Optical Sectioning Techniques Research Articles

Rapid pseudo-H&E imaging using a fluorescence-inbuilt optical coherence microscopic imaging system.

A technique using Linnik-based optical coherence microscopy (OCM), with built-in fluorescence microscopy (FM), is demonstrated here to describe cellular-level morphology for fresh porcine and biobank tissue specimens. The proposed method utilizes color-coding to generate digital pseudo-H&E (p-H&E) images. Using the same camera, colocalized FM images are merged with corresponding morphological OCM images using a 24-bit RGB composition process to generate position-matched p-H&E images. From receipt of dissected fresh tissue piece to generation of stitched images, the total processing time is <15 min for a 1-cm2 specimen, which is on average two times faster than frozen-section H&E process for fatty or water-rich fresh tissue specimens. This technique was successfully used to scan human and animal fresh tissue pieces, demonstrating its applicability for both biobank and veterinary purposes. We provide an in-depth comparison between p-H&E and human frozen-section H&E images acquired from the same metastatic sentinel lymph node slice (∼10 µm thick), and show the differences, like elastic fibers of a tiny blood vessel and cytoplasm of tumor cells. This optical sectioning technique provides histopathologists with a convenient assessment method that outputs large-field H&E-like images of fresh tissue pieces without requiring any physical embedment.

Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER’s sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

High-speed volumetric imaging in vivo based on structured illumination microscopy with interleaved reconstruction.

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies, due to its high imaging speed over large field-of-views. However, WFFM is susceptible to out-of-focus background. To overcome this problem, structured illumination microscopy (SIM) was proposed as a wide-field, optical-sectioning technique, which needs multiple raw images for image reconstruction and thus has a lower imaging speed. Here we propose SIM with interleaved reconstruction, to make SIM of lossless speed. We apply this method in volumetric imaging of neural network dynamics in brains of zebrafish larva in vivo.

Experimental investigation on the effects of glass fiber hybridization on the low-velocity impact response of filament-wound carbon-based composite pipes

Recently, the incorporation of several different types of fibers into a single matrix has led to the development of superior hybrid composite properties at a cheaper cost. Fiber hybridization is one of the active strategies to toughen composites and improve impact damage resistance. However, the extraordinary high strength and stiffness of the carbon fiber as well as its lower damage tolerance make it more susceptible under the impact loading. This article mainly aims to improve impact damage resistance of carbon fiber pipes through fiber hybridization strategy with glass fibers under low-velocity impact. The composite pipes reinforced with thin internal liner of high-density polyethylene were fabricated through filament winding technology. Eight pipe configurations with different stacking sequences and fiber content ratios with a constant winding angle of [Formula: see text] were fabricated and tested under impact energies, 50 and 100 J. The damage characterization was evaluated using the optical imaging and mechanical micrograph sectioning technique. Results indicate that the hybrid configurations showed better energy absorption than reference carbon fibers specimen under 50 J impact energy. Specifically, specimens with glass fibers on the exterior side and alternative configuration of carbon–glass fibers showed better impact resistance with less damage observed. Meanwhile, the specimens with glass fiber on the exterior side suffered from extreme damage with increase in the energy absorption and maximum displacement for both fiber content ratios under 100 J of impact energy.

Using Pixel-Based Microscope Images to Generate 3D Reconstructions of Frozen and Thawed Plant Tissue.

Histological analysis of frozen and thawed plants has been conducted for many years but the observation of individual sections only provides a two-dimensional representation of a three-dimensional phenomenon. Currently available optical sectioning techniques for viewing internal structures in three dimensions are either low in resolution or the instrument cannot penetrate deep enough into the tissue to visualize the whole plant. Methods using higher resolution equipment are expensive and often require time-consuming training. In addition, conventional stains cannot be used for optical sectioning techniques. We present a relatively simple and less expensive technique using pixel-based (JPEG) images of conventionally stained histological sections of an Arabidopsis thaliana plant. The technique uses commercially available software to generate a 3D representation of internal structures.

