Optical sectioning microscopy with planar or structured illumination

While widefield microscopy has long been constrained by out-of-focus scattering, advancements have generated a solution in the form of confocal laser scanning microscopy (cLSM) and optical sectioning microscopy using structured illumination (OSM). In this study, we aim to investigate, using microglia branching, if cLSM and OSM can produce images with comparable morphological characteristics. By imaging the somatosensory microglia from a tissue slice of a 3-week-old mouse and establishing morphological parameters that characterizes the microglial branching pattern, we were able to show that there is no difference in total length of the branch tree, number of branches, mean branch length and number of primary to terminal branches. We did find that area-based parameters such as mean occupied area and mean surveillance area were bigger in cLSM isolated microglia compared to OSM ones. Additionally, by investigating the difference in acquisition time between techniques and personal costs we were able to establish that the amortization could be made in 6.11 ± 2.93 years in the case of countries with a Human Development Index (HDI) = 7-9 and 7.06 ± 3.13 years, respectably, for countries with HDI < 7. As such, OSM systems seem a valid option if one just wants basic histological evaluation, and cLSM should be considered for groups that demand higher resolution or volumetric images.

The studies of early cytologists demonstrated that metaphase chromosomes were not simply aggregations of dispersed interphase chromatin. Well before the turn of this century it had been observed that the positions of mitotic chromosomes correlated with their respective positions in preceding cell divisions. Since these early observations there has been a consistent interest in assessing the relative order of chromosomes in nuclei, especially in interphase, and elaborating on the importance of any observed order with reference to gene expression. A major problem in analysing interphase nuclei has been the difficulty in visualizing chromosomes at this stage in the cell cycle. In recent years significant progress has been made in the rapid analysis of nuclei at metaphase as well as interphase and this was the subject of the workshop. A. J. Hilliker presented the studies from his laboratory on the analysis of relative chromosome positions in cells, in Drosophila melanogaster, which are destined to differentiate into salivary gland cells. The assay measuring the relative positions of chromosomes included irradiating larvae at a stage when progenitor salivary gland cells have undergone their last cell division, to cause cross-linking between chromosome regions that are juxtaposed. The actual positions of the cross-links could be accurately measured cytologically by studying the polytene chromosomes of the fully differentiated salivary gland cells. These cytogenetic studies indicated that the only chromosomal regions that were cross-linked are those from'the same arm of a chromosome. Essentially no crosslinks were found between different chromosome arms leading Hilliker to argue that chromosomes were in clearly defined domains even in interphase when they are in an apparently diffuse stage. This conclusion was also clear from the studies presented by F. M. Waldman using in situ hybridization techniques on human B cell nuclei. Waldman, Pinkel, and Gray have studied the relative positions of specific human chromosomes using fluorescence to detect repetitive, chromosome-specific DNA probes followed by optical sectioning microscopy and computerized image analysis. In the B lymphocytes analysed, the positions of the repetitive sequences on chromosomes Y, 1 , 4 , and 9 were located nearer to the periphery of the nucleus than would be expected from random placement. Probe positions did not appear to alter in the transition from G1 to S phase. The technique of optical sectioning was discussed in more detail by M. Rykowski. The OM1 optical microscope system discussed by Rykowski was designed by John Sedat, David Agard, and colleagues at UCSF. The system combined the use of a cooled CCD (charge-couple device) camera with computational image analysis to yield high quality, three-dimensional (3-D) images of a variety of biological specimens using conventional fluorescence microscopy. The linearity and wide dynamic range of the CCD camera was demonstrated using an in situ hybridization analysis of Drosophila polytene chromosome band 3C7 (the location of the Notch gene) to allow the observation of very fine cytological features. The use of conventional UV light rather than laser light allowed maximum flexibility in choice of fluorochromes with minimal photobleaching. It was evident that the parallel development of computer programmes for image enhancement, analysis, and interpretation is critical to the continued study of the 3-D arrangements of chromosomes in nuclei. J. L. Oud presented studies using the confocal scanning laser microscope, a system that is quite different from the one described by Rykowski. Oud and his colleagues did not use in situ hybridization techniques to identify chromosomes since the cells of interest to them were anaphase cells from Crepis capillaris. In these cells the three chromosome pairs could be readily identified. To estimate the extent to which chromosomes are ordered, 76 Feulgen-stained root-tip anaphases were analysed and the observed chromosome order was compared with that expected from a random ordering model. The work demonstrated a significant surplus of one of the arrangements with a juxtaposition of the two chromosomes with a nucleolar organizer region. Oud suggested that the nucleolus was an important factor in determining the final 3-D arrangement of chromosomes in nuclei. In conclusion, the workshop provided participants with a unique opportunity to see the major techniques available for analysing 3-D arrangements of chromatin-chromosomes in nuclei. It was clear that chromosome domains exist and that the relative arrangement of these domains is dynamic. Observations such as those from Crepis on the preferred juxtaposition of Nor-chromosomes left open the possibility that in specific tissues certain chromosomes may be located in specific positions. Such specific positioning may be limited to defined stages in the cell cycle.

Optical sectioning imaging with high spatial resolution deep inside scattering samples such as mammalian brain is of great interest in biological study. Conventional two-photon microscopy deteriorates in focus when light scattering increases. Here we develop an optical sectioning enhanced two-photon technique which incorporates structured illumination into line-scanning spatial-temporal focusing microscopy (LTSIM), and generate patterned illumination via laser intensity modulation synchronized with scanning. LTSIM brings scattering background elimination and in-focus contrast enhancement, and realizes nearly 2-fold increase in spatial resolution to ∼208 nm laterally and ∼0.94 µm axially. In addition, the intensity modulated line-scanning implementation of LTSIM enables fast and flexible generation of structured illumination, permitting adjustable spatial frequency profiles to optimize image contrast. The highly qualified optical sectioning ability of our system is demonstrated on samples including tissue phantom, C. elegans and mouse brain at depths over hundreds of microns.

