There is a need for new methodologies to investigate cell apoptosis and recovery, cell adhesion, and cell-cell interactions in cellular biology and neurobiology. Such systems should be able to induce localized cell injuries and measure damage responses from single cells. In this regard, pulsed lasers can be used to produce Laser- Induced Shockwaves (LIS), which can cause cell detachments and induce cellular membrane injuries, by applying shear force in order of µN . Furthermore, since the resulting shear force can increase membrane permeability, chemicals and markers can then be transferred into cells non-invasively. Continuous-wave lasers can be used as Optical Tweezers (OT), to apply non-contact delicate forces, as low as 0.1f N , and deliver materials into cells, and also move the cells to different locations. In this paper, we introduce a combination of modalities to apply variable forces, from femto to micro newtons, to cells. Our system consists of a 1060nm continuous laser light source for OT and a 1030nm femtosecond pulsed laser for generating LIS. To have a direct measurement of changes in the cellular thickness and membrane dynamics, the cells are imaged under a Quantitative Phase Microscope (QPM). Our microscope is capable of Differential-Interference Microscopy (DIC) and Phase-Contrast microscopy (PhC) and fluorescent microscopy, making it a unique system for studying cell injuries.
Traumatic brain injury (TBI) occurs when an external shock causes injury to the brain. The mechanism of the disease is not completely understood yet. Studies have shown that astrocytes play various roles following brain injury. However, the exact functional role of them after TBI is still a matter of debate. Laser-induced shock waves (LIS) can create a precise controllable mechanical force that is capable of injuring or lysing cells to simulate the brain injury at the cellular level. Here, we propose a system that enables us to induce injuries in CNS cells with LIS and observe the whole process under a Quantitative phase microscope (QPM). Our system is also capable of adding another laser for optically trapping the cells to keep them at a certain distance from the center of the shockwave, as this distance is one of the important factors which determines the level of injury.
We developed a compact Mach-Zehnder interferometer module to be used as a replacement of the objective lens in a conventional inverted microscope (Nikon, TS100-F) in order to make them quantitative phase microscopes. The module has a 90-degree-flipped U-shape; the dimensions of the module are 160 mm by 120 mm by 40 mm and the weight is 380 grams. The Mach-Zehnder interferometer equipped with the separate reference and sample arms was implemented in this U-shaped housing and the path-length difference between the two arms was manually adjustable. The sample under test was put on the stage of the microscope and a sample light went through it. Both arms had identical achromatic lenses for image formation and the lateral positions of them were also manually adjustable. Therefore, temporally and spatially low coherent illumination was applicable because the users were able to balance precisely the path length of the two arms and to overlap the two wavefronts. In the experiment, spectrally filtered LED light for illumination (wavelength = 633 nm and bandwidth = 3 nm) was input to the interferometer module via a 50 micrometer core optical fiber. We have successfully captured full-field interference images by a camera put on the trinocular tube of the microscope and constructed quantitative phase images of the cultured cells by means of the quarter-wavelength phase shifting algorithm. The resultant quantitative phase images were speckle-free and halo-free due to spectrally and spatially low coherent illumination.
We developed a transportable Linnik-type full-field low-coherent quantitative phase microscope that is able to compensate for optical path length (OPL) disturbance due to environmental mechanical noises. Though two-beam interferometers such as Linnik ones suffer from unstable OPL difference, we overcame this problem with a mechanical feedback system based on digital signal-processing that controls the OPL difference in sub-nanometer resolution precisely with a feedback bandwidth of 4 kHz. The developed setup has a footprint of 200 mm by 200 mm, a height of 500 mm, and a weight of 4.5 kilograms. In the transmission imaging mode, cells were cultured on a reflection-enhanced glass-bottom dish, and we obtained interference images sequentially while performing stepwise quarter-wavelength phase-shifting. Real-time image processing, including retrieval of the unwrapped phase from interference images and its background correction, along with the acquisition of interference images, was performed on a laptop computer. Emulation of the phase contrast (PhC) images and the differential interference contrast (DIC) images was also performed in real time. Moreover, our setup was applied for full-field cell membrane imaging in the reflection mode, where the cells were cultured on an anti-reflection (AR)-coated glass-bottom dish. The phase and intensity of the light reflected by the membrane revealed the outer shape of the cells independent of the refractive index. In this paper, we show imaging results on cultured cells in both transmission and reflection modes.
