Showing papers on "Photoactivated localization microscopy published in 2017"
TL;DR: Recent work on incorporating adaptive optics, a technology originally applied in astronomical telescopes to combat atmospheric aberrations, to improve image quality of fluorescence microscopy for biological imaging is reviewed.
Abstract: This Perspective introduces the development and use of adaptive optics in correcting aberrations in deep optical imaging applications.
396 citations
TL;DR: It is demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells and Notably, the domains during mitosis are observed, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
327 citations
TL;DR: This review evaluates and summarizes especially the data achieved until now in analyzing the organization and function of plant cells, chromosomes and interphase nuclei using super-resolution techniques.
Abstract: Most of the present knowledge about cell organization and function is based on molecular and genetic methods as well as cytological investigations. While electron microscopy allows identifying cell substructures until a resolution of ~1nm, the resolution of fluorescence microscopy is restricted to ~200 nm due to the diffraction limit of light. However, the advantage of this technique is the possibility to identify and co-localize specifically labelled structures and molecules. The recently developed super-resolution microscopy techniques, such as Structured Illumination Microscopy (SIM), Photoactivated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM) and Stimulated Emission Depletion (STED) microscopy allow analysing structures and molecules beyond the diffraction limit of light. Recently, there is an increasing application of these techniques in cell biology. This review evaluates and summarizes especially the data achieved until now in analysing the organization and function of plant cells, chromosomes and interphase nuclei using super-resolution techniques.
69 citations
TL;DR: A multi-color fluorescence smartphone microscope with a single contact lens-like add-on lens and slide-launched total-internal-reflection guided illumination for three common tasks in investigative fluorescence microscopy: autofluorescence, fluorescent stains, and immun ofluorescence is reported.
Abstract: Fluorescence microscopy is an important technique for cellular and microbiological investigations. Translating this technique onto a smartphone can enable particularly powerful applications such as on-site analysis, on-demand monitoring, and point-of-care diagnostics. Current fluorescence smartphone microscope setups require precise illumination and imaging alignment which altogether limit its broad adoption. We report a multi-color fluorescence smartphone microscope with a single contact lens-like add-on lens and slide-launched total-internal-reflection guided illumination for three common tasks in investigative fluorescence microscopy: autofluorescence, fluorescent stains, and immunofluorescence. The open-source, simple and cost-effective design has the potential for do-it-yourself fluorescence smartphone microscopy.
64 citations
TL;DR: Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning.
Abstract: Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.
63 citations
TL;DR: It is shown that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy.
53 citations
TL;DR: Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization and routinely achieves a spatial resolution of 20 to 30 nm, a ten‐fold improvement compared to conventional optical microscopy.
Abstract: Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects—sample preparation, image acquisition, and image reconstruction—to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc.
Keywords:
single molecule localization microscopy (SMLM);
stochastic optical reconstruction microscopy (STORM);
super-resolution fluorescence microscopy
47 citations
TL;DR: The P&D portal is an up-to-date web resource with monthly updates that unifies various commercial and public bioactive compound libraries that helps users identify high-quality chemical tools for use in chemical biology and drug discovery research.
Abstract: attention given to probes and drugs (Supplementary Table 2). Currently, more than 800 distinct probes were acquired from six different library sources; while drugs, extracted from five sources (Supplementary Table 2 and Supplementary Note 3), account for more than 11,000 compounds—5,700 of which are annotated as approved drugs. To ensure that various chemical forms (such as stereoisomers or salts) are assigned to only one unique compound, each compound in the P&D portal is converted into its standardized form (Supplementary Note 2). However, for cases in which different stereoisomers show different biological impact, original forms still remain available. To further support the identification of suitable chemical tools, the annotation of P&D compounds is enriched with additional data, such as the bioactivities, targets and pathways in which these compounds take part. These data, obtained from and linked back to various external sources (Supplementary Table 3), are organized through ontologies. This ensures consistency between sources and contributes to a high data enrichment. A query can be saved simply by bookmarking its URL, and registered users can create custom compound sets from the arbitrary combination of compounds stored in the P&D library (Supplementary Note 8). The P&D portal is an up-to-date web resource with monthly updates that unifies various commercial and public bioactive compound libraries. Through its flexible and powerful filtering system, it helps users identify high-quality chemical tools for use in chemical biology and drug discovery research.
47 citations
TL;DR: The hybrid systems of AFM with optical‐derived microscopies enable to study in detail cell surface properties, their mechanical properties, and allow to gain insight into biological‐related pathways and mechanisms in the complex nanoworld of cells.
Abstract: This review reports on the combined use of the atomic force microscopy (AFM) and several type of optical/fluorescence/laser scanning microscopy for investigating cells. It is shown that the hybrid systems of AFM with optical-derived microscopies enable to study in detail cell surface properties (such as topography), their mechanical properties (e.g., Young's modulus) mechanotransduction phenomena and allow to gain insight into biological-related pathways and mechanisms in the complex nanoworld of cells. Microsc. Res. Tech. 80:109-123, 2017. © 2016 Wiley Periodicals, Inc.
