This PDF file contains the front matter associated with SPIE Proceedings Volume 11521, including the Title Page, Copyright information, and Table of Contents.

Fluorescence light-sheet microscopy is gaining rapid adoption in developmental biology. With irradiation levels well below that of confocal and multi-photon microscopy, it enables the study of intact organs and organisms for prolonged time periods during development. Minimal sample exposure is achieved by selectively illuminating the focal plane with a second objective orthogonal to the detection axis. The light-sheet microscope’s ability to study intact biological samples as and when they grow highlights the importance of imaging deeper into biological samples. Yet, deep-tissue microscopy is hampered by autofluorescence and the scattering of light. Direct observations are therefore limited to highly transparent and thin samples. Here, we show how autofluorescence can be eliminated effectively by relying on reversible photoswitching fluorescence while we propose a way forward to study and control light propagation in optically-thick tissues.

An essential trafficking mechanism of ciliogenesis is intraflagellar transport (IFT). The IFT processes at the ciliary base were largely unknown based on the diffraction-limited kymograph imaging. Here, we optimize single-molecule tracking localization microscopy to study IFT proteins at the ciliary base by observing IFT88-mEOS4b in live human retinal pigment epithelial cells. Surprisingly, we found that IFT88 proteins “switched gears” multiple times from ciliary base to cilium, revealing region-dependent slowdown of IFT proteins at the ciliary base: a slow to relatively fast movement from distal appendages (DAPs) to proximal transition zone (TZ), slow again in the distal TZ, and fastest in the ciliary compartment (CC). Our results further revealed that IFT88 could travel between the DAPs and the axoneme without following DAP structures.

In this study, an integrated theranostic system combining a CW laser diode with optical coherence tomography (OCT)/angiography (OCTA) was utilized for tumor identification and treatment monitoring. To examine the effect of laser exposure on tissue scattering characteristics, the OCT backscattering intensities of non-ablated and ablated tissues were analyzed, and the effect on the skin microvasculature produced by laser ablation was quantitatively evaluated. Moreover, the integrated system and the proposed method were implemented for the treatment of skin and brain tumors on the mouse model. The obtained results indicate that the developed laser ablation system can effectively remove tumor tissues with controllable photodamage under OCT/OCTA guidance and that the system cost may be significantly reduced by using the CW laser diode.

Optical coherence tomography (OCT) is a non-invasive cross-sectional imaging technique with micrometer resolution. We have been investigating ultrahigh-resolution (UHR)-OCT using supercontinuum. The characteristics of OCT imaging depend on the optical wavelength used. In order to investigate the wavelength dependence of UHR-OCT, the wideband, high-power, low-noise supercontinua were generated at wavelengths of 0.8, 1.1, 1.3, and 1.7 um based on ultrashort pulses and nonlinear fibers. The wavelength dependence of OCT imaging was examined quantitatively using biological phantoms and rat lung tissue. Then we developed UHR-spectral domain-OCT and optical coherence microscopy (OCM) at 1.7 um. The high-resolution and high-penetration imaging of mouse brain was demonstrated.

We use patterned light to initiate scattered photons that are obliquely directed to excite voltage indicators, which report changes in the membrane potential. Our proposed scheme reduces the excitation of voltage indicators elsewhere in the cell that constitutes the background signal and therefore enables us to amplify the detected fluorescence carrying the voltage information.

With live-cell imaging using fluorescent proteins, we identified first stem-cell inducing factor which is conserved between both plant and animal kingdoms. We found the accumulation of Cold shock domain protein1 (CSP1) during the stem-cell formation process and the enhanced stem-cell formation caused by overexpression of CSP1 in the moss Physcomitrella patens. These data indicate that CSP1 functions in enhancing the stem-cell formation. CSP1 is the plant homolog of the mammalian induced pluripotent stem cell (iPSC) factor Lin28. In addition, towards high-resolution livecell imaging of biological molecules such as CSP1, we adopted adaptive optics (AO) for microscopy. With AO, we successfully corrected the optical disturbance caused by the artificial sample mimicking the leaf cells of Physcomitrella.

