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Iron gall Inks corrosion causes paper degradation (browning, embrittlement) and treatments were developed to tackle this issue. They often include resizing with gelatin to reinforce the paper and its cellulosic fibers (of diameter approx. 10 µm). This work aimed at measuring the distribution of ink components at the scale of individual paper fibers so as to give a better understanding of the impact of gelatin (re-)sizing on iron gall ink corrosion. For this purpose, scanning transmission X-ray microscopy (STXM) was used at the Canadian light source synchrotron (CLS, Saskatoon). This technique combines nano-scale mapping (resolution of 30 nm) and near edge X-ray absorption fine structure (NEXAFS) analysis. Fe L-edge measurements enabled to map iron distribution and to locate iron(II) and iron(III) rich areas. N K-edge measurement made it possible to map gelatin distribution. C K-edge measurements allowed mapping and discrimination of cellulose, gallic acid, iron gall ink precipitate and gelatin. Three fibers were studied: an inked fiber with no size, a sized fiber that was afterwards inked and an inked fiber sprayed with gelatin. Analysis of gelatin and ink ingredients distribution indicated a lower amount of iron inside the treated cellulosic fiber, which may explain the beneficial effect of gelatin on iron gall ink corrosion.

Significance: The spatial organization of collagen fibers has been used as a biomarker for assessing injury and disease progression. However, quantifying this organization for complex structures is challenging. Aim: To quantify and classify complex collagen fiber organizations. Approach: Using quantitative second-harmonic generation (SHG) microscopy, we show that collagen-fiber orientation can be viewed as pseudovector fields. Subsequently, we analyze them using fluid mechanic metrics, such as energy U, enstrophy E, and tortuosity tau. Results: We show that metrics used in fluid mechanics for analyzing fluid flow can be adapted to analyze complex collagen fiber organization. As examples, we consider SHG images of collagenous tissue for straight, wavy, and circular fiber structures. Conclusions: The results of this study show the utility of the chosen metrics to distinguish diverse and complex collagen organizations. We find that the distribution of values for E and U increases with collagen fiber disorganization, where they divide between low and high values corresponding to uniformly aligned fibers and disorganized collagen fibers, respectively. We also confirm that the values of tau cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Published version

Searching analyzable metaphase chromosomes is a critical step for the diagnosis and treatment of leukemia patients, and the searching efficiency is limited by the difficulty that the conventional microscopic systems have in simultaneously achieving high resolution and a large field of view (FOV). However, this challenge can be addressed by Fourier ptychography microscopy (FPM) technology.The purpose of this study is to investigate the feasibility of utilizing FPM to reconstruct high-resolution chromosome images.An experimental FPM prototype, which was equipped with 4 × / 0.1 NA or 10 × / 0.25 NA objective lenses to achieve a theoretical equivalent NA of 0.48 and 0.63, respectively, was developed. Under these configurations, we first generated the system modulation transfer function (MTF) curves to assess the resolving power. Next, a group of analyzable metaphase chromosomes were imaged by the FPM system, which were acquired from the peripheral blood samples of the leukemia patients. The chromosome feature qualities were evaluated and compared with the results accomplished by the corresponding conventional microscopes.The MTF curve results indicate that the resolving power of the 4 × / 0.1 NA FPM system is equivalent and comparable to the 20 × / 0.4 NA conventional microscope, whereas the performance of the 10 × / 0.25 NA FPM system is close to the 60 × / 0.95 NA conventional microscope. When imaging the chromosomes, the feature qualities of the 4 × / 0.1 NA FPM system are comparable to the results under the conventional 20 × / 0.4 NA lens, whereas the feature qualities of the 10 × / 0.25 NA FPM system are better than the conventional 60 × / 0.95 NA lens and comparable to the conventional 100 × / 1.25 NA lens.This study initially verified that it is feasible to utilize FPM to develop a high-resolution and wide-field chromosome sample scanner.

