Journal of Biophotonics

Structural organization of harmonophores used in hematoxylin (H) and eosin (E) staining is studied with polarimetric multimodal second-harmonic generation (SHG), third-harmonic generation (THG) and multiphoton excitation fluorescence (MPF) microscopy in rat tail tendon histology sections. The polarimetric microscopy imaging reveals that hemalums (complexes of hematoxylin and aluminum) are well aligned with C6h symmetry along the collagen fibers in H-stained tissue, while eosin Y is partially aligned along the fibers in E-stained tissue and also follows organization of C6h symmetry. When both hemalum and eosin are used for H&E staining, the dye molecules interact and align noncentrosymmetrically with C6 symmetry along the collagen fibers, while the stained nuclei appear isotropically organized. The polar alignment of the hemalum and eosin complexes increases the achiral second order susceptibility tensor component ratio [Formula] in H&E-stained tissue. The alignment of hemalum and eosin molecules, and their complexes in collagenous tissue, must be considered in nonlinear microscopy and polarimetric analysis of H&E-stained histopathology.

A multicontrast polarimetric wide-field second harmonic generation (SHG) and multiphoton excitation fluorescence (MPF) microscope is optimized for large area imaging of hematoxylin and eosin (H&E) stained and unstained histology slides. The bleaching kinetics of MPF and SHG are examined with various laser intensities at different pulse repetition rates to determine the optimal wide-filed imaging conditions for H&E stained histology slides. Several polarimetric parameters are used to investigate the organization of extracellular matrix collagen in the histology samples.

We present a light-emitting diode (LED)-based transcranial photoacoustic measurement (LED-trPA) of oxyhemoglobin (HbO2) saturation at superior sagittal sinus (SSS) in hypoxic neonatal piglets. The optimal LED imaging wavelengths and frame averaging scheme were determined based on in vivo characterization of transcranial sensitivity. Based on the framework (690/850 nm with >20 frame averaging), graded hypoxia was successfully identified in neonatal piglets in vivo with less than 10.0 % of root mean squared error (RMSE). This preclinical study suggests the feasibility of a rapid, cost-effective, and safe LED-trPA monitoring of perinatal hypoxia-ischemia and prompt interventions for clinical use.

In new chemical environments or for untested combinations of illumination, unexpected changes to the fluorescent labels photophysical properties, such as photoconversion, can occur. This letter reports on photoblueing of a common cellular probe, LysoTracker Deep Red, in multi-color super-resolution structured illumination microscopy. The dye was found to exhibit a blue shift of its fluorescence spectrum as a step on the photobleaching pathway during such imaging. The observed photoblueing (emission spectrum shifted to lower wavelengths) of this probe is an important finding as many cellular assays rely on the spectral separation of this and such fluorescent cellular markers. No spectral shift of LysoTracker Deep Red was observed for longterm imaging using diffraction limited microscopy requiring significantly lower light dose. We expect that the knowledge of occurrence of photoconversion during super-resolved imaging using this popular dye will help researchers design better imaging experiments and avoid potentially erroneous interpretations in multi-color microscopy.

Full field optical coherence tomography (FF-OCT) enables high-resolution in-depth imaging within turbid media. In this work, we present a simple approach which combines FF-OCT with off-axis interferometry for the reconstruction of the en-face images. With low spatial and temporal coherence illumination, this new method is able to extract an FF-OCT image from only one interference acquisition. This method is described and the proof-of-concept is demonstrated through the observation of scattering samples such as organic and ex-vivo biomedical samples.

As discussed below cataract is the world-leading cause of blindness and impaired vision with only eye surgery as a treatment. There is major requirement for an alternative which this research has provided via Genetically Modified pigs with natural cataract making live animal experiments of LED photobleaching successful monitored by fluorescence spectra. The latter were shown by EBS as spectrally identical in pigs and humans. EBS developed a miniature fluorescence spectrometer via tunable interference filters with fluorescence excited by an LED. A second LED in the blue/violet region provided a 20mW treatment beam which was shown to reverse the fluorescence signal and dramatically improve the transmission of pigs eyes. These results led to permission for early Human Trials. The results were judged by independent optometrists by LOCS and fluorescence in pigs and in Human Trials by LOCS and Visual Acuity. Adequate positive results have led to full length Human Trials being conducted in four European countries. Prospects of worldwide application have been therefore indicated.

We still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machinelearning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.