Fast structured illumination three-dimensional color microscopic imaging method based on Hilbert-transform

As a wide-field microscopy, structured illumination microscopy (SIM) enables super-resolution and three-dimensional (3D) imaging. It has recently received lots of attention due to the advantages of high spatial resolution, short image recording time, and less photobleaching and phototoxicity. The SIM has found numerous important applications in time-lapse imaging of living tissues and cellular structures in the field of biomedical science. Color information is an important physical quantity describing the characteristics of living creatures and reflects the differences in its microstructure and optical property to some extent. Although HSV (hue, saturation, value) color space based structured illumination full-color 3D optical sectioning technique can recover the full color information on the surface of the samples without color distortion. However, for each optical sectioning, three raw images with fixed phase shift are required to calculate the sectioning images by the rootmean square (RMS) algorithm. This will dramatically increase the data acquisition time and data storage space, especially for a large-scaled sample that needs image stitching strategy. The image processing progress operated in HSV color space need to run the RMS algorithm three times in each channel of HSV space for every section, and transform the images between RGB (red-green-blue) space and HSV space twice. This will absolutely extend the data processing time and put forward higher requirements for computer hardware and software for data storage and processing. To this end, in this paper, a fast 3D color optical sectioning SIM algorithm based on Hilbert-transform is proposed. The Hilbert-transform has proved to be a powerful tool in digital signal and image processing and has successfully applied to the SIM. Here, only two raw images with structured illumination are needed to reconstruct a full-color optical sectioned image for each slice. This fast 3D color sectioning method has the advantage of insensitivity to phase-shift error and has better adaptability to noise, high quality color sectioning images can be obtained under the phase-shift error or noise disturbed environment. The image acquisition data are reduced by 1/3 and the color optical sectioning reconstruction time is saved by about 28%, this new method effectively improves the efficiency and speed for 3D color imaging and will bring a wider application range for SIM.

Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins.

.Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of . Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.

Deep learning optical-sectioning method.

Current optical-sectioning methods require complex optical system or considerable computation time to improve imaging quality. Here we propose a deep learning-based method for optical sectioning of wide-field images. This method only needs one pair of contrast images for training to facilitate reconstruction of an optically sectioned image. The removal effect of background information and resolution that is achievable with our technique is similar to traditional optical-sectioning methods, but offers lower noise levels and a higher imaging depth. Moreover, reconstruction speed can be optimized to 14 Hz. This cost-effective and convenient method enables high-throughput optical sectioning techniques to be developed.

Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

The reinvention of twentieth century microscopy for three-dimensional imaging.

In just over a decade, the field of biomedical research has witnessed a radical evolution in technologies for the 3- and 4-dimensional imaging of biological samples. Light sheet fluorescence microscopy is quickly developing into a powerful approach for fast, volumetric imaging of cells, tissues and living organisms. This review touches on the development of 3-dimensional imaging, from its foundations, namely from the invention of confocal microscopy in the twentieth century to more recent examples, notably the IsoView SPIM, the Lattice Light Sheet Microscope and swept confocally aligned planar excitation. These technologies overcome the limitations of conventional optical sectioning techniques and enable unprecedented levels of spatio-temporal resolution with low levels of phototoxicity. Developing in parallel with powerful computational approaches, light sheet based methods promise to completely transform cell biology as we know it today.

Selectable light-sheet uniformity using tuned axial scanning.

Light-sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three-dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light-sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade-off between light-sheet thickness and area over which this thickness is preserved. Recently, an increase in light-sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z-scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light-sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.

