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Label-free optical microscopy utilizes the information encoded in light scattered off unlabeled particles to generate the images. This review article starts off with a discussion on how this light matter interaction gives rise to the issues of poor-contrast and diffraction-limited spatial resolution. Then, this article reviews the various far-field label-free optical microscopy techniques that have been developed, with an emphasis on the physical mechanisms behind the image formation processes in such techniques. Thus the article aims to elucidate the various state-of-the-art label-free techniques and their current applications.

Three-dimensional photoacoustic imaging (PAM) has emerged as a promising technique for non-invasive label-free visualization and characterization of biological tissues with high spatial resolution and functional contrast. The application of PAM and ultrasound as a microscopy technique of study for Atlantic salmon skin is presented here. A custom ultrasound and photoacoustic experimental setup was used for conducting this experiment with a sample preparation method where the salmon skin is embedded in agarose and lifted from the bottom of the petridish. The results of C-scan, B-scan, and overlayed images of ultrasound and photoacoustic are presented. The results are then analyzed for understanding the pigment map and its relation to salmon behavior to external stimuli. The photoacoustic images are compared with the optical images and analyzed further. A custom colormap and alpha map is designed and the matrices responsible for PAM and ultrasound are inserted together to overlay the ultrasound image and PAM image on top of each other. In this study, we propose an approach that combines scanning acoustic microscopy (SAM) images with PAM images for providing a comprehensive understanding of the salmon skin tissue. Overlaying acoustic and photoacoustic images enabled unique visualization of tissue morphology, with respect to identification of structural features in the context of their pigment distribution.

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences.

This review covers the advancements of optical super-resolution microscopy (SRM) on formalin-fixed paraffin-embedded (FFPE) histological samples. We cover the implementation of various SRM strategies in histology, including wide field methods such as structured illumination microscopy, single-molecule localization microscopy and fluorescence fluctuations-based SRM, as well as the point-scanning stimulated emission depletion microscopy. We also cover the recent developments in FFPE-based expansion microscopy. The review highlights the advantages and challenges of these SRM methods in FFPE histology, and provides insights into emerging optical and computational techniques that can potentially open avenues for understanding disease mechanisms, tailoring treatments, and advancing personalized medicine across disciplines. This review article is intended for a broad audience, including histopathologists, biologists, physiologists, and physicists. The review covers cutting-edge advancements in optical super-resolution microscopy (SRM) on formalin-fixed paraffin-embedded (FFPE) histological samples.

High space-bandwidth product with high spatial phase sensitivity is indispensable for a single-shot quantitative phase microscopy (QPM) system. It opens avenue for widespread applications of QPM in the field of biomedical imaging. Temporally low coherence light sources are implemented to achieve high spatial phase sensitivity in QPM at the cost of either reduced temporal resolution or smaller field of view (FOV). In addition, such light sources have low photon degeneracy. On the contrary, high temporal coherence light sources like lasers are capable of exploiting the full FOV of the QPM systems at the expense of less spatial phase sensitivity. In the present work, we demonstrated that use of narrowband partially spatially coherent light source also called pseudo-thermal light source (PTLS) in QPM overcomes the limitations of conventional light sources. The performance of PTLS is compared with conventional light sources in terms of space bandwidth product, phase sensitivity and optical imaging quality. The capabilities of PTLS are demonstrated on both amplitude (USAF resolution chart) and phase (thin optical waveguide, height ~ 8 nm) objects. The spatial phase sensitivity of QPM using PTLS is measured to be equivalent to that for white light source and supports the FOV (18 times more) equivalent to that of laser light source. The high-speed capabilities of PTLS based QPM is demonstrated by imaging live sperm cells that is limited by the camera speed and large FOV is demonstrated by imaging histopathology human placenta tissue samples. Minimal invasive, high-throughput, spatially sensitive and single-shot QPM based on PTLS will enable wider penetration of QPM in life sciences and clinical applications.