Significance: An effective contrast agent for concurrent multimodal photoacoustic (PA) and ultrasound (US) imaging must have both high optical absorption and high echogenicity. Integrating a highly absorbing dye into the lipid shell of gas core nanobubbles (NBs) adds PA contrast to existing US contrast agents but may impact agent ultrasonic response. Aim: We report on the development and ultrasonic characterization of lipid-shell stabilized C3F8 NBs with integrated Sudan Black (SB) B dye in the shell as dual-modal PA-US contrast agents. Approach: Perfluoropropane NBs stabilized with a lipid shell including increasing concentrations of SB B dye were formulated by amalgamation (SBNBs). Physical properties of SBNBs were characterized using resonant mass measurement, transmission electron microscopy and pendant drop tensiometry. Concentrated bubble solutions were imaged for 8 min to assess signal decay. Diluted bubble solutions were stimulated by a focused transducer to determine the response of individual NBs to long cycle (30 cycle) US. For assessment of simultaneous multimodal contrast, bulk populations of SBNBs were imaged using a PA and US imaging platform. Results: We produced high agent yield (∼1011) with a mean diameter of ∼200 to 300 nm depending on SB loading. A 40% decrease in bubble yield was measured for solutions with 0.3 and 0.4 mg/ml SB. The addition of SB to the shell did not substantially affect NB size despite an increase in surface tension by up to 8 mN/m. The bubble decay rate increased after prolonged exposure (8 min) by dyed bubbles in comparison to their undyed counterparts (2.5-fold). SB in bubble shells increased gas exchange across the shell for long cycle US. PA imaging of these agents showed an increase in power (up to 10 dB) with increasing dye. Conclusions: We added PA contrast function to NBs. The addition of SB increased gas exchange across the NB shell. This has important implications in their use as multimodal agents.

Significance: Selective plane illumination microscopy (SPIM) is an emerging fluorescent imaging technique suitable for noninvasive volumetric imaging of C. elegans. These promising microscopy systems, however, are scarce in academic and research institutions due to their high cost and technical complexities. Simple and low-cost solutions that enable conversion of commonplace wide-field microscopes to rapid SPIM platforms promote widespread adoption of SPIM by biologist for studying neuronal expressions of C. elegans. Aim: We sought to develop a simple and low-cost optofluidic add-on device that enables rapid and immobilization-free volumetric SPIM imaging of C. elegans with conventional fluorescent microscopes. Approach: A polydimethylsiloxane (PDMS)-based device with integrated optical and fluidic elements was developed as a low-cost and miniaturized SPIM add-on for the conventional wide-field microscope. The developed optofluidic chip contained an integrated PDMS cylindrical lens for on-chip generation of the light-sheet across a microchannel. Cross-sectional SPIM images of C. elegans were continuously acquired by the native objective of microscope as worms flowed in an L-shape microchannel and through the light sheet. Results: On-chip SPIM imaging of C. elegans strains demonstrated possibility of visualizing the entire neuronal system in few seconds at single-neuron resolution, with high contrast and without worm immobilization. Volumetric visualization of neuronal system from the acquired cross-sectional two-dimensional images is also demonstrated, enabling the standard microscope to acquire three-dimensional fluorescent images of C. elegans. The full-width at half-maximum width of the point spread function was measured as 1.1 and 2.4 μm in the lateral and axial directions, respectively. Conclusion: The developed low-cost optofluidic device is capable of continuous SPIM imaging of C. elegans model organism with a conventional fluorescent microscope, at high speed, and with single neuron resolution.

Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition. Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM. Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM. Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz. Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.

Significance: Two-photon and confocal microscopy can obtain high frame rates; however, mosaic imaging of large tissue specimens remains time-consuming and inefficient, with higher imaging rates leading to a larger fraction of time wasted translating between imaging locations. Strip scanning obtains faster mosaic imaging rates by translating a specimen at constant velocity through a line scanner at the expense of more complex stitching and geometric distortion due to the difficulty of translating at completely constant velocity. Aim: We aim to develop an approach to mosaic imaging that can obtain higher accuracy and faster imaging rates while reducing computational complexity. Approach: We introduce an approach based on scanner-synchronous position sampling that enables subwavelength accurate imaging of specimens moving at a nonuniform velocity, eliminating distortion. Results: We demonstrate that this approach increases mosaic imaging rates while reducing computational complexity, retaining high SNR, and retaining geometric accuracy. Conclusions: Scanner synchronous strip scanning enables accurate, high-speed mosaic imaging of large specimens by reducing acquisition and processing time.