38 citations
TL;DR: By using a high-numerical-aperture objective lens and a custom cryogenic stage, the cPALM technique is able to increase photon yield by 2–3-fold over room temperature, thereby achieving more precise superresolution reconstructions of complex subcellular structures.
Abstract: Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.
29 citations
TL;DR: Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process that reduces phototoxicity and photobleaching, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.
Abstract: Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.
TL;DR: In this paper, a sample-based technique is proposed to calibrate axial detection in 3D single molecule localization microscopy (SMLM) using microspheres coated with fluorescent molecules, which can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns.
Abstract: We propose a straightforward sample-based technique to calibrate the axial detection in 3D single molecule localization microscopy (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.
TL;DR: A framework for determination of the best possible resolution using a localization microscope to image a particular fluorophore is established, and development of probes for use in superresolution localization microscopy must consider the count rate per molecule, the saturation intensity, the photobleaching yield, and, crucially, management of bright/dark state transitions, to optimize image resolution.
TL;DR: Polarized localization microscopy is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales through the theoretical foundation and experimental demonstration provided here.
TL;DR: A novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations and can be advantageously combined with correlative imaging schemes is introduced.
Abstract: Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes.
TL;DR: It is shown that intense 561 nm light typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states, contributing to explain the apparent limited signaling efficiency of this PCFP.
Abstract: Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561 nm light (∼1 kW/cm2) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405 nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of anthozoan origin is substantially more complex than previously thought, and suggests that ...
TL;DR: This work developed an Yb-fiber chirped pulse amplifier, which produces 92-fs 9.0-μJ 1060-nm pulses at a repetition rate of 200 kHz, which enhances the optical sectioning capability of 2PEF-TF microscopy.
Abstract: Temporal focusing (TF) microscopy is a wide-field two-photon excitation fluorescence (2PEF) microscopy technique, the optical sectioning capability of which is lower than that of point-scanning 2PEF microscopy. Here we demonstrate TF microscopy using three-photon excitation fluorescence (3PEF), which enhances the optical sectioning capability. As an excitation light source for the 3PEF, we developed an Yb-fiber chirped pulse amplifier, which produces 92-fs 9.0-μJ 1060-nm pulses at a repetition rate of 200 kHz. The optical sectioning capability was improved by a factor of 1.3 compared with that of 2PEF-TF microscopy. We also demonstrate dual-color imaging with both 2PEF and 3PEF.
TL;DR: This work employs an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz, and shows that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second.
Abstract: Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged.
20 Oct 2017
TL;DR: A novel fast SMLM technique is developed to achieve superresolution imaging within a much shortened duration and relies on computational algorithms to reconstruct a high-density superresolution image from a low-density one using the concept of blind image inpainting.
Abstract: Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy and (fluorescence) photoactivated localization microscopy, has enabled superresolution microscopy beyond the diffraction limit. However, the temporal resolution of SMLM is limited by the time needed to acquire sufficient sparse single-molecule activation events to successfully construct a superresolution image. Here, a novel fast SMLM technique is developed to achieve superresolution imaging within a much shortened duration. This technique does not require a faster switching rate or a higher activation density, which may cause signal degradation or photodamage/bleaching, but relies on computational algorithms to reconstruct a high-density superresolution image from a low-density one using the concept of blind image inpainting. Our results demonstrate that the technique reduces the acquisition time by up to two orders of magnitude compared to the conventional method while achieving the same high resolution. We anticipate our technique to enable future real-time live cell imaging with even higher resolution.
TL;DR: In this article, photoswitchable fluorophores are exploited to boost contrast in light sheet microscopy to reveal structures hidden well below the ambient fluorescent background level by enhancing the contrast by 2 orders of magnitude.
Abstract: Light sheet fluorescence microscopy enables high-resolution imaging of thick biological samples. By restricting the fluorescence excitation to a single plane, rapid wide-field image acquisition is possible with minimal sample exposure. Although light sheet microscopy is able to resolve subcellular features at depth in model organisms, elevated levels of endogenous autofluorescence often preclude acceptable contrast and may obscure features of interest in general samples. Here we demonstrate how photoswitchable fluorophores can be exploited to boost contrast in light sheet microscopy. The novel detection method enables high specificity while maintaining the optical sectioning capability of the light sheet microscope. Our experiments reveal structures hidden well below the ambient fluorescent background level by enhancing the contrast by 2 orders of magnitude.
TL;DR: Saturated virtual fluorescence emission difference microscopy (svFED) as discussed by the authors was proposed to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern.
TL;DR: Using this protocol, single-molecule localizations of synaptic vesicle and active zone proteins in three dimensions within individual synaptic terminals of the striatum in rat brain slices are consistently obtained.
