Optical Sectioning Microscope Research Articles

Quantitative and comparative assessment of dyes and protocols for rapid ex vivo microscopy of fresh tissues

Using ex vivo microscopy, virtual pathology can improve histological procedures by providing pathology images in near real-time without tissue destruction. Several emerging and promising approaches leverage fast-acting small-molecule fluorescent stains to replicate traditional pathology structural contrast, combined with rapid optical sectioning microscopes. However, several vital challenges must be addressed to translate virtual pathology into the clinical environment. One such challenge is selecting robust, reliable, and repeatable staining protocols that can be adopted across institutions. In this work, we addressed the effects of dye selection and staining protocol on image quality in rapid point-of-care imaging settings. For this purpose, we used structured illumination microscopy to evaluate fluorescent dyes currently used in the field of ex vivo virtual pathology, in particular, studying the effects of staining protocol and temporal and photostability on image quality. We observed that DRAQ5 and SYBR gold provide higher image quality than TO-PRO3 and RedDot1 in the nuclear channel and Eosin Y515 in the extracellular/cytoplasmic channel than Atto488. Further, we found that TO-PRO3 and Eosin Y515 are less photostable than other dyes. Finally, we identify the optimal staining protocol for each dye and demonstrate pan-species generalizability.

Smartphone-based optical sectioning (SOS) microscopy with a telecentric design for fluorescence imaging.

We propose a smartphone-based optical sectioning (SOS) microscope based on the HiLo technique, with a single smartphone replacing a high-cost illumination source and a camera sensor. We built our SOS with off-the-shelf optical, mechanical cage systems with 3D-printed adapters to seamlessly integrate the smartphone with the SOS main body. The liquid light guide can be integrated with the adapter, guiding the smartphone's LED light to the digital mirror device (DMD) with neglectable loss. We used an electrically tuneable lens (ETL) instead of a mechanical translation stage to realise low-cost axial scanning. The ETL was conjugated to the objective lens's back pupil plane (BPP) to construct a telecentric design by a 4f configuration to maintain the lateral magnification for different axial positions. SOS has a 571.5µm telecentric scanning range and an 11.7µm axial resolution. The broadband smartphone LED torch can effectively excite fluorescent polystyrene (PS) beads. We successfully used SOS for high-contrast fluorescent PS beads imaging with different wavelengths and optical sectioning imaging of multilayer fluorescent PS beads. To our knowledge, the proposed SOS is the first smartphone-based HiLo optical sectioning microscopy (£1965), which can save around £7035 compared with a traditional HiLo system (£9000). It is a powerful tool for biomedical research in resource-limited areas.

Adaptive structured illumination optical-sectioning microscopy based on the prior knowledge of sample structure

Fast and accurate online detection is of great significance to improve mass production efficiency and reduce the costs of semiconductor chips and other micro-nano devices. Compared with other 3D imaging detection techniques, structured illumination optical-sectioning microscopy has faster 3D imaging because it uses wide-field sinusoidal fringe structured illumination. However, commercial structured illumination optical-sectioning microscopes are designed for general needs, and the orientations of the fringe patterns they project are not optimized for the structures of the sample. To obtain an isotropic result, they need to project multiple sinusoidal fringe patterns with different orientations and perform multiple optical-sectioning imaging, and then average the optical-sectioning results, resulting in low imaging efficiency. This paper proposes an adaptive structured illumination optical-sectioning microscopy. It exploits prior knowledge of the sample structure to design the sinusoidal fringe orientation for each local structure and generates an adaptive structured illumination. In this way, we can achieve the best imaging effect with only one optical-sectioning imaging, thereby improving the imaging efficiency, which is beneficial to the improvement of the mass production efficiency of products. The method proposed in this paper will provide useful guidance on how to correctly and efficiently use structured illumination optical-sectioning microscopy to detect defects of micro-nano devices such as semiconductor chips with known designed structures.

Imaging plant tissues: advances and promising clearing practices.

