in the tibiofemoral joint contains unique tissue microstructures that serve specific functions, including reduction of friction and distributing the dynamic and static cyclic loading at the ends of diarthrodial joints. A proficient understanding of these microstructures can lead to significant clinical advances in diagnosing orthopedic diseases such as osteoarthritis and improving cartilage repairs. The surface of tibiofemoral condyles can be roughly separated into loadbearing and meniscus-covered areas. Due to the difference in mechanical loading between the two regions, we hypothesize that their microstructures differ. To test this hypothesis, we used cartilage punches harvested from the tibial condyle of porcine knee joints as an example tissue and a custom nonlinear optical microscope for performing a dye-free imaging study. The custom nonlinear optical microscope could simultaneously acquire Two-Photon excitation Auto-Fluorescence (TPAF) and Second Harmonic Generation (SHG) images. Through the TPAF channel, elastin fibers are visible along with chondrocytes. The SHG channel was utilized for observing the vast collagen network and its evident orientation throughout the tibial condyle. Images were analyzed by ImageJ to reveal alignment angles of the collagen network and elastin fibers. The load-bearing region exhibits a denser uniform collagen network with minimum elastin fibers. In contrast, the meniscuscovered areas have a distinctive collagen orientation with a greater magnitude of co-localized elastin fibers. The biological differences are likely derived from their different biomechanical environments in the tibiofemoral joint.
Visualization of collagen fibers in cardiac tissues is essential for clinical diagnosis and pathological analysis of cardiac fibrosis. Selecting a proper imaging method is still challenging for researchers and clinicians who want to determine specific information about the collagen network in cardiac tissues. We examined fibrillar collagen network from mouse ventricular myocardium by commonly available light microscopy techniques using our home-built multimodal microscope. Myocardial slices were unstained or stained with either Picrosirius red or collagen type I antibody/dye conjugation, then imaged by polarized light, confocal fluorescence, second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and stimulated emission depletion (STED) microscopy techniques. This study is intended to serve as a reference for basic research and clinical evaluation of fibrillar collage network in cardiac tissues.
Mechanical signaling in vascular tissue can have major effects on remodeling outcomes and the viability of bypass grafts. When a vein is placed into an environment matching that of an artery, the vein begins to remodel to act like an artery. This change is dependent on mechanotransduction pathways that sense stress from the blood flow. To properly study these pathways, vessels need to be studied ex vivo to control the stress patterns vessels will experience within a patient. The mechanical properties of the vessel will then need to be analyzed at a cellular level to correlate the strain of the environment to the cell response. Optical coherence tomography (OCT) is able to capture b-scans of the entire vessel wall to observe changes at different lateral and axial positions. Multiple b-scans can be captured as the vessel experiences a pressure waveform mimicking physiological pressures and digital image correlation (DIC) can be performed to quantify the mechanical response of the tissue at each spot in the b-scan. A custom-built optical coherence tomography system was used to record images of a porcine carotid artery undergoing pressure changes to observe movements inside the vessel wall. DIC was performed to correlate the strain of the tissue to the experienced stress as a means of testing the system. This imaging method will provide valuable mechanical information as a vein is remodeled in a perfusion bioreactor.
Understanding cardiomyocyte-extracellular matrix (ECM) interactions at the molecular level is essential for deeper insights into their mechanical signaling function for cardiac development, homeostasis and remodeling. We report a lab-built microscope integrating two-color STED microscopy with second harmonic generation (SHG) microscopy to investigate the detailed architecture of cardiomyocyte-ECM interactions in murine myocardium at a subdiffractive level. SHG microscopy is used to locate possible interaction sites at the cell-ECM interface through the intrinsic SHG signal generated by collagen assemblies and myosin filaments. Two-color STED microscopy is used to obtain a subdiffractive view of proteins at sites of interest registered by SHG microscopy. Because large field-of-view (FOV) STED microscopy is still challenging, with photobleaching often a major concern, imaging only SHG-registered sites is advantageous. Further, using intrinsic contrast in the study reduces the number of biomarkers for fluorescent staining and thereby the number of detection channels for fluorescent imaging, simplifying sample preparation procedures and STED microscopy architectures. For purpose of demonstration, we show images of immunostained type I collagen, type Ⅳ collagen and laminin as ECM structures of interest in rat ventricular sections without counterstaining.