Significance: High-speed three-dimensional (3D) super-resolution microscopy is a unique tool to investigate various biological phenomena; yet the technology is not broadly adopted due to its high cost and complex system design. Aim: We present a compact, low-cost, and high-speed 3D structured illumination microscopy (SIM) based on a digital micromirror device and binary holography to visualize fast biological events with super-resolution. Approach: The 3D SIM uses a digital micromirror device to generate three laser foci with individually controllable positions, phases, and amplitudes via binary holography at the back aperture of objective lens to form optimal 3D structured patterns. Fifteen raw images are sequentially recorded and processed by the 3D SIM algorithm to reconstruct a super-resolved image. Results: Super-resolution 3D imaging at a speed of 26.7 frames per second is achieved with a lateral and axial resolution of 155 and 487 nm, which corresponds to a 1.65- and 1.63-times resolution enhancement, respectively, comparing with standard deconvolution microscopy. Conclusions: The 3D SIM realizes fast super-resolution imaging with optimal 3D structured illumination, which may find important applications in biophotonics.

Significance Prostate cancer is the most common cancer among men. An accurate diagnosis of its severity at detection plays a major role in improving their survival. Recently, machine learning models using biomarkers identified from Raman micro-spectroscopy discriminated intraductal carcinoma of the prostate (IDC-P) from cancer tissue with a ≥85 % detection accuracy and differentiated high-grade prostatic intraepithelial neoplasia (HGPIN) from IDC-P with a ≥97.8 % accuracy. Aim To improve the classification performance of machine learning models identifying different types of prostate cancer tissue using a new dimensional reduction technique. Approach A radial basis function (RBF) kernel support vector machine (SVM) model was trained on Raman spectra of prostate tissue from a 272-patient cohort (Centre hospitalier de l'Universite de Montreal, CHUM) and tested on two independent cohorts of 76 patients [University Health Network (UHN)] and 135 patients (Centre hospitalier universitaire de Quebec-Universite Laval, CHUQc-UL). Two types of engineered features were used. Individual intensity features, i.e., Raman signal intensity measured at particular wavelengths and novel Raman spectra fitted peak features consisting of peak heights and widths. Results Combining engineered features improved classification performance for the three aforementioned classification tasks. The improvements for IDC-P/cancer classification for the UHN and CHUQc-UL testing sets in accuracy, sensitivity, specificity, and area under the curve (AUC) are (numbers in parenthesis are associated with the CHUQc-UL testing set): +4 % (+8 % ), +7 % (+9 % ), +2 % (6%), +9 (+9) with respect to the current best models. Discrimination between HGPIN and IDC-P was also improved in both testing cohorts: +2.2 % (+1.7 % ), +4.5 % (+3.6 % ), +0 % (+0 % ), +2.3 (+0). While no global improvements were obtained for the normal versus cancer classification task [+0 % (-2 % ), +0 % (-3 % ), +2 % (-2 % ), +4 (+3)], the AUC was improved in both testing sets. Conclusions Combining individual intensity features and novel Raman fitted peak features, improved the classification performance on two independent and multicenter testing sets in comparison to using only individual intensity features.

Significance: Fourier ptychography microscopy (FPM) is a computational optical imaging technology that employs angularly varying illuminations and a phase retrieval algorithm to achieve a wide field of view and high-resolution imaging simultaneously. In the FPM, LED position error will reduce the quality of the reconstructed high-resolution image. To correct the LED positions, current methods consider each of the LED positions as independent and use an optimization algorithm to get each of the positions. When the positional misalignment is large or the search position falls into a local optimal value, the current methods may lack stability and accuracy. Aim: We improve the model of the LED position and propose an accurate and stable two-step correction scheme (tcFPM) to calibrate the LED position error. Approach: The improved LED positions model combines the overall offset, which represents the relative deviation of the LED array and the optical axis, with the slight deviation of each LED’s independent position. In the tcFPM, the overall offset of the LED array is corrected at first, which obtains an approximate value of the overall offset of the LED array. Then the position of each LED is precisely adjusted, which obtains the slight offset of each LED. Results: This LED position error model is more in line with the actual situation. The simulation and experimental results show that the method has high accuracy in correcting the LED position. Furthermore, the reconstruction process of tcFPM is more stable and significantly improves the quality of the reconstruction results, which is compared with some LED position error correction methods. Conclusions: An LED position error correction technology is proposed, which has a stable iterative process and improves the reconstruction accuracy of complex amplitude.

Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode. Aim: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM). Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution. Results: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6 μm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0 μm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM. Conclusions: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.