Phase-contrast imaging of biological tissues is one of the most important techniques used to investigate biological structures and functions. A phase-contrast optical microscope using oblique illumination and active image processing that is a figure-8-shaped spatial-frequency filter with sign changes has been developed to visualize phase structure of living tissues. This microscope offers promising features including separate phase and amplitude-contrast imaging, spherical aberration-free imaging, and reduced multi-scattering light. We reproduced and evaluated phase-contrast images of biological samples. We also extended the technique to the phase-contrast scanning microscope using annular illumination.

Recently, integral (also known as lightfield or plenoptic) imaging concept has been applied successfully to microscopy. The main advantage of lightfield microscopy when compared with conventional 3D imaging techniques is that it offers the possibility of capturing the 3D information of the sample after a single shot. However, integral microscopy is now facing many challenges, like improving the resolution and depth of field of the reconstructed specimens or the development and optimization of specially-adapted reconstruction algorithms. This contribution is devoted to review a new paradigm in lightfield microscopy, namely, the Fourier-domain integral microscope (FiMic), that improves the capabilities of the technique, and to present recent advances and applications of this new architecture.

Quantitative phase microscopy (QPM) has been widely applied to many biological imaging and material metrology applications. Our lab has recently empowered QPM with artificial intelligence (AI), ultrahigh imaging speed, and 3D imaging capability. In one study we have built an artificial neural network for white blood cell (WBC) classification by using thousands of labeled WBC phase images from over 5 healthy human subjects. This ANN has been able to successfully predict the WBC counts of new human subjects with 90% accuracy on average. In the second study, we have implemented high speed digital micro-mirror devices (DMDs) and high-speed image sensor to achieve synthetic aperture phase imaging and 3D imaging of transparent thin material structures with ~200 nm resolution at <500 fps speed, which can be used for cytometry and histopathology applications.

Optical coherence tomography (OCT) is non-invasive biomedical imaging technique, which can provide volumetric imaging of the tissue architectural information. In this talk, I will briefly discuss the preliminary results of several ongoing works in my lab, including the quantitative analysis of the microvasculature with the animal model and investigation of the mouse cochlear anatomy.

This paper reports for high-sensitivity imaging based on Ghost imaging (GI), which is one of the single-pixel imaging. Although the GI is correlation-based imaging between structured illumination lights and detected signals, there is an advantage in detecting weak light intensity such as fluorescence of molecules. Especially, in the case of using extream weak light intensity, a photon signal is useful for imaging. Therefore, we focused on the arrival time of the first photon and used the time as the intensity of the signal. Furthermore, to improve the detection time, we applied machine learning to reduce the measurement number. In this paper, we have proposed the principle and some experimental results.

Acceleration of image acquisition rate in Raman microscopy has been required to fully utilize its analytical advantages for biological/medical applications. By introducing the multiple line illumination and parallel spectral detection capability, image acquisition rate in the Raman microscope was improved < 104 times, compared with the conventional confocal Raman. High-resolution spontaneous Raman imaging of cells/tissues was thus enabled with an image acquisition time of a few minutes. Subsequent high-throughput Raman imaging-based analyses were also performed, including multiplex Raman tag imaging, cell classification, microplastic detection.

We propose a non-invasive and non-contact imaging technique based on the diffuse reflectance spectroscopy to evaluate the concentrations of methemoglobin (metHb), oxygenated hemoglobin (HbO), and deoxygenated hemoglobin (HbR) simultaneously. Experiments with in vivo rat exposed to sodium nitrite (NaNO2) shows the feasibility of the method for monitoring changes in metHb, HbO, and HbR concentrations during methemoglobinemia.

Neuroblastoma has been considered as one of the most common extracranial solid tumors of childhood. In this study, we propose a non-invasive diagnostic measurement in evaluating the malignant level of neuroblastoma utilizing the Mueller matrix decomposition to extract effective optical parameters. The results showed a significant difference in optical properties between good and bad prognosis neuroblastoma samples.