Chondrocyte viability is an important measure to consider when assessing cartilage health. Dye-based cell viability assays are not suitable for in vivo or long-term studies. We have introduced a non-labeling viability assay based on the assessment of high-resolution images of cells and collagen structure using two-photon stimulated autofluorescence and second harmonic generation microscopy. By either the visual or quantitative assessment, we were able to differentiate living from dead chondrocytes in those images. However, both techniques require human participation and have limited throughputs. Throughput can be increased by using methods for automated cell-based image processing. Due to the poor image contrast, traditional image processing methods are ineffective on autofluorescence images produced by nonlinear microscopes. In this work, we examined chondrocyte segmentation and classification using Mask R-CNN, a deep learning approach to implement automated viability analysis. It has been demonstrated an 85% accuracy in chondrocyte viability assessment with proper training. This study demonstrates that automated and highly accurate image analysis is achievable with the use of deep learning methods. This image processing approach can be helpful to other imaging applications in clinical medicine and biological research.
In the recent studies of cartilage imaging with nonlinear optical microscopy, we discovered that autofluorescence of chondrocytes provided useful information for the viability assessment of articular cartilage. However, one of the hurdles to apply this technology in research or clinical applications is the lack of image processing tools that can perform automated and cell-based analysis. In this report, we present our recent effort in the cell segmentation using deep learning algorithms with the second harmonic generation images. Two traditional segmentation methods, adaptive threshold, and watershed, were used to compare the outcomes of different methods. We found that deep learning algorithms did not show a significant advantage over the traditional methods. Once the cellular area is determined, the viability index is calculated as the intensity ratio between two autofluorescence channels in the cellular area. We found the viability index correlated well with the chondrocyte viability. Again, deep learning segmentation did not show a significant difference from the traditional segmentation methods in terms of the correlation.
Using our lab built two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) hybrid confocal imaging system, we observed, for the first time, the dynamic sarcomeric addition process in a rat cardiomyocyte cell culture system; this culture system expressed in vivo-like myofibril structure and mimicked mechanical overload experienced in a heart muscle tissue. Micro-grooved topographic patterned substrates com- bined with electrical stimulation are used to achieve the in vivo-like myofibril structure. After cardiomyocytes aligned, longitudinal and transverse mechanical stretch was applied to cardiomyocytes in parallel or perpendicu- lar, respectively, to the direction of alignment via stretching the substrates to mimic mechanical overload. Z discs, in which alpha-actinin expressed, have been proposed to involve in the process. TPEF detected alpha-actinin that labeled with enhanced yellow florescent protein via plasmid transfection. SHG is intrinsic to noncentrosym- metric structures, thus was used to detect myosin, a polar molecule expressed in myofibril. Pulse splitter system and synchronized recording system was introduced on TPEF-SHG imaging system to reduce the photodamage during live cell imaging. In our study, TPEF-SHG imaging system was used to study the dynamic process of sarcomeric addition in in vivo-like culture model under mechanical overload. This microscopic technique is ideal for tracking sarcomeric components to successively assemble onto pre-exist myofibrils and for revealing the role of Z discs played in sarcomeric addition. Transition of Z discs from continuous to broadened striation and from broadened to uniform striation under stretch has been observed. We concluded that continuous Z discs is the place of new sarcomeric addition.