The study of the organ structure of plants and understanding their physiological complexity requires 3D imaging with subcellular resolution. Most plant organs are highly opaque to light, and their study under optical sectioning microscopes is therefore difficult. In animals, many protocols have been developed to make organs transparent to light using clearing protocols (CPs). By contrast, clearing plant tissues is challenging because of the presence of fibers and pigments. We describe progress in the development of plant CPs over the past 20 years through a modified taxonomy of CPs based on their physical and optical parameters that affect tissue properties. We also discuss successful approaches that combine CPs with new microscopy methods and their future applications in plant science research.

New clearing protocol for tannic roots optical imaging.

The understanding of plant physiological complexity requires 3D imaging of the entire organ with subcellular resolution. However, most plant organs are highly opaque to light and their study under optical sectioning microscopes is difficult. By gathering the latest developments in the field of optical clearing of pigmented plant tissues, it was found that the clearing tannic roots (CTR) protocol drastically reduces light absorption by a depigmentation step and light scattering by the homogenisation of refractive indices through entire grapevine roots.

A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies.

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities can expect a homogenous and time-invariant illumination and an uniform detection sensitivity, which are prerequisites for imaging analysis, tracking or fluorimetric pH or Ca2+ -concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning microscopes calls for a regular calibration with a standardized point source. The test samples required for such measurements are typically different ones, they are often expensive and they depend much on the very microscope technique used. Similarly, the ever-increasing choice among microscope techniques and geometries increases the demand for comparison across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: commercial tissue paper labeled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-μm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

Tissue clearing technique: Recent progress and biomedical applications.

Organisms are inherently three dimensional, thus comprehensive understanding of the complicated biological system requires analysis of organs or even whole bodies in the context of three dimensions. However, this is a tremendous task since the biological specimens are naturally opaque, a major obstacle in whole-body and whole-organ imaging. Tissue clearing technique provides a prospective solution and has become a powerful tool for three-dimensional imaging and quantification of organisms. Tissue clearing technique aims to make tissue transparent by minimizing light scattering and light absorption, thus allowing deep imaging of large volume samples. When combined with diverse molecular labeling methods and high-throughput optical sectioning microscopes, tissue clearing technique enables whole-body and whole-organ imaging at cellular or subcellular resolution, providing detailed and comprehensive information about the intact biological systems. Here, we give an overview of recent progress and biomedical applications of tissue clearing technique. We introduce the mechanisms and basic principles of tissue clearing, and summarize the current tissue clearing methods. Moreover, the available imaging techniques and software packages for data processing are also presented. Finally, we introduce the recent advances in applications of tissue clearing in biomedical fields. Tissue clearing contributes to the investigation of structure-function relationships in intact mammalian organs, and opens new avenues for cellular and molecular mapping of intact human organs. We hope this review contributes to a better understanding of tissue clearing technique and can help researchers to select the best-suited clearing protocol for their experiments.

Real-time video mosaicking to guide handheld in vivo microscopy.

Handheld and endoscopic optical-sectioning microscopes are being developed for noninvasive screening and intraoperative consultation. Imaging a large extent of tissue is often desired, but miniature in vivo microscopes tend to suffer from limited fields of view. To extend the imaging field during clinical use, we have developed a real-time video mosaicking method, which allows users to efficiently survey larger areas of tissue. Here, we modified a previous post-processing mosaicking method so that real-time mosaicking is possible at >30 frames/second when using a device that outputs images that are 400 × 400 pixels in size. Unlike other real-time mosaicking methods, our strategy can accommodate image rotations and deformations that often occur during clinical use of a handheld microscope. We perform a feasibility study to demonstrate that the use of real-time mosaicking is necessary to enable efficient sampling of a desired imaging field when using a handheld dual-axis confocal microscope.

Adaptive optics light-sheet microscopy based on direct wavefront sensing without any guide star.