A single-shot common-path off-axis multi-wavelength digital holographic microscopic (MW-DHM) system for phase imaging of biological specimens is proposed. The DHM system is based on a cube beam splitter which divides the object beam into two beams: one beam is spatially filtered at its Fourier plane to make a reference spherical beam and another beam acts as the object beam. Three wavelengths: 473, 532, and 632.8 nm are used to illuminate the object under observation. The obtained experimental results on various objects corroborate the imaging capability of the proposed single-shot common-path off-axis multi-wavelength MW-DHM system. The proposed system may open new possibilities in bio-imaging and real-time simultaneous measurement of various parameters including the thickness, refractive index, etc.

Spectral reflectance in human skin tissue has been studied through Monte Carlo simulation using the Nine-layered skin tissue model. It is important to estimate absorption and scattering parameters of human skin tissue to know the condition of human skin. In this study, we investigated a method for estimating the absorption and scattering parameters by considering the effect of specific layers on the spectral band in the spectral reflectance database of human skin generated by Monte Carlo simulation.

In this research, we have proposed a multimodal fast fluorescent imaging by an electrical tunable lens, and quantitative phase imaging by digital holography. In conventional confocal microscopy, sectioning in z direction is realized either by moving objective lens or sample stage. In order to speed up the imaging, and utilize in the specific applications such as light stimulation system in optogenetics, the sectioning is realized by changing the focal power of an electrical tunable lens. The preliminary experiment using fluorescence beads and its relation between focal power and depth change is shown.

We proposed an addressable potentiometric sensor for ion imaging using a focused electron beam instead of the light. The electron beam can be easily focused to a spot of several nanometer, and it enable to improve the spatial resolution. The electron beam for imaging the ion concentration distribution is scattered in a sensor substrate and the spatial resolution is reduced. Therefore, it is necessary to reduce the scattering in order to realize a high spatial resolution. In order to reduce scattering, the acceleration voltage of the electron beam and the film thickness of the sensor substrate were examined.

A widefield endoscope with optical sectioning capability using digital micro-mirror device (DMD) is proposed and demonstrated. With the addition of DMD, uniform and grid illumination pattern can be created for HiLo algorithm to remove out-of-focus noise in widefield images. By applying spectral filters to raw images, high frequency in-focus component of uniform illumination image and low frequency in-focus component of grid illumination image can be extracted, respectively. An in-focus image with full resolution can then be reconstructed by combining these two images. To verify the optical sectioning capability of the proposed system, 45μm fluorescent beads and mouse heart tissues were observed. The improvement of signal-to-noise ratio (SNR) can be obviously seen from the results.

We are studying about super-resolution microscopy based on parallel scanning of multiple subdiffraction-limited spots. This paper reports on improvement of signal-to-noise ratio (SNR) of super resolution images by additional digital signal processing. More specifically, the size of the digital pinhole is changed during reconstruction. Experimental results demonstrate that the SNR can be improved by using an appropriate pinhole size.

Multiplicative algebraic reconstruction techniques (MART) is one of the method of projections onto convex sets (POCS) for solving a system of simultaneous equation. The image reconstruction problem can be discretized and depends on seeking the inverse of some matrix. We apply the MART to image reconstruction problems and evaluate the image quality in computer simulations. It is showed that the error decreases with increasing the number of projections and the number of iterations. It is also showed that the error decreases with increasing the value of initial data.

A numerical method to cancel the phase error caused by sample bulk motion from SD-OCT volume data is presented. This method first measures the vectorial gradient field of the phase error. The phase error is then estimated by path integration operation with non-standard integration paths. The performance of this method is validated by assessing the image quality of numerically refocused OCT images which is generated by complex holographic signal processing.

A convolutional neural networks (CNN) based scatterer density estimator for optical coherence tomography (OCT) is presented. In order to train the OCT, small patches of OCT speckle image were numerically generated. In this numerical image generation, the imaging parameters including the resolutions, probe power, signal-to-noise ratio, and scatterer density were randomly defined. So, the CNN was trained to estimate the imaging parameters from the generated OCT image patch. The results showed that our CNN estimator can estimate the parameters from the OCT speckle images.

