Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging

Light‐sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co‐aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2‐photon LSFM (OS‐2P‐LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning‐descanning system. This system also provides improved light‐sheet confinement against scattering by using a 2‐photon Bessel beam. The OS‐2P‐LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high‐speed, translation‐free and scattering‐robust 3D imaging of large biological specimens.      

Quadrature demodulation in line‐scan focal modulation microscopy for imaging three‐dimensional zebrafish neural structure

Visualizing biological processes in neuroscience requires in vivo functional imaging at single‐neuron resolution, high image acquisition speed and strong optical sectioning ability. However, due to light scattering of in tissue, very often conventional wide‐field fluorescence microscopes are unable to resolve cells in the presence of a strong out‐of‐focus background. Line‐scan focal modulation microscopy enables high temporal resolution and good optical sectioning ability at the same time. Here we demonstrate a quadrature demodulation method to extract the focal information with an extended frequency bandwidth and therefore higher spatial resolution. The performance of the demodulation scheme in line‐scan focal modulation microscope has been evaluated by performing imaging experiments with fluorescence beads and zebrafish neural structure. Reduced background, reduced artifacts and more detailed morphological information are evident in the obtained images.      

Development a flexible light‐sheet fluorescence microscope for high‐speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure

We report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Dual light‐emitting diode‐based multichannel microscopy for whole‐slide multiplane,multispectral and phase imaging

We report the development of a multichannel microscopy for whole‐slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single‐frame rapid autofocusing, we place 2 near‐infrared light‐emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near‐infrared light to an autofocusing camera. For multiplane whole‐slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole‐slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport‐of‐intensity equation to recover the phase information. We also provide an open‐source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z‐scanning may also enable fast 3‐dimensional dynamic tracking of various biological samples.      

Ultra‐structural analysis of human spermatozoa by aperture scanning near‐field optical microscopy

Scanning near‐field optical microscopy (SNOM) represents a potential candidate for investigation of ultrastructure in human spermatozoa. It is a noninvasive optical technique that offers two main advantages: minimal sample preparation and simultaneous topographical and optical images acquisition with a spatial resolution beyond the diffraction limit. This enables the combination of surface characterization and information from the inner cellular organization in a single acquisition providing an immediate and comprehensive analysis of the cellular portions. In this work spermatozoa are immobilized on poly‐L‐lysine coated coverslips, fixed according to a standard protocol and imaged by aperture‐SNOM in air. In the SNOM images, all peculiar sperm portions show well‐resolved optical features, which exhibit good similarities with the structures revealed in transmission electron microscopy images, as compared with literature data. The optical features of anomalous spermatozoa are clearly different as respect with those observed for healthy ones. This analysis reveals the potentialities of SNOM and opens to its application to high‐resolution analysis of sperm morphological alterations, which might be relevant in reproductive medicine.     

Pseudo‐real‐time retinal layer segmentation for high‐resolution adaptive optics optical coherence tomography

We present a pseudo‐real‐time retinal layer segmentation for high‐resolution Sensorless Adaptive Optics‐Optical Coherence Tomography (SAO‐OCT). Our pseudo‐real‐time segmentation method is based on Dijkstra's algorithm that uses the intensity of pixels and the vertical gradient of the image to find the minimum cost in a geometric graph formulation within a limited search region. It segments six retinal layer boundaries in an iterative process according to their order of prominence. The segmentation time is strongly correlated to the number of retinal layers to be segmented. Our program permits en face images to be extracted during data acquisition to guide the depth specific focus control and depth dependent aberration correction for high‐resolution SAO‐OCT systems. The average processing times for our entire pipeline for segmenting six layers in a retinal B‐scan of 496 × 400 and 240 × 400 pixels are around 25.60 and 13.76 ms, respectively. When reducing the number of layers segmented to only two layers, the time required for a 240 × 400 pixel image is 8.26 ms.     