Correlative microscopy techniques are used for many different applications in the biological sciences because the comparison of different imaging methods allows researchers to gain more insight and data from samples. Correlative light and electron microscopy (CLEM) methods have been developed to preserve biological samples to withstand the harsh environments necessary for electron microscopy. After first being imaged using widefield (WF) and super-resolution structured illumination fluorescence microscopy (SIM), a NanoSuit chemical treatment was applied to a mammalian testis sample before imaging with scanning electron microscopy (SEM). This was done to compare the image quality and resolution of each technique. SEM yields higher resolution and offers validation of results from SIM.

Non-alcoholic fatty liver disease (NAFLD) is an umbrella term referring to a group of conditions associated to fat deposition and damage of liver tissue. Early detection of fat accumulation is essential to avoid progression of NAFLD to serious pathological stages such as liver cirrhosis and hepatocellular carcinoma. We exploited the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS), which combines the advantages of optical and acoustic contrasts, for an early-stage multi-parametric assessment of NAFLD in mice. The multispectral optoacoustic imaging allowed for spectroscopic differentiation of lipid content, as well as the bio-distributions of oxygenated and deoxygenated hemoglobin in liver tissues in vivo. The pulse-echo (reflection) ultrasound (US) imaging further provided a valuable anatomical reference whilst transmission US facilitated the mapping of speed of sound changes in lipid-rich regions, which was consistent with the presence of macrovesicular hepatic steatosis in the NAFLD livers examined with ex vivo histological staining. The proposed multimodal approach facilitates quantification of liver abnormalities at early stages using a variety of optical and acoustic contrasts, laying the ground for translating the TROPUS approach toward diagnosis and monitoring NAFLD in patients.

Cerebral blood volume (CBV) and blood flow (CBF) constitute key metrics for cerebrovascular monitoring, enabling assessment of stroke severity and risk-prediction, aging-related changes, and neurological diseases. CBF and CBV monitoring are key aspects in diagnosis, treatment triage, and clinical outcome of ischemic and hemorrhagic strokes. In recent years, there have been ongoing efforts toward the development of optical devices for noninvasive monitoring of CBV and CBF. Speckle contrast optical spectroscopy (SCOS) has recently emerged as a strong candidate for clinical translation in monitoring CBF and CBV, due to its affordability, compact and wearable design, and noninvasive nature. However, experimental demonstrations that SCOS can effectively monitor brain hemodynamics remain sparse. This is primarily due to challenges in design experiments that isolate cerebral blood dynamics from those in the scalp and skull. In this paper, we report experiments using SCOS to monitor cerebral hemodynamics in rats during intracerebral blood flow modulation. To modify cerebral blood dynamics, a surgical procedure was performed to insert a catheter for direct injection of flow modulation fluids into the brain. Using the SCOS device, we monitored changes in CBV during deliberate CBF interventions into the brains of five rats. A saline solution was also injected as a sham control of the flow intervention. The results show a significant decrease in CBV during injection, followed by a return to baseline. This behavior is consistent with physiological expectations, as the injected fluids dilute the blood, leading to a transient reduction in blood volume. Notably, the CBV decrease induced by the flow modulation fluid solution required more than twice as long to recover to baseline compared with the saline solution, which is consistent with the delayed clearance of the flow modulation fluid by design. These experimental results demonstrate the effectiveness of SCOS for monitoring cerebral hemodynamics in animal models and highlight its potential for translation to human studies. Moreover, this work paves the way for the testing and characterization of cerebral therapeutic agents intended for blood flow modulation in animal models.

Super resolution techniques are an excellent alternative to wide field microscopy, providing high resolution also in (typically fragile) biological sample. Among the various super resolution techniques, Structured Illumination Microscopy (SIM) improve resolution by employing multiple illumination patterns to be deconvolved with a dedicated software. In the case of blind SIM techniques, unknown patterns, such as speckles, are used, thus providing super resolved images, nearly unaffected by aberrations with a simplified experimental setup. Scattering Assisted Imaging, a special blind SIM technique, exploits an illumination PSF (speckle grains size), smaller than the collection PSF (defined by the collection objectives), to surpass the typical SIM resolution enhancement. However, if SAI is used, it is very difficult to extract the resolution enhancement from a priori considerations. In this paper we propose a protocol and experimental setup for the resolution measurement, demonstrating the resolution enhancement for different collection PSF values.