Differential structured illumination microendoscopy for in vivo imaging of molecular contrast agents

Fiber optic microendoscopy has shown promise for visualization of molecular contrast agents used to study disease in vivo. However, fiber optic microendoscopes have limited optical sectioning capability, and image contrast is limited by out-of-focus light generated in highly scattering tissue. Optical sectioning techniques have been used in microendoscopes to remove out-of-focus light but reduce imaging speed or rely on bulky optical elements that prevent in vivo imaging. Here, we present differential structured illumination microendoscopy (DSIMe), a fiber optic system that can perform structured illumination in real time for optical sectioning without any opto-mechanical components attached to the distal tip of the fiber bundle. We demonstrate the use of DSIMe during in vivo fluorescence imaging in patients undergoing surgery for cervical adenocarcinoma in situ. Images acquired using DSIMe show greater contrast than standard microendoscopy, improving the ability to detect cellular atypia associated with neoplasia.

Multi-beam two-photon imaging of fast Ca2+ signals in the Langendorff mouse heart.

Although the role of calcium (Ca(2+)) in excitation-contraction coupling in the heart can be comprehensively studied at the cellular level, propagation of Ca(2+) signals intercellularly requires tissue-based investigations. To access cells below the epicardium, an optical-sectioning technique is necessary. Multi-photon microscopy allows reliable imaging for penetration to depths of up to 0.5 mm. Here, we provide a protocol that uses multibeam two-photon microscopy for measuring Ca(2+) signals in a Langendorff-perfused mouse heart.

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy

Optical sectioning techniques offer the ability to acquire three-dimensional information from various organ tissues by discriminating between the desired in-focus and out-of-focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community(1). Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities(2,3). A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells(4). However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells(4). Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community. Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies(2,3). Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

Effect of lay-up orientation on ballistic impact behaviors of GLARE 5 FML beams

This paper examines experimental and numerical investigations on ballistic impact behaviors of GLARE 5 (3/2) fiber-metal laminated (FML) beams of various stacking sequences. The ballistic impact tests were conducted using a high-speed gas gun equipped with a high-speed camera. Optical imaging and mechanical sectioning techniques as well as high-speed-camera footage were used to characterize the impact damage in the specimens. The incident projectile impact velocity versus the residual velocity (VI ∼ VR) was plotted and numerically fitted according to the classical Lambert–Jonas equation for the determination of ballistic limit velocity, V50. The results showed that the unidirectional [90°4] specimen offered the least resistance to the projectile perforation. In addition, except for the unidirectional [90°4] specimen, the ballistic limit was almost insensitive with respect to change in stacking sequence. The interfacial delamination/debonding as well as bending and stretching in aluminum layers played significant roles in dissipating the impact energy in the GLARE 5 (3/2) FML beams. The ballistic impact tests were modeled using a 3D finite element (FE) code, LS-DYNA. The FE prediction was validated using the experimentally obtained VI ∼ VR trends, damage patterns and bullet residual length. Good agreement between experimental and numerical results was achieved. Moreover, the FE predicted V50 as a function of specimen stacking sequence was in good agreement with its experimental counterpart. The validated FE model was then utilized to obtain some insights to the ballistic impacts of which experimental results were not available. It was found that the cross-ply specimen offered the highest contact resistance force while the unidirectional [90°4] lay-up orientation offered the lowest. The maximum contact force for the unidirectional [0°4] and quasi-isotropic panels were almost the same. The results showed that when subjected to ballistic impact, the specimen with [0°/90°]s lay-up orientation dissipated more energy compared to the other lay-up orientations.

Long Term Single Molecule TIRF Observation of Biomolecules without Immobilization