Cardiac hypertrophy, a process initiated by mechanical alterations, is hypothesized to cause long-term molecular-level alteration in the sarcomere lattice, which is the main force-generating component in the heart muscle. This molecular-level alteration is beyond the resolving capacity of common light microscopy. Second harmonic generation (SHG) microscopy has unique capability for visualizing ordered molecular structures in biological tissues without labeling. Combined with polarization imaging technique, SHG microscopy is able to extract structural details of myosin at the molecular level so as to reveal molecular-level alterations that occur during hypertrophy. The myosin filaments are believed to possess C6 symmetry; thus, the nonlinear polarization response relationship between generated second harmonic light I^2ωand incident fundamental light I^ω is determined by nonlinear coefficients, χ_15, χ_31 and χ_33. χ_31/χ_15 is believed to be an indicator of the molecular symmetry of myosin filament, whileχ_33/χ_15represents the intramyosin orientation angle of the double helix. By changing the polarization of the incident light and evaluating the corresponding SHG signals, the molecular structure of the myosin, reflected by the χ coefficients, can be revealed. With this method, we studied the structural properties of heart tissues in different conditions, including those in normal, physiologically hypertrophic (heart tissue from postpartum female rats), and pathologically hypertrophic (heart tissue from transverse-aorta constricted rats) conditions. We found that ratios of χ_31/χ_15 showed no significant difference between heart tissues from different conditions; their values were all close to 1, which demonstrated that Kleinman symmetry held for all conditions. Ratios of χ_33/χ_15 from physiologically or pathologically hypertrophic heart tissues were raised and showed significant difference from those from normal heart tissues, which indicated that the intramyosin orientation angle of the double helix was altered when heart tissues hypertrophied. Polarization-resolved SHG microscopy permitted us to study heart tissues at the molecular level and may serve as a diagnostic tool for cardiac hypertrophy.
A fast surface plasmon resonance (SPR) imaging biosensor system based on wavelength interrogation using a liquid crystal tunable filter (LCTF) is presented. The system combines the merits of wide-dynamic detection range offered by the spectral approach and multiplexed high-throughput data collection with a two-dimensional (2-D) biosensor array. The key feature of the reported scheme is a feedback loop that drives the LCTF to achieve fast tracking of the SPR dip movement caused by the binding of target molecules to the sensor surface. Experimental results show that the system is capable of completing an SPR dip measurement within 4 s. Based on using a spectral window of about 100 nm, the experimental dynamic detection range and refractive index resolution are 4.63×10−2 RIU and 5.87×10−6 RIU, respectively. As also demonstrated herein using 2-D microsensor arrays, among the spectral SPR sensors, the reported system is most suitable for multiplexed label-free biosensing applications.
Fluorescence holographic microscope (FINCHSCOPE) is a motionless fluorescence holographic imaging technique based on Fresnel incoherent correlation holography (FINCH) that shows promise in reconstructing three-dimensional fluorescence images of biological specimens with three holograms. We report a developing two-step phase-shifting method that reduces the required number of holograms from three to two. Using this method, we resolved microscopic fluorescent beads that were three-dimensionally distributed at different depths with two interferograms captured by a CCD camera. The method enables the FINCHSCOPE to work in conjunction with the frame-straddling technique and significantly enhance imaging speed.
FINCHSCOPE is a new technology of fluorescence holographic microscopy. It has been successfully applied to recording high-resolution three-dimensional fluorescence images of biological specimens without the need for scanning. In this study, we revealed and analyzed an intrinsic phenomenon, called ghost lens effect, on spatial light modulator which is the core element enabling the incoherent correlation in the FINCHSCOPE. The ghost lens effect can degrade the imaging quality by introducing multiple spherical waves with different focal lengths into the correlation and thus increasing the noise in the recorded holograms.
Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.