We propose an adaptive optics light-sheet fluorescence microscope (AO-LSFM) for closed-loop aberrations' correction at the emission path, providing intrinsic instrumental simplicity and high accuracy when compared to previously reported schemes. The approach is based on direct wavefront sensing, i.e., not on time-consuming iterative algorithms, and does not require the use of any guide star, thus reducing instrumental complexity and/or sample preparation constraints. The design is based on a modified Shack-Hartmann wavefront sensor providing compatibility with extended sources such as images from optical sectioning microscopes. We report an AO-LSFM setup based on such sensors, including characterization of the sensor performance, and demonstrate for the first time to the best of our knowledge a significant contrast improvement on neuronal structures of the ex vivo adult drosophila brain in depth.

Optimizing depth-of-field extension in optical sectioning microscopy techniques using a fast focus-tunable lens.

Volume imaging based on a fast focus-tunable lens (FTL) allows three-dimensional (3D) observation within milliseconds by extending the depth-of-field (DOF) with sub-micrometer transverse resolution on optical sectioning microscopes. However, the previously published DOF extensions were neither axially uniform nor fit with theoretical prediction. In this work, complete theoretical treatments of focus extension with confocal and various multiphoton microscopes are established to correctly explain the previous results. Moreover, by correctly placing the FTL and properly adjusting incident beam diameter, a uniform DOF is achieved in which the actual extension nicely agrees with the theory. Our work not only provides a theoretical platform for volumetric imaging with FTL but also demonstrates the optimized imaging condition.

BiDiFuse: a FIJI plugin for fusing bi-directionally recorded microscopic image volumes

The method is freely available as a plugin for FIJI from https://github.com/JanDetrez/BiDiFuse/ CONTACT: winnok.devos@uantwerpen.be.

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology.

There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device.

Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 106 pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector’s flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.

Note: laser beam scanning using a ferroelectric liquid crystal spatial light modulator.

In this work we describe laser beam scanning using a ferroelectric liquid crystal spatial light modulator. Commercially available ferroelectric liquid crystal spatial light modulators are capable of displaying 85 colored images in 1 s using a time dithering technique. Each colored image, in fact, comprises 24 single bit (black and white) images displayed sequentially. We have used each single bit image to write a binary phase hologram. For a collimated laser beam incident on the hologram, one of the diffracted beams can be made to travel along a user defined direction. We have constructed a beam scanner employing the above arrangement and demonstrated its use to scan a single laser beam in a laser scanning optical sectioning microscope setup.

A diffuser-based optical sectioning fluorescence microscope

A novel design for an optical sectioning microscope is described. The microscope uses laser illumination of a diffuser to illuminate a sample with a rapidly-changing image-plane speckle pattern. The fluorescent light from the sample is then imaged back onto the same diffuser before being imaged onto an integrating detector array. The diffuser is moved so as to illuminate each part of the specimen with a very large number of independent speckles while an image is captured. This arrangement employs a simple rotating diffuser and should allow a wide-field optically-sectioned image to be recorded in a shorter time than can be achieved with a scanning confocal system. An analysis is presented to show that the arrangement can produce an image with a sectioning capability similar to that of a conventional scanning confocal microscope. The quality of the images produced is assessed through computer simulation.

A liquid optical phantom with tissue-like heterogeneities for confocal microscopy

Phantoms play an important role in the development, standardization, and calibration of biomedical imaging devices in laboratory and clinical settings, serving as standards to assess the performance of such devices. Here we present the design of a liquid optical phantom to facilitate the assessment of optical-sectioning microscopes that are being developed to enable point-of-care pathology. This phantom, composed of silica microbeads in an Intralipid base, is specifically designed to characterize a reflectance-based dual-axis confocal (DAC) microscope for skin imaging. The phantom mimics the scattering properties of normal human epithelial tissue in terms of an effective scattering coefficient and a depth-dependent degradation in spatial resolution due to beam steering caused by tissue micro-architectural heterogeneities.

