Conventional Confocal Microscopy Research Articles

Development of novel two-photon microscopy for living brain and neuron.

"In vivo" two-photon microscopy (TPLSM) has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. To break the limit of this penetration depth, we introduced a novel light source based on a semiconductor laser [1]. The light source successfully visualized not only cortex layer V pyramidal neurons spreading to all cortex layers at a superior S/N ratio, but visualize hippocampal CA1 neurons in young adult mice [2]. These results indicate that the penetration depth of this laser was ∼1.4 mm. In vivo TPLSM with a laser emitting a longer wavelength might give us insights on activities of neurons in the cortex or the hippocampus. This deep imaging method could be applicable to other living organs including tumor tissues. In addition, we developed liquid crystal devices to convert linearly polarized beams (LP) to vector beams [3]. A liquid device generated a vector beam called higher-order radially polarized (HRP) beam, which enabled that each of the aggregated 0.17 m beads was distinguished individually, whereas in conventional confocal microscopy or TPLSM they could not. We also visualized the finer structures of networks of filamentous cytoskeleton microtubule fluorescently-labeled in the COS-7, and primary culture of mouse neurons. Moreover, by taking an advantage of the LCDs that can utilize various wavelengths including near-infrared, we could employ an HRP beam for improving TPLSM. An HRP beam visualized fine intracellular structures not only in fixed cells stained with various dyes, but also in living cells expressing a fluorescent protein [4]. HRP beam also visualized finer structures of microtubules in fixed cells. Here, we will discuss these improvements and future application on the basis of our recent data.jmicro;63/suppl_1/i7/DFU087F1F1DFU087F1Fig. 1."in vivo" imaging of living mouse brain (H-line).

Point spread function analysis with saturable and reverse saturable scattering.

Nonlinear plasmonics has attracted a lot of interests due to its wide applications. Recently, we demonstrated saturation and reverse saturation of scattering from a single plasmonic nanoparticle, which exhibits extremely narrow side lobes and central peaks in scattering images [ACS Photonics 1(1), 32 (2014)]. It is desirable to extract the reversed saturated part to further enhance optical resolution. However, such separation is not possible with conventional confocal microscope. Here we combine reverse saturable scattering and saturated excitation (SAX) microscopy. With quantitative analyses of amplitude and phase of SAX signals, unexpectedly high-order nonlinearities are revealed. Our result provides greatly reduced width in point spread function of scattering-based optical microscopy. It will find applications in not only nonlinear material analysis, but also high-resolution biomedical microscopy.

Glucagon-like peptide 1 and peptide YY are in separate storage organelles in enteroendocrine cells.

A sub-group of enteroendocrine cells (L cells) release gastrointestinal hormones, GLP-1 and PYY, which have different but overlapping physiological effects, in response to intraluminal nutrients. Whilst their release profiles are not identical, how the plasma levels of these two hormones are differentially regulated is not well understood. We investigate the possibility that GLP-1 and PYY are in separate storage vesicles. In this study, the subcellular location of GLP-1 and PYY storage organelles is investigated using double-labelling immunohistochemistry, super resolution microscopy and high-resolution confocal microscopy. In all species tested, human, pig, rat and mouse, most cytoplasmic stores that exhibited GLP-1 or PYY immunofluorescence were distinct from each other. The volume occupancy, determined by 3D analysis, overlapped by only about 10∼20%. At the lower resolution achieved by conventional confocal microscopy, there was also evidence of GLP-1 and PYY being in separate storage compartments but, in subcellular regions where there were many storage vesicles, separate storage could not be resolved. The results indicate that different storage vesicles in L cells contain predominantly GLP-1 or predominantly PYY. Whether GLP-1 and PYY storage vesicles are selectively mobilised and their products are selectively released needs to be determined.

Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405nm, 488nm, 555nm, and 639nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.

Nanoscopic spine localization of Norbin, an mGluR5 accessory protein.