We developed a novel addressable multiregional multiphoton microscope that employs a fast one-dimensional discrete-line scanning approach based on a spatial light modulator (SLM). The phase-only SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple specific discrete-lines, which are designed according to the sizes and locations of the target samples. Only the target-sample areas of are scanned
one-dimensionally, resulting in an efficient use of the laser’s power. Compared with conventional multiphoton microscopies, this technique shortens scanning time and minimizes photodamage by concentrating scanning energy and dwell time on the areas of interest. Additionally, our discrete-line-focus design eliminates the cross-talk that occurs in conventional one-dimensional line-scanning multiphoton microscopes, thus enhancing the lateral and axial resolutions of the line-scanning imaging system.
We developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain neurons and spinal cord neurons. Observation of neurons under a high-magnification microscope, which we obtained from these two cell types, showed no difference in morphology. However, when flowing in the microfluidic channel and simultaneously being laser guided, the two cell types gained quite different guidance speeds under the same experimental conditions. The results demonstrate that different cell types with the same morphology (e.g., size, shape, etc.) can be effectively distinguished from each other by measuring the difference in guidance speeds (the maximum flow speed minus the initial flow speed). This technique is expected to provide a new approach to high-throughput, label-free cell sorting with high sensitivity.
In this study, we developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain
neurons and chick embryonic spinal cord neurons. The neurons we obtained from these two cell types have no difference in
morphology as observed under a high-magnification microscope. However, when flowing in the microfluidic channel and
simultaneously being laser-guided, the two cell types gained quite different guidance velocities under the same
experimental conditions. The experimental results demonstrate that different cell types with the same morphology (e.g.,
size, shape, etc.) can be effectively distinguished from each other by measuring the difference of guidance velocities (the
maximum flow velocities minus the initial flow velocities). This technique is expected to provide a new approach to
high-throughput, label-free cell sorting with sensitivity.
Congenital Heart Disease (CHD) is the most common congenital malformation in newborns in the US. Although
knowledge of CHD is limited, altered hemodynamic conditions are suspected as the factor that stimulates cardiovascular
cell response, resulting in the heart morphology remodeling that ultimately causes CHDs. Therefore, one of recent efforts
in CHD study is to develop high-speed imaging tools to correlate the rapidly changing hemodynamic condition and the
morphological adaptations of an embryonic heart in vivo. We have developed a high-speed streak mode OCT that works
at the center wavelength of 830 nm and is capable of providing images (292x220 μm2) of the outflow tract of an
embryonic chick heart at the rate of 1000 Hz. The modality can provide a voxel resolution in the range of 10 μm3, and
the spectral resolution allows a depth range of 1.63 mm. In the study reported here, each of the 4D images of an outflow
tract was recorded for 2 seconds. The recording was conducted every 2 hours (HH17 to HH18), 3 hours (HH14 to HH17),
and 4 hours (HH18 to HH19). Because of the fast scan speed, there is no need for postacquisition processing such as use
of gating techniques to provide a fine 3D structure. In addition, more details of the outflow tract are preserved in the
recorded images. The 4D images can be used in the future to determine the role of blood flow in CHD development.
Using hybrid TPEF-SHG imaging and immunocytological techniques, we studied dedifferentiation of adult
cardiomyocytes. First, the myofibrils shrank to shorten the sarcomere length. At the cell ends, the striated pattern of
myosin filaments began to dissociate; at the center of the cell, the striated pattern of alpha-actinin first faded away and
reappeared near the cell membrane during dedifferentiation. The results suggest that when freshly isolated adult
cardiomyocytes are used to model cardiac muscle, the end-to-end connection may be important to maintain their striated
myofibrillar structure and rod-shape morphology.
Through a combination of a deflective phase-only diffractive spatial light modulator (SLM) and galvo scanners, an addressable multiregional and multifocal multiphoton microscope (AM-MMM) is developed. The SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple beamlet arrays with addressable shapes and sizes that match the regions of interest on the sample. Compared with conventional multifocal multiphoton microscope (MMM), AM-MMM achieves the effective use of the laser power with an increase of imaging rate and a decrease of photodamage without sacrifice of resolution.