Micro-optical coherence tomography ({micro}OCT) improves the spatial resolution of in vivo OCT imaging by utilizing sophisticated focusing schemes and broadband illumination. This study explores the safety of coronary and trachea tissue exposure to {micro}OCT illumination. (C) 2021 The Authors

Multimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and require complex alignment procedures, as well as daily maintenance by properly trained personnel for optimal performance. Here, we describe a hybrid system we constructed to overcome these disadvantages by modifying a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo samples. The system is highly stable and endowed with remote alignment hardware that ensures simplified operability for non-expert users. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging.

SignificanceOf the most compelling unsolved issues in the paradigm to create success in the field of breast cancer infrared imaging is localization of direct internal heat of the tumor. The contribution of differential heat production related to metabolism versus perfusion is not understood. Previous work until now has not shown progress beyond identifying veins which are fed by the hot cancer. Employing signal analysis techniques, we probe important questions which may lead to further understanding pathophysiology of heat transfer occurring in the setting of malignancy. AimWhen using thermal imaging to detect breast cancer, the dominant heat signature is that of indirect heat transported in gradient away from the tumor location. Unprocessed images strikingly display vasculature which acts to direct excess heat superficially towards the skin surface before dissipating. In current clinical use, interpretation of thermogram images considers abnormal vascular patterns and overall temperature as indicators of disease. The goal of this work is to present a processing method for dynamic external stimulus thermogram images to isolate and separate the indirect vascular heat while revealing the desired direct heat from the tumor. ApproachIn dynamic thermal imaging of the breast, a timed series of images are taken following application of external temperature stimulus (most often cooled air). While the tumor heat response is thought to be independent of the external stimulus, the secondary heat of the veins is known to be affected by vasomodulation. The recorded data is analyzed using independent component analysis (ICA) and principal component analysis (PCA) methods. ICA separates the image sequence into new independent images having a common characteristic time behavior. Resulting individual components are analyzed for correspondence to the presence or lack of vasomodulation. ResultsUsing the Brazilian visual lab mastology data set containing dynamic thermograms, applying components analysis resulted in three corresponding images: 1. Minimum change as a function of applied temperature or time (suggests correlation with the cancer generated heat), 2. Moderate temperature dependence (suggests correlation with veins affected by vasomodulation) and 3. Complex time behavior (suggests correlation with heat absorption due to high tumor perfusion). All components appear clear and distinct. ConclusionsApplying signal processing methods to the dynamic infrared data, we found three distinct components with correspondence to understood physiologic processes. The two cases shown are self-evident of the capability of the method but are lacking supporting ground truth that is unavailable with such a limited data set. Validation of this proposed paradigm and studying furthering clinical applications has potential to create significant achievement for IR modality in diagnostic imaging.

Photobiomodulation (PBM) is a novel technique that is actively studied for neuromodulation. However, despite the many in vivo studies, the stimulation protocols for PBM vary amongst studies, and the current understanding of neuromodulation via PBM is limited in terms of the extent of light penetration into the brain and its dosage dependence. Moreover, as near-infrared light can be absorbed by melanin in the skin, skin tone is a highly relevant but under-studied variable of interest. In this study, to address these gaps, we use Monte Carlo simulations (with MCX) of a single laser source for transcranial (tPBM) and intranasal (iPBM, nostril position) irradiated on a healthy human brain model. We investigate wavelengths of 670, 810 and 1064 nm in combination with light ("Caucasian"), medium ("Asian") and dark ("African") skin tones. Our simulations show that a maximum of 15% of the incidental energy for tPBM and 1% for iPBM reaches the cortex from the light source at the skin level. The rostral dorsal prefrontal cortex in tPBM and the ventromedial prefrontal cortex for iPBM accumulates the highest highest light energy, respectively for both wavelengths. Specifically, the 810 nm wavelength for tPBM and 1064 nm wavelength for iPBM produced the highest energy accumulation. Optical power density was found to be linearly correlated with energy. Moreover, we show that "Caucasian" skin allows the accumulation of higher light energy than other two skin colours. This study is the first to account for skin colour as a PBM dosing consideration, and provides evidence for hypothesis generation in in vivo studies of PBM.