光片荧光显微镜特点及其应用

传统荧光显微镜由于对某些荧光分子存在光毒性、光损伤等方面的缺陷,无法满足对部分活体样本进行长时间观测的需求。光片荧光显微镜（light sheet fluorescence microscope,LSFM）是一种新型荧光显微镜,有别于激光共聚焦显微镜,其特殊的正交光路设计和高效的信号采集装置,使其具备低光毒性、低光漂白、低光损伤和高时空分辨率等优良特性,从而能对细胞及大尺度生物组织样本进行时空连续性较好的记录,尤其适宜于活体生物样品。基于此,概述了光片荧光显微镜的成像原理、成像优势、成像效果的改进与优化历程及其在生命科学领域应用所取得的研究成果,重点对近三年相关应用进行了汇总,并简要介绍了其在神经生物学、发育生物学、动物细胞生物学和植物科学领域中一部分代表性研究内容,最后,总结了光片荧光显微镜的优点与发展至今仍存在的不足,并对其在光遗传学和多组学研究中的潜在应用进行了展望,以期为研究人员提供较为系统的光片荧光显微镜相关基础知识、最新的研究应用进展以及未来的潜在应用方向,为研究人员提供参考。     

Illuminating the dark depths inside coral

The ability to observe in situ 3D distribution and dynamics of endosymbionts in corals is crucial for gaining a mechanistic understanding of coral bleaching and reef degradation. Here, we report the development of a tissue clearing (TC) coupled with light sheet fluorescence microscopy (LSFM) method for 3D imaging of the coral holobiont at single‐cell resolution. The initial applications have demonstrated the ability of this technique to provide high spatial resolution quantitative information of endosymbiont abundance and distribution within corals. With specific fluorescent probes or assays, TC‐LSFM also revealed spatial distribution and dynamics of physiological conditions (such as cell proliferation, apoptosis, and hypoxia response) in both corals and their endosymbionts. This tool is highly promising for in situ and in‐depth data acquisition to illuminate coral symbiosis and health conditions in the changing marine environment, providing fundamental information for coral reef conservation and restoration.     

Light Sheet Fluorescence Microscopy: A Review

Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.     

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label‐free multiphoton microscopic images

In the current clinical care practice, Gleason grading system is one of the most powerful prognostic predictors for prostate cancer (PCa). The grading system is based on the architectural pattern of cancerous epithelium in histological images. However, the standard procedure of histological examination often involves complicated tissue fixation and staining, which are time‐consuming and may delay the diagnosis and surgery. In this study, label‐free multiphoton microscopy (MPM) was used to acquire subcellular‐resolution images of unstained prostate tissues. Then, a deep learning architecture (U‐net) was introduced for epithelium segmentation of prostate tissues in MPM images. The obtained segmentation results were then merged with the original MPM images to train a classification network (AlexNet) for automated Gleason grading. The developed method achieved an overall pixel accuracy of 92.3% with a mean F1 score of 0.839 for epithelium segmentation. By merging the segmentation results with the MPM images, the accuracy of Gleason grading was improved from 72.42% to 81.13% in hold‐out test set. Our results suggest that MPM in combination with deep learning holds the potential to be used as a fast and powerful clinical tool for PCa diagnosis.     

Telecentric design for digital-scanning-based HiLo optical sectioning endomicroscopy with an electrically tunable lens

Confocal endoscopy has been widely used to obtain fine optically sectioned images. However, confocal endomicroscopic images are formed by point-by-point scanning in both lateral and axial directions, which results in long image acquisition time. Here, an endomicroscope with telecentric configuration is presented to achieve nonmechanical and rapid axial scanning for volumetric fluorescence imaging. In our system, optical sectioning in wide-field fashion is obtained through HiLo imaging with a digital micromirror device. Axial scanning, without mechanical moving parts, is conducted by digital focus adjustment using an electrically tunable lens, offering constant magnification and contrast. We demonstrate imaging performance of our system with optically sectioned images using fluorescently labeled beads, as well as ex vivo mice cardiac tissue samples. Our system provides multiple advantages, in terms of improved scanning range, and reduced image acquisition time, which shows great potentials for three-dimensional biopsies of volumetric biological samples.     

Axial resolution enhancement of light‐sheet microscopy by double scanning of Bessel beam and its complementary beam

The side lobes of Bessel beam will create significant out‐of‐focus background when scanned in light‐sheet fluorescence microscopy (LSFM), limiting the axial resolution of the imaging system. Here, we propose to overcome this issue by scanning the sample twice with zeroth‐order Bessel beam and another type of propagation‐invariant beam, complementary to the zeroth‐order Bessel beam, which greatly reduces the out‐of‐focus background created in the first scan. The axial resolution can be improved from 1.68 μm of the Bessel light‐sheet to 1.07 μm by subtraction of the two scanned images across a whole field‐of‐view of up to 300 μm × 200 μm × 200 μm. The optimization procedure to create the complementary beam is described in detail and it is experimentally generated with a spatial light modulator. The imaging performance is validated experimentally with fluorescent beads as well as eGFP‐labeled mouse brain neurons.      