We developed a novel addressable scanless multifocal multiphoton microscope. This microscope works in a fast scanless
mode. Subjectively selected sample (or multiple samples located in separated areas) in a large field of view can be
imaged by illuminating only the area (or areas) where the target sample (or samples) locate(s). In this way, by precisely
designing the multiple foci according to the size and position of the area of interest, we can concentrate all the laser
energy and dwell time on that area of the sample, making full use of the available laser power and avoiding photodamage
in other areas. Since no scanning is involved, the acquisition time of a multiphoton image is decided only by the
sensitivity and readout time of the CCD camera. Moreover, the interfocal distance of the multiple foci matches the lateral
resolution of the imaging system, so that the two-photon image was recorded with high lateral resolution. However,
crosstalk (spatial interference) on out-of-focus planes occurs between adjacent points when they are too close, degrading
the resolution, especially the axial resolution of the imaging system.
Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second
harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes
during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of
existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed
mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled
onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an
important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length
during maturation.
Doppler Fourier domain optical coherence tomography is able to be used for in vivo blood flow measurement. In
conventional methods, the highest velocity that can be measured is limited to the range the phase shift between two
successively recorded depth profiles at the same probe-beam location, which cannot exceed (-π, π), otherwise phase
wrapping will occur. This phase-wrapping limit is determined by the time interval between two consecutive A-scans. We
present a novel approach to shorten the time interval between two consecutive A-scans and thus increase the phase-wrapping
limit by using an area scan camera to record the interference spectrum in a streak mode. To demonstrate the
effectiveness of this method, the blood flows in HH18 and HH19 chick hearts were imaged and phase wrapping free
Doppler images were obtained.
Recently, we developed the streak-mode Fourier domain optical coherence tomography (OCT) technique in which an
area-scan camera is used in a streak-mode to record the OCT spectrum. Here we report the application of this technique
to in ovo imaging HH18 embryonic chick hearts with an ultrahigh speed of 1,016,000 axial scans per second. The high-scan
rate enables the acquisition of high temporal resolution 2D datasets (1,000 frames per second or 1 ms between
frames) and 3D datasets (10 volumes per second), without use of prospective or retrospective gating technique. This
marks the first time that the embryonic animal heart has been 4D imaged using a megahertz OCT.
We developed a hybrid two-photon excitation fluorescence-second harmonic generation (TPEF-SHG) imaging system with an on-stage incubator for long-term live-cell imaging. Using the imaging system, we observed the addition of new sarcomeres during myofibrillogenesis while a cardiomyocyte was spreading on the substrate. The results suggest that the TPEF-SHG imaging system with an on-stage incubator is an effective tool for investigation of dynamic myofibrillogenesis.
We propose a novel optical asymmetric cryptosystem based on a phase-truncated Fourier transform. Two decryption keys independent of each other are generated. They are referred to as universal key and special key, respectively. Each of them can be used for decryption independently in absence of the other. The universal key is applicable to decrypt any ciphertext encoded by the same encryption key, but with poor legibility. On the contrary, the special key is adequate for legible decryption, but only valid for one ciphertext corresponding to the specified plaintext. A set of simulation results show the interesting performance of two types of decryption keys.
Here we present an ultrahigh-speed Fourier-domain optical coherence tomography (OCT) that records the OCT spectrum in streak mode with a high-speed area scan camera, which allows higher OCT imaging speed than can be achieved with a line-scan camera. Unlike parallel OCT techniques that also use area scan cameras, the conventional single-mode fiber-based point-scanning mechanism is retained to provide a confocal gate that rejects multiply scattered photons from the sample. When using a 1000 Hz resonant scanner as the streak scanner, 1,016,000 A-scans have been obtained in 1 s. This method's effectiveness has been demonstrated by recording in vivo OCT-image sequences of embryonic chick hearts at 1000 frames/s. In addition, 2-megahertz OCT data have been obtained with another high speed camera.