Spectral information plays a crucial role in biological imaging, yet conventional epifluorescence and histological techniques often rely on RGB image acquisition, limiting the resolution of spectrally overlapping components. Here, we present a phasor-based spectral analysis framework adapted for RGB images, enabling unsupervised segmentation and unmixing without the need for hyperspectral systems or sequential acquisition. By applying a discrete Fourier transform to the red, green, and blue intensities at each pixel, we generate a two-dimensional phasor plot where spectral relationships are encoded in modulation and phase. We demonstrate the utility of this approach across three distinct applications: segmentation of lung histology images stained with hematoxylin and eosin to quantify alveolar collapse, analysis of autofluorescence in skin lesions (nevi and melanoma) to highlight pathological spectral signatures, and spectral unmixing in multicolor-labeled U2OS cells to resolve overlapping fluorophores. Our method improves signal separation, reduces noise, and enhances biological interpretability using standard RGB acquisition. These findings establish RGB phasor analysis as a practical and powerful tool for spectral decomposition and segmentation in microscopy, bridging the gap between conventional imaging and advanced spectral analysis.

Single-molecule localization microscopy can decipher fine details that are otherwise not possible using diffraction-limited microscopy. Often the reconstructed super-resolved image contains unwanted noise, random background and is prone to false detections. This cause spurious data that necessitates several trials, multiple experimentations, and repeated preparation of specimens. Moreover, this is not suitable for experiments that require time-lapse imaging and real-time microscopy. To overcome these limitations, we propose a technique (corrSMLM) that can recognize and detect fortunate molecules (molecules with long fluorescence cycles) from the recorded data. The technique uses correlation between two or more consecutive frames to extract fortunate molecules that blink for longer than the standard blinking time. Accordingly, strongly-correlated spots (single molecule signatures) are compared in consecutive frames, followed by data integration (mean centroid position and the total number of photons) and estimation of critical parameters (position and localization precision). The technique addresses two major problems that plague SMLM : (1) random noise due to false detection that contributes to strong background, and (2) poor localization precision offered by standard SMLM techniques. On the brighter side, corrSMLM allows only fortunate molecules contribute to the super-resolved image, thereby suppressing the background and improving localization precision by a factor of 2-4 times as compared to standard SMLM. To substantiate, corrSMLM is used for imaging fixed cell samples (Dendra2-Actin and Dendra2-Tubulin transfected NIH3T3 cells). Results show multi-fold reduction in noise and localization precision with a marked improvement in overall resolution and SBR. We anticipate corrSMLM to improve overall image quality and offer a better understanding of single molecule dynamics in cell biology.

In this paper, we aim to present a new intravital dye, called ABDS, which can be prepared using a marker pen and is useful for eukaryotic cell research. ABDS binds to the cytoplasmic membrane of living cells and can be used for their visualization in biophysical experiments, including Z-stack tomography, cell proliferation monitoring, and adhesion studies. Important properties of ABDS are its availability, bright stable fluorescence, manufacturing simplicity, and safety for living cells in vitro. The paper includes a method for preparing ABDS, a data set with its characteristics, as well as examples of ABDS use in cells investigation.

Polarimetric second harmonic generation (SHG) microscopy is employed to study partially oriented fibrillar structures. The polarimetric SHG parameters are influenced by three-dimensional (3D) configuration of C6 symmetry fibrilar structures in the focal volume (voxel) of a microscope. The achiral and chiral susceptibility tensor components ratios (R and C, respectively) are extracted from the linear polarization-in polarization-out (PIPO) measurements. The analytical derivations along with the polarimetric SHG microscopy results obtained from rat tail tendon, rabbit cornea, pig cartilage and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) cylindrical aggregates demonstrate that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and R and C ratio values change by tilting the fibers out of image plane, as well as by crossing the fibers in 2D and 3D. The polarimetric microscopy results are consistent with the digital microscopy modeling of fibrillar structures. These results facilitate the interpretation of polarimetric SHG microscopy images in terms of 3D organization of fibrilar structures in each voxel of the samples. Statement of SignificancePolarimetric second harmonic generation (SHG) microscopy is used to study partially oriented C6 symmetry chiral fibrillar structures. The linear polarization-in polarization-out (PIPO) SHG imaging is performed on rat tail tendon, rabbit cornea, pig cartilage tissues and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) cylindrical aggregates. The study demonstrates that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and the achiral and chiral susceptibility component ratio values change by tilting the fibers out of image plane, as well as by crossing the fibers in 2D and 3D. The polarimetric microscopy results are consistent with the digital microscopy modeling of fibrillar structures. These results facilitate the interpretation of polarimetric SHG microscopy images in terms of 3D organization of fibrillar structures in each voxel of the samples.

Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.