A new method for quantitative assessment of tissue dynamics and activity is presented. The method is based on polarizationsensitive optical coherence tomography. Temporal variance of birefringence and temporal polarization uniformity are used to assess the tissue dynamics. These methods are applied to hourly time-course evaluation of tissue activity of ex-vivo dissected mouse heart.

We have developed one-shot RGB-spectroscopic full-field OCT (FF-OCT). In this system, red (R), green (G), and blue (B) lights emitted from LED light sources are synthesized into the light incident to a Michelson interferometer. The interference image at the detecting plane are separated into RGB images by a Bayer filter on a single-panel CMOS color camera.We show the possibility of RGB-spectroscopic OCT imaging in the one-shot operation in this study.

Due to asynchronization between the acquisition trigger and K-clock trigger in a swept source optical coherence tomography (SS-OCT) system, trigger jitter causes the spectrum a temporal shift in the spectral domain and thus corrupts the measurement. We study ternary distribution of the jitter signal by measuring TiO₂ phantom using a SS-OCT system, and it shows one-pixel spectral shift in the spectral domain.

We previously proposed a three-dimensional microscopic imaging system for objects hidden behind scattering media using in-line phase-shift digital holography, which simultaneously captures amplitude and phase information. However, as the thickness of the scattering medium increases, the influence of scattering is enhanced, and the reconstructed image of the object behind the scattering medium deteriorates. In this paper, we report the evaluation of this image using a near-infrared light source with a wavelength of 780 nm that is capable of deep tissue penetration. A favorable microscopic image of the object behind the rat-skin sample of 912 μm-thickness was successfully reconstructed.

We report the three-dimensional (3D) trajectory of a Volvox moving in water was recorded by parallel phase-shifting digital holographic microscope providing 10X magnification. The recording frame rate, the shutter speed, and the total recording time were 1000 fps, 0.25 ms, and 2.1 s, respectively. In the reconstructed phase image of the Volvox, the shape of the Volvox is regarded as a circle. The lateral coordinates of the Volvox were determined as the center of the circle. The depth coordinates of the Volvox were determined as the propagation distance where the edge of the Volvox in the reconstructed amplitude image was clearest while the propagation distance was varied. We successfully demonstrated the 3D tracking of curvedly moving Volvox.

In this study, we report spatial resolution enhancement and image quality improvement of a digital holographic microscopy using speckle patterns generated from a moving diffuser. In this method, speckle patterns are produced by moving a diffuser in the in-plane directions and incident on an object. In comparison with other methods for generating speckle illuminations, it realizes a simple and low-cost optical setup in digital holographic microscopy.

Phase-shifting digital holography has a feature that a hologram can be acquired with a spatial resolution of an image sensor. The interference images should be acquired at multiple times while the phase-shifting is given to the reference light. Therefore, some idea for imaging a moving object have been discussed. The phase-shifting digital holography with burst-imaging method is proposed for imaging a moving object. The phase-shifting digital holography at 460 μs is demonstrated by using a high-speed camera and a high-speed phase modulation based on a piezoelectric element.

We report imaging of a sound field radiated from a sound source by parallel phase-shifting digital holography. We used a Nd:YVO₄ laser emitting light with a wavelength of 532 nm as a light source and a polarization imaging camera to record holograms. The holograms were recorded 40000 Hz sound with 100000 frame per second. To adjust one wavelength of sound to the recordable area of the image sensor, we introduced a demagnification optical system in the path of the object beam. The phase difference images were calculated from the recorded holograms. Thus, we observed propagation of periodical phase distributions of sound and succeeded in sound field imaging.

A new type of reference mirror constructed by a half mirror and a full reflective mirror was introduced for the lowcoherence interferometer. The interference pattern recorded by the camera consisted of the interference of the object with different reflectance orders of the reference mirror, it allowed reconstructing the profile of the object whose depth was many times longer than the coherence length of the light source in a single-short. Fast Fourier transform (FFT) of the recorded hologram and the spatial filters were applied to obtain the complex amplitude for each object parts those were then combined to obtain the completed 3D information of the object.