The optical cryptosystem based on phase-shifting interferometry (PSI) is one of the most interesting optical cryptographic schemes in recent years. However, we find that the PSI technique provides an attractive method to record the ciphertext, but contributes little to the security level of the cryptosystem. From the cryptanalysis point of view, in a certain simplified case, an attacker is only required to crack two equivalent decryption keys instead of the original random phase keys and geometric key. Moreover, a chosen-plaintext attack method is proposed, in which an impulse function is chosen as a known plaintext. By using this attack, the attacker can effectively recover any plaintext from the corresponding ciphertext. The validity of the attack is verified by computer simulations.
We report a technique, which uses an area-scan camera to record the interference spectrum. Traditional point-scanning is
remained in this streak-mode FDOCT so that the small aperture of the single-mode fiber functions as a confocal gate and
screens multiply scattered photons very well. While the sample beam is scanning the specimen laterally, the interference
spectrum is physically scanned on the area scan camera using a streak scanner. Therefore, pixels of the camera are
illuminated by the spectrum of OCT signal row by row, corresponding to each A-scan at different lateral position. A
unidirectional B-scan of 700 lines is obtained in 1 ms; thus, an A-scan time of 1.4 μs is achieved. A Day 4 chick embryo
sampled is imaged using this method. This technique is highly potential for multi-Megahertz OCT imaging.
We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire
laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement
high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The
SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this
imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF
in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG
and TPEF in the research of myofibrillogensis.
A rotational microelectromechanical(MEMS) motor based common-path Fourier-domain OCT for endoscopic imaging,
which uses the interface between the index-match oil and distal-end surface of the fiber as a self-aligned reference
mirror, is reported. The reference intensity is easy to be tuned by altering the index of the match oil to optimize the signal
to noise ratio of the system. An external Michelson interferometer is used to compensate for the optical path difference
and dispersion mismatch to the index-match oil and the GRIN lens. Due to this common-path design, the OCT signal is
immune to bending or stretching of the endoscopic catheter. The outer diameter of the probe is 3 mm, and 22
circumferential-scans and 50,000 lines A-scans are obtained in one second.
A common-path Fourier-domain OCT for endoscopic imaging, which uses the distal-end surface of the fiber as a selfaligned
reference mirror, is reported. A miniaturized probe is designed for this OCT system. A reference Michelson
interferometer is used to compensate for the optical path difference and mismatch of dispersion and polarization states
due to the miniaturized probe. This configuration allows arbitrary probe fiber length and provides sufficient working
space for imaging optics and their package, and thus is suitable for OCT imaging of lumens of various sizes.
Additionally, the reference intensity is able to be tuned by index match oil to optimize the signal to noise ratio of the
system. Due to this common-path configuration, the OCT signal is immune to the bending or handling of the fiber
connecting with the probe.
Laser guidance is the technique that uses a weakly convergent laser beam to trap particles radially in the center of the
beam and simultaneously propel them along the beam propagation axis with a travelling distance over millimeters. In
this paper, we describe the applications of laser guidance to detect different cell types, including those of phenotypically
transformed or gene-modified cells, especially for situations in which fluorescent markers used in flow cytometry for cell
detection are not available or their application is contraindicated by clinical restriction. The optical force, which
determines the guidance speed of the cell, is dependent on the characteristics, such as size, shape, composition and
refractive index, of the cell being guided. Therefore, by measuring the guidance speed of the cell along the laser beam,
cells with different properties can be effectively distinguished. We report two experimental results: 1) the laser-guidance
system could significantly distinguish the metastatic cancer cell type 4T1 from its non-metastatic counterpart 4T07,
which could not be achieved by using a high magnification microscope; 2) The laser-guidance experiment demonstrated
that only one gene modification between L-10 and TC-1 cells resulted in ~40% difference in guidance speed. These
experimental data indicate that laser guidance can be used to detect subtle differences between sub-cell types.
