This PDF file contains the front matter associated with SPIE Proceedings Volume 12630, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

We present color third-order sum-frequency generation (color TSFG) microscopy, a multiphoton imaging strategy based on the simultaneous detection of several third-order coherent signals produced by two synchronized femtosecond pulse trains. We demonstrate that it can be used to obtain red blood cell (RBC)-specific label-free contrast in live zebrafish and is a promising tool for probing RBC oxygenation.

Temporal focusing multiphoton excitation microscopy (TFMPEM) can rapidly provide 3D imaging in neuroscience; however, due to the widefield illumination and the use of camera detector, the strong scattering of emission photons through biotissue will degrade the image quality and reduce the penetration depth. As a result, TFMPEM images suffers from poor spatial resolution and low signal-to-noise ratio (SNR), burying weak fluorescent signals of small structures such as neurons in calyx part, especially for deep layers under fast acquisition rate. In the study, we present a prediction learning model with depth information to overcome. First, a point-scanning multiphoton excitation microscopy (PSMPEM) image as the gold standard was precisely registered to the corresponding TFMPEM image via a linear affine transformation and an unsupervised VoxelMorph network. Then, a multi-stage 3D U-Net model with cross-stage feature fusion mechanism and self-supervised attention module has been developed to restore shallow layers of drosophila mushroom body under cross-modality training. Furthermore, a convolutional long short-term memory (ConvLSTM)- based network with PhyCell, which is designed to forecast the deeper information according to previous 3D information, is introduced for the prediction of depth information.

Coherent anti-Stokes Raman Scattering (CARS) microscopy is a label-free vibrational imaging technique that delivers chemical maps of cells and tissues. CARS employs two narrowband picosecond pulses (pump and Stokes) that are spatiotemporally superimposed at the sample plane to probe a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite many technological advancements, BCARS microscopes still struggle to image biological samples spanning the entire Raman active region of biological samples (400-3100 cm^-1). Here, we demonstrate a novel BCARS method to answer this need. Our experimental setup is based on a femtosecond fiber laser at 1035 nm and 2 MHz repetition rate, thus delivering high energy pulses used for generating sub-20 fs broadband Stokes pulses by white-light continuum in a bulk YAG crystal, a compact and alignment-insensitive technique. Combining them with narrowband picosecond pulses, we can generate a CARS signal with high (< 10 cm^-1) spectral resolution in the entire Raman window exploiting both two-color and three-color excitation mechanisms. The system is equipped with a home-made transmission microscope to image cells and tissue at high-speed (< 3 ms) and large field of views. Using a post-processing pipeline, we deliver high-quality chemical maps, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, unveiling the path for applications in histopathological settings.

We analyze the tissue penetration of a steroid drug commonly used for the treatment of dermal and eye inflammations using phase-modulated stimulated Raman scattering (PM-SRS) microscopy. Depth-resolved imaging and penetration profile analysis reveal a clear difference in the drug penetration between the skin epithelial and the corneal epithelial tissue models due to the different barrier properties. The results suggest that PM-SRS imaging can be used to visualize the penetration pathway of topical drugs based on the spatial information of the cell and tissue structures, which should contribute to the design of drug functions.

We describe a 3D holographic two-photon optogenetics setup based on acousto-optic deflectors combined with a custom-made compact light-sheet microscope setup. The system allows performing 3D-targeted perturbations of neuronal activity while simultaneously imaging the whole brain neuronal response over large field-of-view. This random-access holographic light patterning method offers high-speed sequential activation of dense neuronal assemblies spread over large areas while maintaining constant lateral and axial resolution. Here, we report on a systematic characterization of the stimulated neurons response and perform functional imaging on zebrafish (Danio Rerio) larvae while stimulating single neurons.

We developed a small form factor, microscope objective like, wavefront shaping assisted optical device (Wavelens) to produce and control illumination patterns for use on Light-Sheet Fluorescence Microcopy (LSFM). We characterize it and demonstrate its capability to perform in vivo imaging using fluorescently labeled specimens, outperforming conventional optics.