This work was supported by the EPSRC (Grant No. EP/K503162/1). Significance : Optical microscopy is characterized by the ability to get high resolution, below 1 μm, high contrast, functional and quantitative images. The use of shaped illumination, such as with lightsheet microscopy, has led to greater three-dimensional isotropic resolution with low phototoxicity. However, in most complex samples and tissues, optical imaging is limited by scattering. Many solutions to this issue have been proposed, from using passive approaches such as Bessel beam illumination to active methods incorporating aberration correction, but making fair comparisons between different approaches has proven to be challenging. Aim : We present a phase-encoded Monte Carlo radiation transfer algorithm (φMC) capable of comparing the merits of different illumination strategies or predicting the performance of an individual approach. Approach : We show that φMC is capable of modeling interference phenomena such as Gaussian or Bessel beams and compare the model with experiment. Results : Using this verified model, we show that, for a sample with homogeneously distributed scatterers, there is no inherent advantage to illuminating a sample with a conical wave (Bessel beam) instead of a spherical wave (Gaussian beam), except for maintaining a greater depth of focus. Conclusion : φMC is adaptable to any illumination geometry, sample property, or beam type (such as fractal or layered scatterer distribution) and as such provides a powerful predictive tool for optical imaging in thick samples. Publisher PDF

Significance: Artificial skin (AS) is widely used in dermatology, pharmacology, and toxicology, and has great potential in transplant medicine, burn wound care, and chronic wound treatment. There is a great demand for high-quality AS product and a non-invasive detection method is highly desirable. Aim: To quantify the constructure parameters (i.e., thickness and surface roughness) of AS samples in the culture cycle and explore the growth regularities using optical coherent tomography (OCT). Approach: An adaptive interface detection algorithm is developed to recognize surface points in each A-scan, offering a rapid method to calculate parameters without constructing OCT B-scan pictures and further achieving realizing real-time quantification of AS thickness and surface roughness. Experiments on standard roughness plates and H&E-staining microscopy were performed as a verification. Results: As applied on the whole cycle of AS culture, our method’s results show that during the air–liquid culture, the surface roughness of the skin first decreases and then exhibits an increase, which implies coincidence with the degree of keratinization under a microscope. And normal and typical abnormal samples can be differentiated by thickness and roughness parameters during the culture cycle. Conclusions: The adaptive interface detection algorithm is suitable for high-sensitivity, fast detection, and quantification of the interface with layered characteristic tissues, and can be used for non-destructive detection of the growth regularity of AS sample thickness and roughness during the culture cycle.

Significance: We introduce a model for better calibration of the trapping force using an equal but oppositely directed drag force acting on a trapped red blood cell (RBC). We demonstrate this approach by studying RBCs’ elastic properties from deidentified sickle cell anemia (SCA) and sickle cell trait (SCT) blood samples. Aim: A laser trapping (LT) force was formulated and analytically calculated in a cylindrical model. Using this trapping force relative percent difference, the maximum (longitudinal) and minimum (transverse) radius rate and stiffness were used to study the elasticity. Approach: The elastic property of SCA and SCT RBCs was analyzed using LT technique with computer controlled piezo-driven stage, in order to trap and stretch the RBCs. Results: For all parameters, the results show that the SCT RBC samples have higher elastic property than the SCA RBCs. The higher rigidity in the SCA cell may be due to the lipid composition of the membrane, which was affected by the cholesterol concentration. Conclusions: By developing a theoretical model for different trapping forces, we have also studied the elasticity of RBCs in SCT (with hemoglobin type HbAS) and in SCA (with hemoglobin type HbSS). The results for the quantities describing the elasticity of the cells consistently showed that the RBCs in the SCT display lower rigidity and higher deformability than the RBCs with SCA.

Significance: The morphological properties and hemoglobin (Hb) content of red blood cells (RBCs) are essential biomarkers to diagnose or monitor various types of hematological disorders. Label-free mass mapping approaches enable accurate Hb quantification from individual cells, serving as promising alternatives to conventional hematology analyzers. Deep ultraviolet (UV) microscopy is one such technique that allows high-resolution, molecular imaging, and absorption-based mass mapping. Aim: To compare UV absorption-based mass mapping at four UV wavelengths and understand variations across wavelengths and any assumptions necessary for accurate Hb quantification. Approach: Whole blood smears are imaged with a multispectral UV microscopy system, and the RBCs’ dry masses are computed. This approach is compared to quantitative phase imaging-based mass mapping using data from an interferometric UV imaging system. Results: Consistent Hb mass and mean corpuscular Hb values are obtained at all wavelengths, with the precision of the single-cell mass measurements being nearly identical at 220, 260, and 280 nm but slightly lower at 300 nm. Conclusions: A full hematological analysis (including white blood cell identification and characterization, and Hb quantification) may be achieved using a single UV illumination wavelength, thereby improving the speed and cost-effectiveness.