Abstract: Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless-steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single-molecule localizations of synaptic vesicle and active zone proteins in three dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging.
TL;DR: A non-iterative state space-based localization method which combines the detection and estimation steps for localizing fluorophores and it is demonstrated that the estimated locations obtained can be used as initial conditions in an estimation routine to potentially obtain improved location estimates.
Abstract: Single molecule super-resolution microscopy enables imaging at sub-diffraction-limit resolution by producing images of subsets of stochastically photoactivated fluorophores over a sequence of frames. In each frame of the sequence, the fluorophores are accurately localized, and the estimated locations are used to construct a high-resolution image of the cellular structures labeled by the fluorophores. Many methods have been developed for localizing fluorophores from the images. The majority of these methods comprise two separate steps: detection and estimation. In the detection step, fluorophores are identified. In the estimation step, the locations of the identified fluorophores are estimated through an iterative approach. Here, we propose a non-iterative state space-based localization method which combines the detection and estimation steps. We demonstrate that the estimated locations obtained from the proposed method can be used as initial conditions in an estimation routine to potentially obtain improved location estimates. The proposed method models the given image as the frequency response of a multi-order system obtained with a balanced state space realization algorithm based on the singular value decomposition of a Hankel matrix. The locations of the poles of the resulting system determine the peak locations in the frequency domain, and the locations of the most significant peaks correspond to the single molecule locations in the original image. The performance of the method is validated using both simulated and experimental data.
TL;DR: This chapter details the method the team developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus) and the key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure.
Abstract: There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. This chapter details the method we developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus). The key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure. Cells are cryofixed using high pressure freezing for optimal structural preservation and then freeze substituted in tannic acid, which preserves the photoswitching ability of the fluorescent proteins and is essential for high-quality SMLM imaging. Resin sections are then imaged using SMLM, achieving a structural resolution of 40–50 nm and a localization precision of ∼17 nm, followed by transmission electron microscopy. This produces high quality correlative images without the use of specialized fluorescent proteins or antibodies.
01 Jan 2017
TL;DR: In this paper, isolated single molecule images can be localized, using a variety of algorithms based on knowledge of the point spread function, with precisions of 10-20nm, which is exploited in localization microscopy to reconstruct super-resolution images from many such localizations.
Abstract: In conventional fluorescence microscopy, diffraction in the imaging system causes the light from a point source, such as a single molecule, to spread out. The width of this distribution, known as the point spread function, limits the spatial resolution of the system, typically to around 200–250 nm. However, isolated single molecule images can be localized, using a variety of algorithms based on knowledge of the point spread function, with precisions of 10–20 nm. This is exploited in localization microscopy to reconstruct super-resolution images from many such localizations.
TL;DR: A simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM) is described, and the spatial relationship of proteins with the bacterial chromosome can be studied.
Abstract: Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.
TL;DR: In this paper, four microscopy resolution enhancement methods based on parallel detection were investigated: confocal microscopy with four pinhole sizes, fluorescence emission difference microscopy (FED), Airyscan microscopy, and virtual k-vector modulation optical microscopy.
TL;DR: Several commonly used super-resolution techniques are reviewed, explicating their basic principles and applications in biological science, especially in neuroscience, and characteristics and limitations of each technique are compared to provide a guidance for biologists to choose the most suitable tool.
Abstract: Optical microscopy promises researchers to see most tiny substances directly. However, the resolution of conventional microscopy is restricted by the diffraction limit. This makes it a challenge to observe subcellular processes happened in nanoscale. The development of super-resolution microscopy provides a solution to this challenge. Here, we briefly review several commonly used super-resolution techniques, explicating their basic principles and applications in biological science, especially in neuroscience. In addition, characteristics and limitations of each technique are compared to provide a guidance for biologists to choose the most suitable tool.
TL;DR: The paper presents a classification of such fluorophores and describes their photoswitching mechanisms and successful practical applications, and discusses recent progress and prospects for the development of new effective labels suitable for SMLM.
Abstract: Super-resolution fluorescence microscopy allows for obtaining images with a resolution of 10–20 nm, far exceeding the diffraction limit of conventional optical microscopy (200–350 nm), and provides an opportunity to study in detail the subcellular structures and individual proteins in both living and fixed cells Among these methods, single-molecule localization microscopy (SMLM) has become widespread SMLM techniques are based on special fluorophores capable of photoswitching The paper presents a classification of such fluorophores and describes their photoswitching mechanisms and successful practical applications We discuss recent progress and prospects for the development of new effective labels suitable for SMLM
TL;DR: In this paper, an integrated label-free dual approach that combines 3D-Polarized light imaging with two-photon fluorescence microscopy was employed to study the mixture of various fiber orientations within the sample of interest.
Abstract: In this work, we employ an integrated label-free dual approach that combines 3D-Polarized light imaging with two-photon fluorescence microscopy to study the mixture of various fiber orientations within the sample of interest.