We successfully induced intracellular ion concentration changes in live culture cells using mid-infrared laser irradiation. The laser used for irradiation was a quantum cascade laser with a wavelength of 6.1 micrometers. We tuned the power of the laser to be between 30 to 60 mW at the sample. Cell lines, namely HeLa and Chinese hamster ovary cell lines, were used. They were cultured on specially fabricated silicon-bottom dishes. Live cells were stained using ion-sensitive dyes such as Calcium Green-1. The mid-infrared light was incident on the cell samples from the bottom of the dish through the silicon plate, and fluorescence imaging of the ion concentrations was performed using an upright fluorescence microscope placed on top of the sample stage. The mid-infrared lasers were operated in the continuous wave mode and light irradiations onto the cells were temporally controlled using a mechanical shutter in a periodical on-and-off pattern in the second timescale. The cells showed oscillations in their ionic concentration, which was synchronized with the periodical mid-infrared irradiation, and the threshold power needed for evoking the ion concentration change was dependent on the cell types and ion species. These results demonstrated that mid-infrared light directly changed the ionic response within cells and had the ability to change cell functions.
We developed a portable and user-friendly prototype of low-coherent reflection-type quantitative phase microscope (QPM). Our setup is based on the full-field Linnik type phase-shifting interference microscope and is optimized for surface profiling of living cell’s membrane. Unlike commonly available transmission-type quantitative phase microscopes which reveal the optical thickness, our reflection-type setup can obtain the geometrical thickness (real shape) of the sample, decoupled with the refractive index. The coherence length of our imaging light source (halogen lamp) was approximately 1 micrometer so that we can selectively obtain the interference of the light reflected from the cell membranes whose reflectivity in culture medium is only on the order of 0.1%. Moreover our setup has a feedback controlled path-length stabilization circuit so that users can implement accurate phase shifting interferometry with one nanometer of reproducibility. The stabilization circuit allows installing our setup even in noisy environments such as biology labs without an optical bench. In this paper, we will also show our studies of recent biomedical applications, including imaging of cell plasma membrane and phase-resolved 3D tomography of living cells.
We have reported a new molecular-targeted cancer phototherapy, photoimmunotherapy (PIT), which
killed implanted tumors in mice without side-effects. To understand the mechanism of cell killing
with PIT, three-dimentional dynamic low-coherence quantitative phase microscopy (3D LC-QPM),
a device developed by Hamamatsu Photonics K.K, was used to detect morphologic changes in
cancer cells during PIT. 3T3/HER2 cells were incubated with anti-HER2 trastuzumab-IR700 (10
μg/mL, 0.1 μM as IR700) for 24 hours, then, three-dimensionally imaged with the LC-QPM during
the exposure of two different optically filtered lights for excitation of IR700 (500-780 nm) and
imaging (780-950 nm). For comparison with traditional PDT, the same experiments were performed
with Photofrin (10 and 1 μM).
Serial changes in the cell membrane were readily visualized on 3D LC-QPM. 3T3/HER2 cells began
to swell rapidly after exposure to 500-780 nm light excitation. The cell volume reached a maximum
within 1 min after continuous exposure, and then the cells appeared to burst. This finding suggests
that PIT damages the cell membrane by photo-reaction inducing an influx of water into the cell
causing swelling and bursting of the cells. Interestingly, even after only 5 seconds of light exposure,
the cells demonstrated swelling and bursting albeit more slowly, implying that sufficient cumulative
damage occurs on the cell membrane to induce lethal damage to cells even at minimal light exposure.