Single-molecules fluorescence techniques have yielded valuable structural and dynamical information about molecular biological machines. The most common approaches used to observe single molecules in biology are confocal spectroscopy of freely diffusing molecules and total internal reflection (TIR) microscopy of surface attached molecules. With the latter technique it is possible to perform long-term observation of individual molecules, but at the cost of complex immobilization procedures that cannot be generally applied and often perturb the sample. Here we demonstrate a versatile technology that combines the advantages of both existing approaches, i.e. freely diffusing molecules and long-term observation with high signal to noise, in a simple, easy to handle, low-cost nanofluidic platform. Biomolecules flow through channels that are less than 100 nm deep, which keeps them within the TIR field, as required to fully exploit the high signal to noise possible with this optical sectioning technique. Rather than employing typical expensive nanofluidic technology, the nanochannels are generated using controlled channel collapse on a microfluidic device made from PDMS, ensuring easy fabrication and handling. We demonstrate that biomolecules labeled with single fluorophores can be imaged with millisecond time-resolution for several seconds. In addition, the design of the control channel of the device makes it possible to achieve high fluorophore photostability by removing oxygen without any need to add chemicals. By having a continuous stream of biomolecules flowing through the nanochannels, thousands of biomolecules can be measured each hour, illustrating the potential for automated experiments. We demonstrate the compatibility of the device with large biological complexes by showing multi-channel imaging of freely diffusing single nucleosomes.

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

Developmental Programming: Reproductive Endocrinopathies in the Adult Female Sheep After Prenatal Testosterone Treatment Are Reflected in Altered Ontogeny of GnRH Afferents

The GnRH system represents a useful model of long-term neural plasticity. An unexplored facet of this plasticity relates to the ontogeny of GnRH neural afferents during critical periods when the hypothalamic-pituitary-gonadal axis is highly susceptible to perturbation by sex steroids. Sheep treated with testosterone (T) in utero exhibit profound reproductive neuroendocrine dysfunctions during their lifespan. The current study tested the hypothesis that these changes are associated with alterations in the normal ontogeny of GnRH afferents and glial associations. Adult pregnant sheep (n=50) were treated with vehicle [control (CONT)] or T daily from gestational day (GD)30 to GD90. CONT and T fetuses (n=4-6/treatment per age group) were removed by cesarean section on GD90 and GD140 and the brains frozen at -80°C. Brains were also collected from CONT and T females at 20-23 wk (prepubertal), 10 months (normal onset of puberty and oligo-anovulation), and 21 months (oligo-anovulation in T females). Tissue was analyzed for GnRH immunoreactivity (ir), total GnRH afferents (Synapsin-I ir), glutamate [vesicular glutamate transporter-2 (VGLUT2)-ir], and γ-aminobutyric acid [GABA, vesicular GABA transporter (VGAT)-ir] afferents and glial associations (glial fibrillary acidic protein-ir) with GnRH neurons using optical sectioning techniques. The results revealed that: 1) GnRH soma size was slightly reduced by T, 2) the total (Synapsin-I) GnRH afferents onto both somas and dendrites increased significantly with age and was reduced by T, 3) numbers of both VGAT and VGLUT inputs increased significantly with age and were also reduced by T, and 4) glial associations with GnRH neurons were reduced (<10%) by T. Together, these findings reveal a previously unknown developmental plasticity in the GnRH system of the sheep. The altered developmental trajectory of GnRH afferents after T reinforces the notion that prenatal programming plays an important role in the normal development of the reproductive neuroendocrine axis.

Advances in Microscopy Techniques

Advances in microscopy enable visualization of a broad range of new morphologic features. To review and illustrate advances in microscopy with relevance to pathologists. Literature review and new observations. Fluorescence microscopy enables multiantigen detection; allows novel optical-sectioning techniques, with some advantages compared to paraffin sectioning; and permits live-cell imaging. Live-cell imaging allows pathologists to move from a period when all diagnostic expertise was reliant on interpreting static images to a period when cellular dynamics can play a role in diagnosis. New techniques have bypassed by about 100-fold what had long been believed to be a limit to the resolution of light microscopy. Fluorescence resonance energy transfer (FRET) appears capable of visualizing diagnostically relevant molecular events in living or fixed cells that are immeasurable by other molecular techniques. We describe applications of 2-photon microscopy, FRET, structured illumination, and the subdiffraction techniques of near-field microscopy, photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy. New microscopy techniques present opportunities for pathologists to develop improved diagnostic tests.