To perform real-time stimulation of neurons and simultaneous observation of the neural connectome, a deep learning-based computer-generated holography (DeepCGH) system has been developed. This system utilized a neural network to generate a hologram, which is then real-time projected onto a high refresh rate spatial light modulator (SLM) to generate fast 3D micropatterns. However, DeepCGH had two limitations: the computation time is increased as the number of input layers grew, and it cannot reconstruct arbitrary 3D micropatterns within the same model. To address these issues, integrated a digital propagation matrix (DPM) into the DeepCGH data preprocessing to generate arbitrary 3D micropatterns within the same model and reduce the computation time. Furthermore, to incorporate temporal focusing confinement (TFC), the axial resolution (FWHM) is improved from 30 μm to 6 μm, and then it can avoid to excite other cells. As a result, the DeepCGH with DPM system is able to timely generate customized micropatterns within a 150-μm volume with high accuracy. With DPM, the DeepCGH was able to generate arbitrary 3D micropatterns and further save 50% computation time. Additionally, the DeepCGH holograms achieve superior results in optical reconstruction and have high accuracy in both position and depth as combined with TFC.

The system presented here is a further development of the recently introduced spectro-temporal laser imaging by diffractive excitation (SLIDE) microscopy technique. To excite endogenous fluorescence, a new flexible and fibre-based laser source at 780 nm has been developed. The fiber-based FDML-MOPA was amplified to high peak and average powers using rare earth erbium fiber amplifiers. Broadband quasi-phase-matched frequency doubling using a fan-out PPLN crystal was then employed. The output is a 10 nm wide swept pulsed laser at 780 nm with a peak pulse power of 150 W, a pulse duration of 44 ps and a pulse repetition rate of 82 MHz (250 pulses at a sweep rate of 347 kHz). The sweep rate is converted into line scans by a diffraction grating and sent to a microscope for two-photon excitation of UV-excited dyes or endogenous autofluorescence. For detection, the signal is captured with a 4 GS/s high-speed digitisation card, resulting in 2kHz fluorescence lifetime imaging (FLIM) acquisition. In this paper we present the first images of 780nm SLIDE obtained at 2kHz frame rate. Through the additional use of a piezo objective scanner, we are able to perform 3D imaging at 20Hz volume rate. We have also used this novel system for high-speed LiDAR imaging at 2kHz using the recently introduced SLIDE-based time-stretch LiDAR approach.

We have constructed a hyperspectral confocal microscope for high-speed and high-resolution readout of spectra from bio-integrated microlasers. We demonstrate its advanced performance in multiplexed cell tracking and dynamic sensing experiments.

The recently introduced high-speed multiphoton microscopy technique of Spectro Temporal Laser Imaging by Diffractive Excitation (SLIDE) provides fluorescence microscopy and fluorescence lifetime imaging (FLIM) at kilohertz frame-rates. Combined with flow cytometry, these speeds enable acquisitions of high resolution microscopy images of cells in flow with high sensitivity at specificity. Until now, SLIDE used infrared laser light for nonlinear two-photon imaging. Here we present SLIDE in the Visible by frequency-doubling of the 1064 nm Fourier Domain Mode Locked (FDML) laser. This new one-photon SLIDE system centered around 532 nm was used to image autofluorescent cells using fluorescence and FLIM imaging, as well as acquiring transmission images for a morphological imaging channel. The high-speed and multi-modal acquisition will permit applications in high-throughput cell analysis and cell sorting applications.

We explored the capabilities of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) in combination with machine learning (ML) approaches for the characterization and classification of urine sediments. Bright-field images and off-axis holograms of a liquid control for urine analysis were acquired with a modular DHM system. From the retrieved images, particle morphology parameters were extracted by segmentation procedures. In addition, the ability of supervised ML-algorithms to classify and identify urine sediment components based on biophysical parameters was evaluated. The results demonstrate DHM in combination with ML as a prospective tool for urine analysis.

Structured illumination microscopy (SIM) is a most popular super-resolution technique used in cell biology and bio-imaging. Here, we present a novel approach to realize multiscale super-resolution SIM by swapping the non-linearity between instrumentation and reconstruction algorithm to achieve super-resolution. Our goal is to overcome two conventional limitations of SIM i.e., fixed resolution and the need of precise knowledge of illumination pattern. The optical system encodes higher order frequencies of the sample by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in SIM. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. Our approach eliminates the need of clean peaks in the illumination pattern, which have multiple advantages i.e., simple instrumentation and the flexibility of using different collection lenses. The reconstruction algorithm used in the proposed work does not require known illumination. Finally, we reduce the sensitivity of reconstruction algorithm to the signal to background ratio. Here, we acquired patterned illumination images of the same sample using different collection objective lenses, and obtained diffraction limited as well as super-resolved images, supporting 4 different resolution in the same system through SIM. Our experimental results with multiple collection objective lens show wider applicability of the proposed system at signal to background ration as small as <3.