Optical sectioning microscope with a binary hologram based beam scanning

We describe the development of a beam scanning microscope that can perform optical sectioning based on the principle of confocal microscopy. The scanning is performed by a laser beam diffracted from a dynamic binary hologram implemented using a liquid crystal spatial light modulator. Using the proposed scanning mechanism, unlike the conventional confocal microscopes, scanning over a two-dimensional area of the sample can be obtained without the use of a pair of galvo mirror scanners. The proposed microscope has a number of advantages, such as superior frame to frame repeatability, simpler optical arrangement, increased pixel dwell time relative to the time between two pixels, illumination of only the sample points without pulsing the laser, and absolute control over the amplitude and phase of the illumination beam on a pixel to pixel basis. The proposed microscope can be particularly useful for applications requiring very long exposure time or very large working distance objective lenses. In this paper we present experimental implementation of the setup using a nematic liquid crystal spatial light modulator and proof-of-concept experimental results.

Experimental demonstration of an Optical-Sectioning Compressive Sensing Microscope (CSM)

In this paper we present the design and implementation of a Compressive Sensing Microscopy (CSM) imaging system, which uses the Compressive Sensing (CS) method to realize optical-sectioning imaging. The theoretical aspect of the proposed system is investigated using the mathematical model of the CS method and an experimental prototype is constructed to verify the CSM design. Compared to conventional optical-sectioning microscopes (such as Laser Scanning Confocal Microscopes (LSCMs) or Programmable Array Microscopes (PAMs)), the CSM system realizes optical-sectioning imaging using a single-pixel photo detector and without any mechanical scanning process. The complete information of the imaging scene is reconstructed from the CS measurements numerically.

Characterization of deformable mirrors for spherical aberration correction in optical sectioning microscopy

In this paper we describe the wavefront aberrations that arise when imaging biological specimens using an optical sectioning microscope and generate simulated wavefronts for a planar refractive index mismatch. We then investigate the capability of two deformable mirrors for correcting spherical aberration at different focusing depths for three different microscope objective lenses. Along with measurement and analysis of the mirror influence functions we determine the optimum mirror pupil size and number of spatial modes included in the wavefront expansion and we present measurements of actuator linearity and hysteresis. We find that both mirrors are capable of correcting the wavefront aberration to improve imaging and greatly extend the depth at which diffraction limited imaging is possible.

Single Molecule Tracking With Light Sheet-Based Microscopy In Vivo

Autofluorescence, rapid photobleaching and high particle concentrations present restrictions to single molecule observation in biological systems by epi-illumination. Light sheet-based microscopy overcomes these limitations [1]. By combining the speed of parallel image acquisition and the optical sectioning produced by light sheet illumination, we created a powerful tool to study single molecule dynamics on a millisecond timescale [2].We illuminated the sample perpendicular to the detection axis with a thin light sheet (FWHM 2-3μm). In this manner a simple optical sectioning microscope is created, because only the focal plane of the detection optics is illuminated and no out-of-focus fluorescence is generated. The background fluorescence is strongly reduced and the signal-to-noise-ratio (SNR) greatly improved.We constructed a miniaturized glass specimen chamber, which can be illuminated from the side in a very flexible manner and directly be mounted on a commercial inverse microscope. The specimen is easily accessible for micromanipulation and can be observed via the 0.17mm thin glass bottom of the chamber using high NA objective lenses.With this setup it was straightforward to observe trajectories of single protein molecules in aqueous solution with a D = 90μm2/s, and also in the cellular interior. By analysis of the diffusion behavior of single fluorescent dextran molecules we determined the viscosity of living C. tentans salivary gland cell nuclei. Similarly, molecular dynamics in adherent cells can be observed with greatly improved contrast.With this new experimental setup we use the ideal imaging scheme for single molecule visualization and push the limit of sensitivity far beyond the potential of conventional epi-illumination.[1] Huisken, J. et al. (2004). Science 305(5686): 1007-9.[2] Ritter, J.G. et al. (2008). Opt Express 16(10): 7142-52.