We present experimental demonstration of tilt-mirror assisted transmission structured illumination microscopy (tSIM) that offers a large field of view super resolution imaging. An assembly of custom-designed tilt-mirrors are employed as the illumination module where the sample is excited with the interference of two beams reflected from the opposite pair of mirror facets. Tunable frequency structured patterns are generated by changing the mirror-tilt angle and the hexagonal-symmetric arrangement is considered for the isotropic resolution in three orientations. Utilizing high numerical aperture (NA) objective in standard SIM provides super-resolution compromising with the field-of-view (FOV). Employing low NA (20X/0.4) objective lens detection, we experimentally demonstrate ∼ (0.56 mm × 0.35 mm) size single FOV image with ∼ 1.7- and ∼ 2.4-fold resolution improvement (exploiting various illumination by tuning tilt-mirrors) over the diffraction limit. The results are verified both for the fluorescent beads as well as biological samples. The tSIM geometry decouples the illumination and the collection light paths consequently enabling free change of the imaging objective lens without influencing the spatial frequency of the illumination pattern that are defined by the tilt-mirrors. The large and scalable FOV supported by tSIM will find usage for applications where scanning large areas are necessary as in pathology and applications where images must be correlated both in space and time.

The morphology and molecular study of the penguin brain are crucial to define its survival in the extreme conditions of Antarctica. The present study focusses on extracting different optical parameters of the penguin brain using label-free optical imaging and spectroscopic techniques. In label-free optical imaging, we have used quantitative phase imaging, which provides morphological information about the neurons in brain tissue, giving the quantitative phase value of 5 to 20 radians corresponding to the 8 µm tissue section. In label-free spectroscopic techniques, we have used autofluorescence and Raman spectroscopy. Autofluorescence spectroscopy provides molecular information about nicotinamide dinucleotide, flavins, lipofuscins, and porphyrins in the brain’s spectral range of 420 nm to 700 nm. Raman spectroscopy provides multiple peaks associated with different molecules in the brain; among them, few signals are observed at approximately 1305 cm −1 , 1448 cm −1 , and 1661 cm −1 , which correspond to vibrational modes indicative of vibrational features within lipids and protein structures, as well as the presence of amide groups within brain tissue constituents. All these techniques provide the microscopic and molecular fingerprint of the penguin brain, which can be useful for understanding penguin’s anatomical, physiological, and social behavior.

ObjectiveTo assess the rate, location, risk factors, management, and outcomes of neonatal thrombosis (NT).DesignA retrospective study investigating infants admitted to NICUs in Canadian Neonatal Network between January 2014 and December 2016 and diagnosed with NT. Each infant with NT was matched with an infant without NT.ResultsOf 39,971 infants, 587 (1.5%) were diagnosed with NT: 440 (75%) venous, 112 (19%) arterial, 29 (5%) both. NT rate was 1.4% in full-term and 1.7% in preterm infants. Venous thrombi occurred most commonly in the portal vein and arterial thrombi in the cerebral artery. Conservative management and low molecular weight heparin were the most common treatment modalities. Hospital stay was longer (p < 0.001) in the NT patients, but mortality was similar.ConclusionsNT was diagnosed in ~15/1000 NICU admissions and most commonly in the portal vein and cerebral arteries. Management varied based on the type and location of thrombi. Large multicenter trials are needed to address the best management strategies.

This study aims to demonstrate the generation and detection of Scholte waves inside polystyrene microparticles. This was proven using both experimental analysis and COMSOL simulation. Microspheres of different sizes were excited optically with a pulsed laser (532 nm), and the acoustic signals were detected using a transducer (40 MHz). On analyzing the laser-generated ultrasound signals, the results obtained experimentally and from COMSOL are in close agreement both in the time and frequency domain. A simplified analysis of Scholte wave generation by laser irradiation for homogeneous, isotropic microspheres is presented. The theoretical wave velocity of the Scholte wave was calculated and found close to our experimental results. A representation of pressure wave motion showing the Scholte wave generation is presented at different times.