Subcellular Localization of Disease-Associated Prion Protein in the Human Brain

BackgroundSome plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.ResultsHere, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).ConclusionsThe study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.

This chapter discusses the stratagems and approaches to imaging that are available to reduce blurring in the final image or are required to collect blur-free 2D and 3D image data sets. It explains how the point-scanning confocal and other optical sectioning microscopes work. The single-beam point-scanning confocal is the most widespread and popular of all the optical sectioning fluorescence microscopes, and several designs are available on the market. The confocal microscope is so-called because the illumination pinhole, the plane of focus within the specimen and the detector pinhole are all situated at conjugate focal planes. The chapter shows the generic features of a typical laser-scanning confocal microscope. Confocal microscopes clearly offer considerable advantages over widefield fluorescence microscopy. Programmable array and line-scanning microscopes are designs that attempt to increase the frame rate, and thus the temporal resolution, of the laser-scanning confocal microscope.

Grating-based X-ray differential phase contrast imaging provides excellent image contrast for low-Z objects that cannot be acquired by conventional X-ray imaging, which has great potential applications in the early diagnosis of cancer and non-destructive detections of low-Z materials and devices. Large field of view imaging is a crucial factor for this technology from the laboratory to practical application. For the objective need of large field of view, on the basis of the Fresnel diffraction theory and the structure characteristics of gratings, we establish a quantitative physical model to analyze the factors that affect the imaging field of view and give a feasible way for large imaging field of view. This work provides a theoretical basis for the large field of view grating-based X-ray differential phase contrast imaging in the future.

This short review begins with the theories of Airy, Rayleigh and Abbe on microscope resolution. Next, the principal developments in microscopy in the last half-century are examined for relevance to ophthalmology: confocal microscopy, photoactivation light microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), structured illumination (SI), 2-photon and multiphoton excitation microscopy with a focused beam. Except for confocal, these are difficult to apply to the eye in vivo, as are the interference methods available in microscopes. However, interferometry in the form of coherence tomography is now a major ophthalmic method which has diverged from microscopy. Multiphoton excitation microscopy with an unfocussed beam is a new, low-damage microscope method so-far not exploited in ophthalmoscopy. The Mesolens, which throws off the historic limitations in microscopy set by the human eye, is described as a possible future aid to ophthalmology of the anterior eye.

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed.

Confocal fluorescence microscopy is a well‐established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal‐to‐noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to “virtually” perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super‐resolution, and for this reason we call this method SPLIT‐pinhole (SPLIT‐PIN). We show with simulated data that the SPLIT‐PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT‐PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT‐PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium.Research HighlightsWe describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor‐based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT‐pinhole (SPLIT‐PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope.

Smart devices have been fabricated based on design concept of multiple layer structures which require through silicon vias to transfer electric signals between stacked layers. Because even a single defect leads to fail of the packaged devices, the dimensions of the through silicon vias are needed to be measured through whole sampling inspection process. For that, a novel hybrid optical probe working based on optical interferometry, confocal microscopy and optical microscopy was proposed and realized for enhancing inspection efficiency in this report. The optical microscope was utilized for coarsely monitoring the specimen in a large field of view, and the other methods of interferometry and confocal microscopy were used to measure dimensions of small features with high speed by eliminating time-consuming process of the vertical scanning. Owing to the importance of the reliability, the uncertainty evaluation of the proposed method was fulfilled, which offers a practical example for estimating the performance of inspection machines operating with numerous principles at semiconductor manufacturing sites. According to the measurement results, the mean values of the diameter and depth were 40.420 µm and 5.954 µm with the expanded uncertainty of 0.050 µm (k = 2) and 0.208 µm (k = 2), respectively.

This paper presents a new technique for studying the growth and morphology of fatigue cracks. The technique allows short fatigue crack growth, crack depth, aspect ratio (crack depth/half crack length), and crack front configuration to be measured using a Confocal Scanning Laser Microscope (CSLM). CSLM measurements of the initial stage of crack growth in Al 2024-T351 revealed that microstructurally short fatigue cracks grew initially along a plane inclined to the applied stress. The angle of the inclined plane (Stage I crack growth) was found to be about 45 degrees to the axis of the applied tensile load. Aspect ratio and the angle of maximum shear plane (Mode II), obtained using the CSLM technique, showed a good agreement with those obtained using a Surface Removal (SR) technique. The aspect ratios obtained using the CSLM technique were found to remain constant with increasing crack length in Al 2024-T351 and SAE 1045 Steel at 0.83 and 0.80, respectively. Optical sectioning along the length of a crack revealed that the crack front in the interior of the materials has a semi-elliptical shape. These results are in good agreement with results obtained using the SR technique. The CSLM technique was employed to characterize the fracture surface of fatigue cracks in an SAE 1045 Steel. CSLM image processing of the fracture surface near the crack tip constructed a three dimensional profile of fracture surface asperities. The heights of asperities were obtained from this profile. Optical sectioning from a post-image-processed crack provided crack depth and crack mouth width at every point along the crack length for each load level. The crack opening stress was taken as the stress level at which the crack depth stopped increasing with increases in applied stress.

Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.

Bringing Histology to the Bedside