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1. Deep-3D microscope: 3D volumetric microscopy of thick scattering samples using a wide-field microscope and machine learning

   https://doi.org/10.1364/BOE.444488  

   Li, Bowen ; Tan, Shiyu ; Dong, Jiuyang ; Lian, Xiaocong ; Zhang, Yongbing ; Ji, Xiangyang ; Veeraraghavan, Ashok ( December 2021 , Biomedical Optics Express)

   Confocal microscopy is a standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering samples. Unfortunately, a confocal microscope is quite expensive compared to traditional microscopes. In addition, the point scanning in confocal microscopy leads to slow imaging speed and photobleaching due to the high dose of laser energy. In this paper, we demonstrate how the advances in machine learning can be exploited to teach a traditional wide-field microscope, one that’s available in every lab, into producing 3D volumetric images like a confocal microscope. The key idea is to obtain multiple images with different focus settings using a wide-field microscope and use a 3D generative adversarial network (GAN) based neural network to learn the mapping between the blurry low-contrast image stacks obtained using a wide-field microscope and the sharp, high-contrast image stacks obtained using a confocal microscope. After training the network with widefield-confocal stack pairs, the network can reliably and accurately reconstruct 3D volumetric images that rival confocal images in terms of its lateral resolution, z-sectioning and image contrast. Our experimental results demonstrate generalization ability to handle unseen data, stability in the reconstruction results, high spatial resolution even when imagingmore »thick (∼40 microns) highly-scattering samples. We believe that such learning-based microscopes have the potential to bring confocal imaging quality to every lab that has a wide-field microscope.

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2. Polarization Sensitive Photothermal Mid-Infrared Spectroscopic Imaging of Human Bone Marrow Tissue

   https://doi.org/10.1177/00037028211063513  

   Mankar, Rupali ; Gajjela, Chalapathi C. ; Bueso-Ramos, Carlos E. ; Yin, C. Cameron ; Mayerich, David ; Reddy, Rohith K. ( March 2022 , Applied Spectroscopy)

   Collagen quantity and integrity play an important role in understanding diseases such as myelofibrosis (MF). Label-free mid-infrared spectroscopic imaging (MIRSI) has the potential to quantify collagen while minimizing the subjective variance observed with conventional histopathology. Infrared (IR) spectroscopy with polarization sensitivity provides chemical information while also estimating tissue dichroism. This can potentially aid MF grading by revealing the structure and orientation of collagen fibers. Simultaneous measurement of collagen structure and biochemical properties can translate clinically into improved diagnosis and enhance our understanding of disease progression. In this paper, we present the first report of polarization-dependent spectroscopic variations in collagen from human bone marrow samples. We build on prior work with animal models and extend it to human clinical biopsies with a practical method for high-resolution chemical and structural imaging of bone marrow on clinical glass slides. This is done using a new polarization-sensitive photothermal mid-infrared spectroscopic imaging scheme that enables sample and source independent polarization control. This technology provides 0.5 µm spatial resolution, enabling the identification of thin (≈1 µm) collagen fibers that were not separable using Fourier Transform Infrared (FT-IR) imaging in the fingerprint region at diffraction-limited resolution ( ≈ 5 µm). Finally, we propose quantitative metrics to identify fiber orientation frommore »discrete band images (amide I and amide II) measured under three polarizations. Previous studies have used a pair of orthogonal polarization measurements, which is insufficient for clinical samples since human bone biopsies contain collagen fibers with multiple orientations. Here, we address this challenge and demonstrate that three polarization measurements are necessary to resolve orientation ambiguity in clinical bone marrow samples. This is also the first study to demonstrate the ability to spectroscopically identify thin collagen fibers (≈1 µm diameter) and their orientations, which is critical for accurate grading of human bone marrow fibrosis.

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3. Quantification of Errors in Applying DIC to Fiber Networks Imaged by Confocal Microscopy

   https://doi.org/10.1007/s11340-022-00870-6  

   Sarkar, M. ; Notbohm, J. ( January 2022 , Experimental Mechanics)

   Background. An assumption of Digital Image Correlation (DIC) is that the displacement field within each subset is relatively smooth, captured with reasonable accuracy by, for example, linear or quadratic shape functions. Although this assumption works well for many materials, it becomes problematic for heterogeneous materials, such as fiber networks, wherein the length scale of heterogeneity matches the size of a subset. Objective. Here we applied DIC to fibrous networks made of collagen, for which displacements at the scale of a subset are highly heterogeneous, but errors caused by the heterogeneity are difficult to quantify. We developed a method to quantify such errors. Methods. We began by generating a synthetic three-dimensional fiber network with structure matching that of gels made of fibrous collagen. We then formulated an algorithm to mimic the way in which a confocal microscope images the fibers at its focal plane, thereby generating synthetic images similar to those obtained in experiments. Displacement boundary conditions were applied to the synthetic fiber networks, and the resulting displacement fields were computed using a finite element solver. DIC was applied to the synthetic images, and displacements were compared to the data from the finite element method, enabling rigorous quantification of error. Results.more »Point-wise errors in the DIC-measured displacements were substantial, often exceeding 40%, but over regions far larger than the length scales of heterogeneity or the DIC subset size, errors were modest, e.g., ≤15%. Conclusions. Although DIC can accurately measure displacements of fiber networks at length scales larger than the subset window, quantification of mechanical behavior at the scale of material heterogeneity will require new methods to complement or replace the use of DIC.« less
4. Resolution enhancement of 2-photon microscopy using high-refractive index microspheres

   https://doi.org/10.1117/12.2290613  

   Tehrani, Kayvan F. ; Darafsheh, Arash ; Phang, Sendy ; Mortensen, Luke J. ( February 2018 , Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII)

   Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves featuresmore »of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.« less
5. Quantitative Histopathology of Stained Tissues using Color Spatial Light Interference Microscopy (cSLIM)

   https://doi.org/10.1038/s41598-019-50143-x  

   Majeed, Hassaan ; Keikhosravi, Adib ; Kandel, Mikhail E. ; Nguyen, Tan H. ; Liu, Yuming ; Kajdacsy-Balla, Andre ; Tangella, Krishnarao ; Eliceiri, Kevin W. ; Popescu, Gabriel ( October 2019 , Scientific Reports)

   Tissue biopsy evaluation in the clinic is in need of quantitative disease markers for diagnosis and, most importantly, prognosis. Among the new technologies, quantitative phase imaging (QPI) has demonstrated promise for histopathology because it reveals intrinsic tissue nanoarchitecture through the refractive index. However, a vast majority of past QPI investigations have relied on imaging unstained tissues, which disrupts the established specimen processing. Here we present color spatial light interference microscopy (cSLIM) as a new whole-slide imaging modality that performs interferometric imaging on stained tissue, with a color detector array. As a result, cSLIM yields in a single scan both the intrinsic tissue phase map and the standard color bright-field image, familiar to the pathologist. Our results on 196 breast cancer patients indicate that cSLIM can provide stain-independent prognostic information from the alignment of collagen fibers in the tumor microenvironment. The effects of staining on the tissue phase maps were corrected by a mathematical normalization. These characteristics are likely to reduce barriers to clinical translation for the new cSLIM technology.