Significance: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood. Aim: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy. Approach: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used. Results: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37 deg of a single monomer with fluorescence microscopy. Conclusions: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

This work reports a modification of a fibrous cellulose material (paper) by the addition of polyacrylonitrile (PAN) fibres doped with 10,12–pentacosadiynoic acid (PDA). The fibres are sensitive to ultraviolet (UV) light. When the paper containing PAN–PDA is irradiated with UV light it changes colour to blue as a consequence of interaction of the light with PDA. The colour intensity is related to the absorbed dose, content of PAN–PDA fibres in the paper and the wavelength of UV radiation. The features of the paper are summarised after reflectance spectrophotometry and scanning microscopy analyses. All the properties of the modified paper were tested in accordance with adequate ISO standards. Moreover, a unique method for assessing the unevenness of the paper surface and the quality of printing was proposed by using a Python script (RGBreader) for the analysis of RGB colour channels. The modification applied to the paper can serve as a paper security system. The modified paper can act also as a UV radiation indicator.

Currently, there is an increasing interest in the use of biopolymers in industrial applications to replace petroleum-based additives, since they are abundantly available, renewable and sustainable. Cottonseed protein is a biopolymer that, when used as a modifier, has shown improved performance for wood adhesives and paper products. Thus, it would be useful to explore the feasibility of using cellulose nanomaterials to further improve the performance of cottonseed protein as a paper strength agent. This research characterized the performance of cottonseed protein isolate with/without cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs) to increase the dry strength of filter paper. An application of 10% protein solution with CNCs (10:1) or CNFs (50:1) improved the elongation at break, tensile strength and modulus of treated paper products compared to the improved performance of cottonseed protein alone. Further analysis using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) indicated that the cottonseed protein/nanocellulose composites interacted with the filter paper fibers, imparting an increased dry strength.

Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored. Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition). Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids. Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group. Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.

Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth. Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices. Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μm inside the biological tissue and obtain a 60% signal increase after correction. Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue.

Significance: Coherent Raman scattering (CRS) microscopy is an optical imaging technique with capabilities that could benefit a broad range of biomedical research studies. Aim: We reflect on the birth, rapid rise, and inescapable growing pains of the technique and look back on nearly four decades of developments to examine where the CRS imaging approach might be headed in the next decade to come. Approach: We provide a brief historical account of CRS microscopy, followed by a discussion of the challenges to disseminate the technique to a larger audience. We then highlight recent progress in expanding the capabilities of the CRS microscope and assess its current appeal as a practical imaging tool. Results: New developments in Raman tagging have improved the specificity and sensitivity of the CRS technique. In addition, technical advances have led to CRS microscopes that can capture hyperspectral data cubes at practical acquisition times. These improvements have broadened the application space of the technique. Conclusion: The technical performance of the CRS microscope has improved dramatically since its inception, but these advances have not yet translated into a substantial user base beyond a strong core of enthusiasts. Nonetheless, new developments are poised to move the unique capabilities of the technique into the hands of more users.

Significance: Spectral-domain optical coherence tomography (SD-OCT) offers depth-resolved imaging of optical scattering contrast but is limited in sensitivity to optical absorption. Dual-modality imaging combined with the noncontact absorption contrast of photoacoustic remote sensing (PARS) microscopy can augment SD-OCT applications with specific molecular and functional contrasts in an all-optical, fiber-based platform. Aim: To develop a fiber-based multimodal PARS and SD-OCT imaging system, which efficiently uses a common 1050-nm light source for SD-OCT and PARS interrogation. Approach: PARS microscopy has predominantly utilized a 1310-nm interrogation light source to date. Hence, a recent dual-modality PARS and 1050-nm SD-OCT imaging system required three distinct wavelengths including a 532-nm PARS excitation, necessitating a free-space optical architecture with discrete subsystems. Here, we validate the first use of a 1050-nm interrogation wavelength for PARS. This enables the transition to fiber-based interferometry as is standard in modern SD-OCT systems, though infeasible with inclusion of an additional 1310-nm wavelength. PARS interrogation functionality is integrated using a broadband optical circulator. Results: Dual-modality imaging is demonstrated in carbon fiber phantoms and a mouse ear in vivo. SD-OCT provided a 4.5-μm lateral resolution, 8.8-μm axial resolution in air, and >101 dB of sensitivity, and PARS contributed 532-nm optical absorption contrast with a 47-dB SNR, and lateral and axial resolutions of 2.4 and 35 μm, respectively. Total interrogation power was reduced from 90% to 58% of the ANSI limit compared to a previous three-wavelength approach. Conclusions: Adapting PARS to use the 1050-nm SD-OCT light source for interrogation enabled implementation of a fiber-based dual-modality system configuration, with image quality maintained. This will facilitate development of potential applications demanding handheld, catheter-based, or endoscopic form factors.