To precisely control the position of multiple types of cells in a coculture for the study of cell-cell interactions, we have developed a laser micropatterning technique. The technique employs the optical forces generated by a weakly focused laser beam. In the beam's focal region, the optical force draws microparticles, such as cells, into the center of the beam, propels them along the beam axis, and guides them onto a target surface. Specific patterns are created through computercontrolled micromanipulation of the substrate relative to the laser beam. Preliminary data have demonstrated cell viability after laser guidance. This project was designed to systematically vary the controllable laser parameters, namely, intensity and exposure time of the laser on single cells, and thus determine the laser parameters that allow negligible cell damage with functional cellular position control. To accomplish this goal, embryonic day 7 (E7) chick forebrain neurons were cultured in 35 mm petri dishes. Control and test cells were selected one hour after cell placement to allow cell
attachment. Test cells were subjected to the laser at the focal region. The experimental parameters were chosen as: wavelength - 800 nm, intensities - 100 mW, 200 mW, and 300 mW, and exposure times - 10 s and 60 s. Results were analyzed based on neurite outgrowth and the Live/Dead assay (Viability/Cytoxicity kit from Molecular Probes). No statistical difference (p >> 0.1, student t-test) in viability or function was found between the control neurons and those exposed to the laser. This confirms that laser guidance seems to be a promising method for cellular manipulation.
In this article, we report the development of a novel system for distinguishing various types of cell based on the differences in the values of optical forces experienced by them. The laser induced optical forces arise when molecules comprising the cell interact with the photons of the laser beam resulting in an exchange of momentum. The basic principle is based on the phenomenon of laser cell guidance: a cell, in proximity of a weakly focused laser beam experiences a net force which attracts it to the centre of the beam and propels it along the axis of the beam. The 'guidance' is thus a direct result of the optical forces experienced by the cell. The guidance force experienced by the cell is directly related to the amount of momentum exchange, which in turn depends on factors such as the cell shape and size, the refractive index difference between the materials composing the cell and the materials in the culture media, and the properties of the beam. Since different cell types have different physical properties, they experience different optical (guiding) forces when they interact with the same laser beam. Since guidance force causes the cells to be propelled along the axis of the beam, the force with which the cell is propelled will directly determine the velocity with which the cell will move along the beam. Hence different types of cells will move with different velocities. Thus by studying this motion of cell, we in turn study the forces experienced by the cell. We propose that by analyzing the velocity with which cells are being guided by a laser beam, we can develop a technique to distinguish various cell types. Extensive GLMT theories calculate and predict theoretically the forces experienced by particles of a given size and refractive index for a given set of beam parameters. These theories are used as the basis to make judgments about the validity of our velocity and force calculation. The velocity assessments are conducted for a statistically significant number of cells from various cell types to explore the systematic difference among various groups of cells and, accordingly, the reliability of the developed method is discussed.
The various cell mechanisms, including cell-cell interactions, in native tissue could be better understood by engineering a cell coculture with a micro environment that closely mimics the natural cell arrangement. To this end, we developed a cell micropatterning system that uses a weakly focused laser beam to trap individual cells at the center of the beam and propel them forward onto an appropriate substrate. The optimal methods of introducing different cell types to be patterned into the patterning system and preventing cells from randomly falling onto the pattern were issues to be addressed with this system. Here, we report the development of a multi-chamber, multi-beam laser cell micropatterning system, in which the delivery of specific cells into the beam can be controlled using secondary laser beams. This permits consecutive creation of a pattern involving multiple cell types at specific relative positions. As examples, various patterns of fibroblasts have been created on collagen coated coverslips. In addition, two asynchronously beating clusters of cardiomyocytes were connected with fibroblasts of cardiac origin, yielding a deeper insight into the electrophysiological role of fibroblasts in conduction of the action potentials among cardiomyocytes.
Tissue engineering has shown great potential for solving health problems through replacing or repairing malfunctioning tissue with functional constructs of living cells and associated molecules. To realize this potential, complicated cell-cell interactions both in the macro scale and micro scale need to be understood.
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