Quantitative phase microscope (QPM) is used for the quantitative information and dynamic phase imaging of biological specimen, which provides wide application in biomedical sciences. High temporal phase stability of the QPM system is the primary requirement for accurate phase measurement. We have developed a common-path QPM geometry based on beam displacer and pinhole unit to achieve high temporal stability. The convenient adjustment of reference and object beams makes optical system compact and low-cost. The membrane fluctuations and qualitative phase are measured to demonstrate the capability and applicability of the system.

The recently developed SLIDE microscope enables rapid imaging in nonlinear two-photon microscopy, where frame rates of 4 kHz are achieved. Such fast acquisition speeds coupled with the molecular specificity of fluorescence markers and the high optical resolution in the sub-μm range allow volume scan rates at 40 Hz. A commercially available Fourier Domain Mode Locked Laser system (Optores GmbH, Munich, Germany) was used as the light source emitting at 1060 nm (Bandwidth 15 nm). An electro-optical Modulator (EOM) splits the light of a single sweep duration up into 600 pulses with 30 ps pulse duration each. Each of it is then spatial separated by a diffractive grating. Only one scanner is needed for beam steering to excite the slow axis resulting to a frame rate of 4 kHz. Using a piezo driver for the objective of the microscope at a frequency of 20 Hz, a live 4D volume scan of 40 Hz with 600 x 400 x 100 voxel is possible. Until now, SLIDE systems were bulky and bound onto a fixed optical desk. The Medical Laser Centre Lübeck developed a transportable and reliable SLIDE system, so that this new and highly innovative technology can be made available to various biological laboratories in Europe. This work was conducted in the framework of the EU project “Faircharm.”

Coherent confocal light absorption and scattering spectroscopic (C-CLASS) microscopy, which extends the principles of light scattering spectroscopy to subcellular imaging, can be used to reveal biological structures well beyond the diffraction limit. Here we show that high-resolution C-CLASS microscopy can be used to detect nanoscale changes in chromatin structure. Unlike most methods for chromatin monitoring, C-CLASS microscopy can be used label-free in live cells. Live differentiating hiPSC organoids were measured over the space of sixteen days and characteristic chromatin changes were observed.

White light phase shifting is an important technique in interferometry to extract the high-resolution quantitative phase images with high spatial phase sensitivity i.e., of the order of sub nanometer. The dynamical information about a biological sample is limited in white light phase-shifting interferometry (WL-PSI) due to multiple frame requirement. The multiple frame requirement with controlled phase-shift is the key limitation for the high-resolution phase information extraction about the sample. A high-cost piezoelectric transducer (PZT) is required to introduce equal phase-shifts between the frames in WL-PSI. Here, we introduce a deep learning (DL)-based phase-shifter instead of PZT to introduce equal phase-shift in WL-PSI. We use deep neural network to introduce the equal phase-shift between the data frames. The idea is to train the network with multiple equal phase-shifted frames for training and learn the basic application of a PZT. After sufficient training of the network, it will generate multiple phase-shifted frames. Our study is validated by simulating step-like object with equal phase-shifts for training and testing of the network. The network is trained for a total 4 phase-shifted frame generation from a single interferogram. Further, the line profile of DL-based phase-shifter generated data frames are compared with the line profile of simulated data frames. Finally, generated phase-shifted data frames are used for the final phase reconstruction of the sample and compared with the phase reconstruction from simulated data frames.

Existing optical tissue phantoms are usually designed for wide field imaging systems and not readily usable for microscopic or endoscopic systems, especially such without any z-stage. Therefore a fs-laser microstructured artificial tissue phantom with adaptable geometric, tissue-optical and localized fluorescence properties enabling comparison and testing of different microscopic/endoscopic systems was designed, characterized and tested.