Isotropic quantitative differential phase contrast (iDPC) microscopy based on pupil engineering has made significant improvement in reconstructing phase image of weak phase objects. To further enhance acquisition speed for phase recovery in iDPC, we adapt deep neural networks to achieve isotropic phase retrieval from half-pupil based quantitative differential phase contrast (qDPC) microscopy. We proposed to utilize U-net model for transforming phase distribution from 2-axis reconstruction to 6-axis one. The results show that deep neural network we proposed works as well as we expected. The final loss value of our model after 500 epochs of training can achieve about 5.7e-5 after normalized.

Photoacoustic microscopy with large depth of focus (DoF) is significant to the biomedical research. Here, we developed a virtual multi-focus optical-resolution photoacoustic microscope with extended depth of field by using block Discrete Cosine Transform (DCT) fusion. The source images from different focus is first 8 × 8 partitioned, and then DCT coefficients of each block can be calculated by using DCT transformation, and then the variance values of the corresponding blocks are calculated through DCT coefficients. The variance values are used as the activity level measures, blocks with large variances are selected. Finally, the fused image with virtual multi-focus is made up of blocks with the larger variances. Simulation and the in vivo imaging of zebra fish were performed to demonstrate that this method can extend the depth of field of PAM two times without the sacrifice of lateral resolution.

We used only a narrow range, 1397-1501 cm^-1, for high-throughput analysis of cancerous and noncancerous human cell lines by spontaneous Raman microscopy. With baseline-corrected cellwise spectra in this range, two cell lines were discriminated at accuracy higher than 90%. This narrowband measurement allowed reduction of the signal readout time by 24-folds in comparison to a correspondent wideband measurement detecting 536-3132 cm^-1, enabling cell analysis at 2.5 cells/min. To further improve the throughput, we employed detector binning, which allowed reduction not just of the readout time but also of the signal accumulation time with maintaining signal-to-background ratio and the accuracy. Improvement of the imaging speed by this approach reached at 4-folds, enabling a high-throughout analysis at 10 cells/min.

Similarity in skin reflectance spectra with different combination of absorption and scattering conditions makes erroneous estimation of parameters for any measured spectrum through the database containing simulated spectra. In this study, such similar reflectance spectra are investigated by Monte Carlo simulation and phantom experiment.

The circular polarization (CP) of light scattered by biological tissues provides valuable information about the structural changes in tissues. We investigate the spatial discrimination of cancer using CP light scattering within the in-plane and along the depth direction. In-plane spatial resolution was investigated using experiments on sliced biological tissues, which show a noticeable difference in polarization values between healthy and cancerous parts in a wide angular range. The resolution in the depth direction is examined with the Monte Carlo calculation method on pseudo-tissues having thin cancerous layers on healthy tissues. The calculation results suggest that the thickness of cancer can be estimated by detecting the degree of circular polarization values with different detection angles. The in-plane and depth resolutions are approximately 0.3 mm and 0.6 mm, respectively.

Lensless digital holography can reconstruct the intensity and phase of an object located behind a diffuser. However, the image quality of reconstructed images is dependent on the distance between the diffuser and the image sensor or between the diffuser and the sample. For this study, we reconstruct the object intensity by changing the distance between the sample and the diffuser. Then we investigate the image quality of reconstructed images.

Computational imaging through diffusing or scattering media has attracted attention in many fields. Deep learning has been used in recent years to reconstruct images behind scattering media. As described in this paper, we investigate image reconstruction through a diffuser by changing the distance separating the diffuser and the lens. We also investigate aperture diameter effects on scattered light removal by spatial filtering.

Visible-wavelength two-photon excitation (v2PE) is a powerful technique for simultaneous multicolor fluorescence imaging via simultaneous excitation of fluorescent proteins (FPs) with different emission wavelengths. We implemented v2PE into a slit-scanning confocal microscope in order to realize faster simultaneous multicolor fluorescence imaging with utilizing the capability of spectral detection. We demonstrated simultaneous multicolor imaging of living HeLa cells with expressing three types of FPs with different emission wavelengths localized at different intracellular structures. Linear un-mixing of hyperspectral images successfully separated the distribution of multiple FPs expressed in the sample.