Similar but non-selective membrane damage was shown in PDT-treated cells Photofrin.
Thus, PIT induces sufficient damage to the cell membrane within 5 seconds to induce rapid necrotic
cell death which can be observed directly with 3D LC-QPM. Further investigation is needed to
evaluate the biochemical mechanisms underlying PIT-induced cellular membrane damage.
There is an increasing need for non-invasive imaging techniques in the field of stem cell research. Label-free techniques are the best choice for assessment of stem cells because the cells remain intact after imaging and can be used for further studies such as differentiation induction. To develop a high-resolution label-free imaging system, we have been working on a low-coherence quantitative phase microscope (LC-QPM). LC-QPM is a Linnik-type interference microscope equipped with nanometer-resolution optical-path-length control and capable of obtaining three-dimensional volumetric images. The lateral and vertical resolutions of our system are respectively 0.5 and 0.93 μm and this performance allows capturing sub-cellular morphological features of live cells without labeling. Utilizing LC-QPM, we reported on three-dimensional imaging of membrane fluctuations, dynamics of filopodia, and motions of intracellular organelles. In this presentation, we report three-dimensional morphological imaging of human induced pluripotent stem cells (hiPS cells). Two groups of monolayer hiPS cell cultures were prepared so that one group was cultured in a suitable culture medium that kept the cells undifferentiated, and the other group was cultured in a medium supplemented with retinoic acid, which forces the stem cells to differentiate. The volumetric images of the 2 groups show distinctive differences, especially in surface roughness. We believe that our LC-QPM system will prove useful in assessing many other stem cell conditions.
We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called ”membrane nanotubes” or “tunneling nanotubes.” However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.
Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common
with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells,
undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered
EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence
quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic
technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the
light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera.
P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in
diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers.
The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the
sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional
structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The
results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of
cell properties including their pluripotency.
Surface topography and its dynamic fluctuations in live cultured cells were obtained by low-coherent quantitative phase
microscopy (LC-QPM), using a reflection-type interference microscope employing the digital holographic technique
with a low-coherent light source. Owing to the low coherency of the light-source, only the light reflected at a specific
sectioning height of the sample generates interference fringes on the CCD camera. Because the digital holographic
technique enables us to quantitatively measure the intensity and phase of the optical field, a nanometer-scale surface
profile of a living cell can be obtained by capturing the light reflected by the cell membrane. The lateral and the vertical
spatial resolution was 0.56 microns and 0.93 microns, respectively, and the mechanical stability of the phase
measurement was better than 2 nanometers. The measurements were made at fast (21 frames/sec) and slow (2
frames/sec, time-lapse) frame rates and the slow measurements were performed over a period of 10 minutes. The
temporal fluctuations of the cell membrane were analyzed by the mean-square-displacement (MSD) as a function of the
time-difference τ. By merging the fast and slow data, the MSDs from τ = 50 msec to τ = 300 sec were obtained and
wide-dynamic-range measurements of the MSDs from 2 nm2 to over 100000 nm2 were demonstrated. The results show
significant differences among different cell types under various conditions.
We demonstrate tomographic imaging of intracellular activity of living cells by a low-coherent quantitative phase
microscope. The intracellular organelles, such as the nucleus, nucleolus, and mitochondria, are moving around inside
living cells, driven by the cellular physiological activity. In order to visualize the intracellular motility in a label-free
manner we have developed a reflection-type quantitative phase microscope which employs the phase shifting
interferometric technique with a low-coherent light source. The phase shifting interferometry enables us to quantitatively
measure the intensity and phase of the optical field, and the low-coherence interferometry makes it possible to selectively
probe a specific sectioning plane in the cell volume. The results quantitatively revealed the depth-resolved fluctuations of
intracellular surfaces so that the plasma membrane and the membranes of intracellular organelles were independently
measured. The transversal and the vertical spatial resolutions were 0.56 μm and 0.93 μm, respectively, and the
mechanical sensitivity of the phase measurement was 1.2 nanometers. The mean-squared displacement was applied as a
statistical tool to analyze the temporal fluctuation of the intracellular organelles. To the best of our knowledge, our
system visualized depth-resolved intracellular organelles motion for the first time in sub-micrometer resolution without
contrast agents.