BackgroundNorbin is a neuron-specific, cytosolic protein that interacts with the metabotropic glutamate receptor 5 (mGluR5) and has a profound impact on mGluR5 signaling. Yet, little is known about its synaptic distribution.ResultsHere we have analyzed the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), actin and mGluR5 in spines using super-resolution microscopy. Norbin was found to have a high degree of colocalization with actin and a lower degree of colocalization with PSD-95. Co-immunoprecipitation studies confirmed that interaction occurs between Norbin and actin, but not between Norbin and PSD-95. Norbin was also found to have a high degree of colocalization with the perisynaptically located mGluR5. Findings based on structured illumination microscopy (3D-SIM) of exogenous expressed Norbin-GFP were confirmed by stimulated emission depletion microscopy (STED) of immunolabeled endogenous Norbin.ConclusionsNorbin associates with actin rather than with PSD-95 in dendritic spines. Results regarding protein localization and colocalization performed with conventional confocal microscopy must be interpreted with great caution. The now available super-resolution microscopy techniques provide more accurate information about sub-cellular protein localization than previously was possible.

Focus on photonics and biomedical optics

Focus on photonics and biomedical optics

Microsphere-Coupled Scanning Laser Confocal Nanoscope for Sub-Diffraction-Limited Imaging at 25 nm Lateral Resolution in the Visible Spectrum

We report a direct optical super-resolution imaging approach with 25 nm (∼ λ/17) lateral resolution under 408 nm wavelength illumination by combining fused silica and polystyrene microspheres with a conventional scanning laser confocal microscope (SLCM). The microsphere deposited on the target surface generates a nanoscale central lobe illuminating a sub-diffraction-limited cross-section located on the target surface. The SLCM confocal pinhole isolates the reflected light from the near-field subdiffractive cross-section and suppresses the noises from the side lobe and the far-field paraxial focal point. The structural detail of the subdiffractive cross-section is therefore captured, and the 2D target surface near the bottom of microspheres can be imaged by intensity-based point scanning.

Confocal supercritical angle microscopy for cell membrane imaging

We demonstrate subwavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fluorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel. Confocal-supercritical angular fluorescence microscopy would be a powerful tool to achieve high-resolution surface imaging, especially for membrane imaging in biological samples.

Imaging Through the Pupal Case of <em>Drosophila melanogaster</em>

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of <em>Drosophila melanogaster</em>

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Focal Adhesion Axial Topography by the Z-Phasor Approach in Confocal Microscopy

The protein lateral and axial organization within focal adhesions has been studied by state of the art super resolution methods due to its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation requiring sample fixation that limits their application. In this report we present a phasor-based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that when about a total of 4000 photon counts are collected along a z-scan, axial localization precision close to 10 nm is achievable. We show at 10nm resolution that axial localization of paxillin, FAK and talin is similar at different focal adhesion sites, while F-actin shows a sharp increase in height towards the cell center. We further demonstrated that using line scans we can obtain axial resolution of 10 nm with a temporal resolution of less than a second of the changes of topography along the line. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.

Superresolution imaging of DNA tetrahedral nanostructures in cells by STED method with continuous wave lasers

DNA tetrahedral nanostructures are considered to be new nanocarriers because they can be precisely controlled and hold excellent penetration ability to the cellular membrane. Although the DNA tetrahedral nanostructure is extensively studied in biology and medicine, its behavior in the cells with nanoscale resolution is not understood clearly. In this letter, we demonstrate superresolution fluorescence imaging of the distribution of DNA tetrahedral nanostructures in the cell with a simulated emission depletion (STED) microscope, which is built based on a conventional confocal microscope and can provide a resolution of 70 nm.

Ultra Fast Raman Hyperspectral Imaging using Bragg Tunable Filters and a High Performance Emccd Camera

Because of its high specificity to a variety of molecular processes and its low sensitivity to the presence of water, Raman hyperspectral imaging is regarded as a very promising technique to help pathologists improve the accuracy of medical diagnostics when compared to conventional histopathological analysis. However, since on average approximately one photon per million undergoes Raman scattering, acquisition time per hyperspectral image is very long, typically of about 6 hours. This significantly reduces the appeal of this technique for ex-vivo diagnostics and makes in-vivo applications impracticable. To increase acquisition speed, a Raman hyperspectral imager based on holographic Bragg tunable filters was used and images of carbon nanotubes could be acquired 30 times faster than with a conventional confocal microscope optimised for fast mapping. This speed gain over traditional methods was further enhanced when also using a low-noise EMCCD camera, resulting in measurements performed 150 times faster. These results thus indicate that in vivo and ex vivo applications of wide-field Raman hyperspectral imaging are now at reach, hence paving the way for real time tumor detection during surgery.