Using water as immersion medium has many advantages compared to oil as it is extremely cheap and non-toxic. This work explores the possibility of using water as immersion medium in Scanning Laser Optical Tomography

Imaging of multicellular tumor spheroids (MCTS) is of high relevance since MCTS recapitulate tumor environment characteristics more closely compared to 2D cell cultures. In particular, fluorescence microscopy imaging after DNA staining by DAPI allows observation of cell nuclei in MCTS. Limited depth-of-field (DOF) however affects the capability of visualizing the whole sample all-in-focus in a single shot. In order to overcome limited DOF while achieving 3D visualization of MCTS a custom-built fluorescence microscope including an electrically focus-tunable lens was developed. Once the multifocus sequence is acquired, image registration is applied in order to correct for changes in the field of view between images of the stack. Then post-processing in Fourier domain of the z-stack allows for DoF extension along with synthesis of novel viewpoints from which a 3D visualization of the MCTS can be reconstructed.

Phase imaging is a solution for the reconstruction of phase information from intensity observations. To make phase imaging possible, sophisticated extra systems are embedded into the existing imaging systems. Contrary, we propose a phase problem solution by DCNN-based framework, which is simple in terms of an optical system. We propose to replace optical lenses with computational algorithms such as CNN phase reconstruction and wavefront propagation. The framework is tested in simulation and real-life experimental phase imaging. To have real experiments with objects close to real-life biological cells, we simulated experimental training datasets on a phase-only spatial light modulator, where phase objects are modeled with corresponding phase distribution to biological cells.

Interaction of polarized light with tissue samples is an emergent and powerful tool for biomedical diagnosis due to the possibility of providing quantitative information through Stokes parameters and Mueller matrix elements. Division of Focal Plane polarimetric sensing in particular has recently become popular due to advances in technology and commercial availability of microgrid polarizers integrated onto the sensing chip of a camera. After calibration of the system, intensity measures from these sensors can be mapped to the State of Polarization (SoP) of the input. A microscopy setup incorporating DoFP sensing can allow then to obtain Stokes parameters corresponding to a given Field of View (FoV) of a tissue sample. By illuminating with linearly independent SoP in the input, Mueller matrix elements can also be retrieved for the FoV of the sample. Finally, FoV extension to whole-slide Mueller matrix imaging can be achieved through stitching of multiple images taken by means of a motorized XY stage. Validation results for whole-slide Mueller matrix elements of tissue samples are presented.

Lensless digital holography is a low-cost imaging technique with promising applications in a resource deficit environment. It can be used for the diagnosis and analysis of RBCs. Modifications in their morphology and other physical parameters are indicative of several diseases. The sparsity constraint imposed by the twin image phenomenon in inline holography allows the implementation of compressive sensing based holographic reconstruction. It allows the estimation of parameters like size, thickness, volume, sphericity index etc. form the extracted phase information. In this work this approach is used to study RBC in healthy and anemic states.

The potential of laser-induced thermal therapy can be reassessed in treating abnormal mucosal tissues with advances in fiber optics, diode laser technology, and optical imaging modalities. In this context, studies optimizing a large parameter matrix (e.g., laser power, surface scanning speed, beam diameter, and irradiation duration) may be of interest. This study presents an artificial intelligence algorithm utilizing a generative adversarial network that predicts dark-field microscopy images from bright-field images of H&E-stained esophageal specimens. The calculated structural similarity index measurement between ground truth and the predicted dark-field image reaches an average of 74%. Also, the mean squared error is 0.7%.

A novel method of artificial intelligence (AI) classification is proposed for hepatitis B virus (HBV) detection based on the Mueller matrix imaging system. The feasibility of the proposed technique is demonstrated by measuring the optical properties of non-infected and infected HBV blood samples. Furthermore, different AI classifier techniques namely Yolo5, Yolo5-Restnet101, Yolo5-EfficientnetB0, and Yolo5-MobilenetV2 have been employed to classify the HBV samples. The results show that the proposed method provides 99% accuracy for HBV classification. In general, the proposed technique provides reliable and simple devices for HBV diagnosis applications.

We present a laser speckle contrast imaging (LSCI) device equipped with an image conduit to image microvascular blood flow in remote tissues like ear, nose, throat (ENT) and cervical region. The system is validated using a tissue mimicking microfluidic flow phantom with different widths and flow speeds. The proposed system is being developed as a point of care testing (POCT) device best suited for at-home self-monitoring in resource-limited areas as it is non-invasive, portable, affordable and real time.