Wide-field reflection phase microscopy is highly desired for depth-resolved measurement of cellular structures without
the need for raster scanning. We report a low coherence reflection phase microscope based on time-domain optical
coherence tomography and off-axis interferometry. The setup uniquely provides the desired angular shift to the reference
beam for off-axis interferometry while promising equal path length across the whole reference beam. We show sub-nanometer
path-length sensitivity of our instrument and demonstrate high-speed imaging of membrane fluctuations in
eukaryotic cells.
Multiple scattering is a significant obstacle in the optical imaging of biological samples. However, it is possible to
reverse its effects through optical phase conjugation (OPC) of the scattered field. We perform digital OPC (DOPC)
utilizing a spatial light modulator (SLM) and a Sagnac interferometer geometry. This design permits a simple and robust
DOPC implementation, which we demonstrate experimentally. We exploit the beam-shaping flexibility of the SLM to
demonstrate the possibility to enhance either the optical power transmission or the light focusing ability of the DOPC
process.
We report a practical method for label-free quantification of specific molecules using spectroscopic imaging of sampleinduced
phase shifts (for the detail, please see the Ref. [1]). Diffraction phase microscopy equipped with various
wavelengths of light source is used to record wavelength-dependent phase images. We first perform dispersion
measurements on pure solutions of single molecular species present in the cells, such as albumin and hemoglobin (Hb).
With this prior calibration of molecular specific dispersion, we demonstrate the extraction of Hb concentration from
individual red blood cells (RBCs). The end point of this study is
non-invasive monitoring of physiological states of intact
living cells.
Cell membrane motions of living cells are quantitatively measured in nanometer resolution by low-coherent full-field
quantitative phase microscopy. Our setup is based on a full-field phase shifting interference microscope with a very lowcoherent
light source. The reflection mode configuration and the low-coherent illumination make it possible to
differentiate the weak reflection light from the cell membrane from the strong reflection from the glass substrate. Two
cell populations are quantitatively assessed by the power spectral density of the cell surface motion and show different
trends.
We report a full-field, phase-shifting microscope with precise control of the optical path difference (OPD) and show
topographic phase images of living cells. Our system is based on a Linnik interference microscope with Kohler
illumination of a halogen lamp for the imaging lightsource. Phase-sensitive active-stabilization of the OPD is employed
with an infrared laser whose optical path is the same as that of halogen light. We previously reported the results of
cultured cell topography with this stabilization scheme; however the phase stability and the image quality were
insufficient. We have improved the noise-cancellation system and achieved control of the OPD with stability down to
0.7 nm in the bandwidth of 500 Hz. Quarter wavelength phase-shifting was carried out with sub-nanometer accuracy,
and clear topographic phase images of cultured single-layer cells were obtained. The Kohler illumination with the
halogen lamp, whose coherence length is 2 &mgr;m, enables homogenous illumination and suppression of artifact signals
arising from optical components not associated with the surface of interest.
We describe the mechanism of distribution measurement of fiber Brillouin spectrum by Brillouin Optical Correlation Domain Analysis (BOCDA), and numerically simulate the distribution of the Brillouin gain spectrum, which is expected to be measured in experiments. The simulation results agree well with the experimental results. We show the verification of the theoretical formulation of spatial resolution, by using the numerical simulations.
We propose and demonstrate a novel method of distribution sensing of Doppler-Shift frequency, by applying Synthesis of delta-function-like Optical Coherence Function (SOCF) to laser Doppler velocimetry. SOCF is a technique to resolve longitudinal position information along one arm of a laser-interferometric system. Distribution of the Doppler frequency is successfully measured in experiments.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.