Derivation of analytical coherent transfer function for a confocal microscope with arbitrary Toraldo filter

The Toraldo filter is one of the most widely investigated and used pupil filters for super-resolution focusing and imaging in both conventional wide-field microscopy and confocal scanning microscopy. The analytical coherent transfer function is therefore derived from convolution decomposition of a confocal microscope with arbitrary Toraldo filter. It is demonstrated using two typical examples that the mechanism of super-resolution or resolution improvement can be easily evaluated without numerical convolution calculation.

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.

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined.

Extraordinary Transmission‐based Plasmonic Nanoarrays for Axially Super‐Resolved Cell Imaging

Extraordinary transmission based axial imaging (EOT‐AIM) for cell microscopy is reported. EOT‐AIM uses linear arrays of nanoapertures, each of which samples target fluorescence up to a preset axial distance from surface, in combination with wide‐field microscopy for acquisition of lateral images. Current design of nanoapertures provides EOT‐AIM with axial super‐resolution that is as small as 20 nm for a depth range of 500 nm. Experiments were performed for the measurement of the axial distribution of ganglioside in mouse macrophage (RAW264.7) cells using FITC‐conjugated cholera toxin subunit B. The results were successfully confirmed with conventional confocal and total internal reflection fluorescence microscopy.

Plasmon-Assisted Super-Resolution Axial Distance Sensitivity in Fluorescence Cell Imaging

There is currently a great need to develop live-cell compatible optical microscopy tools that can provide super-resolution information on biomolecules, in particular for the study of membrane receptors. We present a novel imaging technique, which employs a nanoplasmonic substrate in combination with conventional confocal fluorescence lifetime microscopy, to deliver an axial position sensitivity of order 10 nm in whole cell imaging. The technique exploits the Purcell effect experienced by fluorescent molecules in the vicinity of noble metal nanoparticles, leading to a reduction of the radiative lifetime and a commensurate increase in fluorescence intensity. We employ this technique to map the topography of the cellular membrane, by imaging the fluorescent protein eGFP labeled to the receptor CXCR4, and further investigate receptor-mediated endocytosis in carcinoma cells. These results demonstrate a new approach in biological cell imaging, using bespoke plasmonic nanostructures to provide axial super-resolution sensitivity, while retaining compatibility with conventional fluorescence microscopy techniques.

Imaging the Effects of Prostaglandin Analogues on Cultured Trabecular Meshwork Cells by Coherent Anti-Stokes Raman Scattering

The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 μg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 μg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo.

Monitoring Tectal Neuronal Activities and Motor Behavior in Zebrafish Larvae

To understand how visuomotor behaviors are controlled by the nervous system, it is necessary to monitor the activity of large populations of neurons with single-cell resolution over a large area of the brain in a relatively simple, behaving organism. The zebrafish larva, a small lower vertebrate with transparent skin, serves as an excellent model for this purpose. Immediately after the larva hatches, it needs to catch prey and avoid predators. This strong evolutionary pressure leads to the rapid development of functional sensory systems, particularly vision. By 5 d postfertilization (dpf), tectal cells show distinct visually evoked patterns of activation, and the larvae are able to perform a variety of visuomotor behaviors. During the early larval stage, zebrafish breathe mainly through the skin and can be restrained under the microscope using a drop of low-melting-point agarose, without the use of anesthetics. Moreover, the transparency of the skin, the small diameter of the neurons (4-5 µm), and the high-neuronal density enable the use of in vivo noninvasive imaging techniques to monitor neuronal activities of up to ∼500 cells within the central nervous system, still with single-cell resolution. This article describes a method for simultaneously monitoring spontaneous and visually evoked activities of large populations of neurons in the optic tectum of the zebrafish larva, using a synthetic calcium dye (Oregon Green BAPTA-1 AM) and a conventional confocal or two-photon scanning fluorescence microscope, together with a method for measuring the tail motor behavior of the head-immobilized zebrafish larva.