A robust co-localisation measurement utilising z-stack image intensity similarities for biological studies

Background

Co-localisation is a widely used measurement in immunohistochemical analysis to determine if fluorescently labelled biological entities, such as cells, proteins or molecules share a same location. However the measurement of co-localisation is challenging due to the complex nature of such fluorescent images, especially when multiple focal planes are captured. The current state-of-art co-localisation measurements of 3-dimensional (3D) image stacks are biased by noise and cross-overs from non-consecutive planes.

Method

In this study, we have developed Co-localisation Intensity Coefficients (CICs) and Co-localisation Binary Coefficients (CBCs), which uses rich z-stack data from neighbouring focal planes to identify similarities between image intensities of two and potentially more fluorescently-labelled biological entities. This was developed using z-stack images from murine organotypic slice cultures from central nervous system tissue, and two sets of pseudo-data. A large amount of non-specific cross-over situations are excluded using this method. This proposed method is also proven to be robust in recognising co-localisations even when images are polluted with a range of noises.

Results

The proposed CBCs and CICs produce robust co-localisation measurements which are easy to interpret, resilient to noise and capable of removing a large amount of false positivity, such as non-specific cross-overs. Performance of this method of measurement is significantly more accurate than existing measurements, as determined statistically using pseudo datasets of known values. This method provides an important and reliable tool for fluorescent 3D neurobiological studies, and will benefit other biological studies which measure fluorescence co-localisation in 3D.     

Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second

Non‐invasive biological imaging is crucial for understanding in vivo structure and function. Optical coherence tomography (OCT) and reflectance confocal microscopy are two of the most widely used optical modalities for exogenous contrast‐free, high‐resolution, three‐dimensional imaging in non‐fluorescent scattering tissues. However, sample motion remains a critical barrier to raster‐scanned acquisition and reconstruction of wide‐field anatomically accurate volumetric datasets. We introduce spectrally encoded coherence tomography and reflectometry (SECTR), a high‐speed, multimodality system for simultaneous OCT and spectrally encoded reflectance (SER) imaging. SECTR utilizes a robust system design consisting of shared optical relays, scanning mirrors, swept laser and digitizer to achieve the fastest reported in vivo multimodal imaging rate of 2 gigapixels per second. Our optical design and acquisition scheme enable spatiotemporally co‐registered acquisition of OCT cross‐sections simultaneously with en face SER images for multivolumetric mosaicking. Complementary axial and lateral translation and rotation are extracted from OCT and SER data, respectively, for full volumetric estimation of sample motion with micron spatial and millisecond temporal resolution.      

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.     

Enhanced spatial resolution for snapshot hyperspectral imaging of blood perfusion and melanin information within human tissue

We report a reconstruction method to achieve high spatial resolution for hyperspectral imaging of chromophore features in skin in vivo. The method utilizes an established structure‐adaptive normalized convolution algorithm to reconstruct high spatial resolution of hyperspectral images from snapshot low‐resolution hyperspectral image sequences captured by a snapshot spectral camera. The reconstructed images at chromophore‐sensitive wavebands are used to map the skin features of interest. We demonstrate the method experimentally by mapping the blood perfusion and melanin features (moles) on the facial skin. The method relaxes the constrains of the relatively low spatial resolution in the snapshot hyperspectral camera, making it more usable in imaging applications.     

Dual‐color STED super‐resolution microscope using a single laser source

STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies，due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples.     

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.     

Restored interlaced volumetric imaging increases image quality and scanning speed during intravital imaging in living mice

Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living organisms and is influenced primarily by several factors: motion artifacts, optical properties and spatial resolution. Conventional imaging quality within a volume, however, is degraded by involuntary movements and trades off between the imaged volume, imaging speed and quality. To balance such trade‐offs incurred by two‐photon excitation microscopy during intravital imaging, we developed a unique combination of interlaced scanning and a simple image restoration algorithm based on biological signal sparsity and a graph Laplacian matrix. This method increases the scanning speed by a factor of four for a field size of 212 μm × 106 μm × 130 μm, and significantly improves the quality of four‐dimensional dynamic volumetric data by preventing irregular artifacts due to the movement observed with conventional methods. Our data suggest this method is robust enough to be applied to multiple types of soft tissue.