Significance: Light-sheet fluorescence microscopy (LSFM) is a powerful technique for highspeed volumetric functional imaging. However, in typical light-sheet microscopes, the illumination and collection optics impose significant constraints upon the imaging of non-transparent brain tissues. We demonstrate that these constraints can be surmounted using a new class of implantable photonic neural probes.Aim: Mass manufacturable, silicon-based light-sheet photonic neural probes can generate planar patterned illumination at arbitrary depths in brain tissues without any additional micro-optic components.Approach: We develop implantable photonic neural probes that generate light sheets in tissue. The probes were fabricated in a photonics foundry on 200-mm-diameter silicon wafers. The light sheets were characterized in fluorescein and in free space. The probe-enabled imaging approach was tested in fixed, in vitro, and in vivo mouse brain tissues. Imaging tests were also performed using fluorescent beads suspended in agarose.Results: The probes had 5 to 10 addressable sheets and average sheet thicknesses Conclusions: The neural probes can lead to new variants of LSFM for deep brain imaging and experiments in freely moving animals.

Significance: To explore brain architecture and pathology, a consistent and reliable methodology to visualize the three-dimensional cerebral microvasculature is beneficial. Perfusion-based vascular labeling is quick and easily deliverable. However, the quality of vascular labeling can vary with perfusion-based labels due to aggregate formation, leakage, rapid photobleaching, and incomplete perfusion. Aim: We describe a simple, two-day protocol combining perfusion-based labeling with a two-day clearing step that facilitates whole-brain, three-dimensional microvascular imaging and characterization. Approach: The combination of retro-orbital injection of Lectin-Dylight-649 to label the vasculature, the clearing process of a modified iDISCO+ protocol, and light-sheet imaging collectively enables a comprehensive view of the cerebrovasculature. Results: We observed ∼threefold increase in contrast-to-background ratio of Lectin-Dylight-649 vascular labeling over endogenous green fluorescent protein fluorescence from a transgenic mouse model. With light-sheet microscopy, we demonstrate sharp visualization of cerebral microvasculature throughout the intact mouse brain. Conclusions: Our tissue preparation protocol requires fairly routine processing steps and is compatible with multiple types of optical microscopy.

Significance: Digital holographic microscopy is widely used to get the quantitative phase information of transparent cells. Aim: However, the sample phase is superimposed with aberrations. To quantify the phase information, aberrations need to be fully compensated. Approach: We propose a technique to obtain aberration-free phase imaging, using the derivative-based principal component analysis (dPCA). Results: With dPCA, almost all aberrations can be extracted and compensated without requirements on background segmentation, making it efficient and convenient. Conclusions: It solves the problem that the conventional principal component analysis (PCA) algorithm cannot compensate the common but intricate higher order cross-term aberrations, such as astigmatism and coma. Moreover, the dPCA strategy proposed here is not only suitable for aberration compensation but also applicable for other cases where there exist cross-terms that cannot be analyzed with the PCA algorithm.

Significance: Myelin breakdown is likely a key factor in the loss of cognitive and motor function associated with many neurodegenerative diseases. Aim: New methods for imaging myelin structure are needed to characterize and quantify the degradation of myelin in standard whole-brain sections of nonhuman primates and in human brain. Approach: Quantitative birefringence microscopy (qBRM) is a label-free technique for rapid histopathological assessment of myelin structural breakdown following cortical injury in rhesus monkeys. Results: We validate birefringence microscopy for structural imaging of myelin in rhesus monkey brain sections, and we demonstrate the power of qBRM by characterizing the breakdown of myelin following cortical injury, as a model of stroke, in the motor cortex. Conclusions: Birefringence microscopy is a valuable tool for histopathology of myelin and for quantitative assessment of myelin structure. Compared to conventional methods, this label-free technique is sensitive to subtle changes in myelin structure, is fast, and enables more quantitative assessment, without the variability inherent in labeling procedures such as immunohistochemistry.