Conventional Confocal Microscopy Research Articles

Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart

Background & aimsTwo-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging. MethodsDiscrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy. ResultsFluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation. ConclusionOur results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties.

3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy

Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy.

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

Scattered Light Imaging Enables Real-Time Monitoring of Label-Free Nanoparticles and Fluorescent Biomolecules in Live Cells.

Simultaneously monitoring label-free nanoparticles (NPs) and fluorescent biomolecules inside the live cell in real time is challenging because both imaging methods require different instrumentation and measuring principles. Here we report a novel scattered light imaging (SLi) technique that allows label-free NPs to be monitored using a conventional confocal microscope. The method shows ahigh spatial resolution and can distinguish label-free silver nanoparticles (AgNPs) with a 10 nm size difference in live cells. We performed SLi to observe the uptake, movement, distribution, and transformation of AgNPs in live cells at a single-particle level. The method is applicable to accurately track the localization of a variety of nanomaterials inside the cell. With this approach, label-free NP and fluorescent-labeled biomolecules are imaged simultaneously making it possible to real-time monitor nanobio interactions.

Area scanning method for 3D surface profilometry based on an adaptive confocal microscope

Conventional confocal 3D microscopes suffer from a slow measurement speed due to its requirement of scanning in three directions. Although lateral slit scanning has been proven to perform similarly to single point scanning, a direct area confocal scanning microscope is commonly considered impossible as the system is reduced into a wide-field microscope and loses its depth discerning capability. In this article, a direct area scanning method is proposed for 3D surface profilometry based on a tilted focal field. To demonstrate the underlying principle, theoretical analysis is conducted with simulation result, showing that depth discerning capability can be maintained in area scanning when the tilting angle is specifically chosen according to the numerical aperture of the system. An adaptive experimental setup is constructed based on a programmable light source and a programmable array microscope with chromatic axial encoding. Measurement result of a test target using the proposed method is comparable to the result of conventional array scanning. Although the axial uncertainty is increased by a factor of approximately 2.5, the direct area scanning method is more than 300 times faster than the conventional array scanning mode of the same setup.

Extension of longitudinal measuring range of a confocal surface profilometer using a gradient-intensity probe beam

A confocal surface profilometer with a long vertical range was developed by using a gradient-intensity probe beam. In conventional confocal microscopy, vertical scanning of a tested surface by either stage height movement or the shifting of objective focus is time-consuming, thus making unacceptable measurement efficiency for in situ inspection. To overcome this, in this research, a quasi-Bessel beams (QBB) with the properties of narrow lateral spot and gradient-intensity axial distribution is adopted as a probe beam to generate an accurate intensity-to-height conversion for 3-D profile measurement. The influences of the incident Gaussian beam and the real axicon lens with an oblate-tip shape on the axial probe beam are calculated and analyzed. By switching the axicon lenses with different base angles, the measurable height of the surface profilometer can be ranged from a few millimeters to several hundred millimeters. According to the measured results, it was found that by using the axicon lens with a base angle of 20°, the measurable height reached at least 2 mm. By integrating a high-speed acquisition unit, the proposed profilometer can further increase the measurement efficiency, especially fitting in situ automatic optical inspection (AOI).

Super-resolution fluorescence imaging of carbon nanotubes using a nonlinear excitonic process.

Highly efficient exciton-exciton annihilation process unique to one-dimensional systems is utilized for super-resolution imaging of air-suspended carbon nanotubes. Through the comparison of fluorescence signals in linear and sublinear regimes at different excitation powers, we extract the efficiency of the annihilation processes using conventional confocal microscopy. Spatial images of the annihilation rate of the excitons have resolution beyond the diffraction limit. We investigate excitation power dependence of the annihilation processes by experiment and Monte Carlo simulation, and the resolution improvement of the annihilation images can be quantitatively explained by the superlinearity of the annihilation process. We have also developed another method in which the cubic dependence of the annihilation rate on exciton density is utilized to achieve further sharpening of single nanotube images.

Rapid Determination of the Distribution of Cellulose Nanomaterial Aggregates in Composites Enabled by Multi-Channel Spectral Confocal Microscopy.

There is increased interest in the use of cellulose nanomaterials for the mechanical reinforcement of composites due to their high stiffness and strength. However, challenges remain in accurately determining their distribution within composite microstructures. We report the use of a range of techniques used to image aggregates of cellulose nanocrystals (CNCs) greater than 10 µm2 within a model thermoplastic polymer. While Raman imaging accurately determines CNC aggregate size, it requires extended periods of analysis and the limited observable area results in poor reproducibility. In contrast, staining the CNCs with a fluorophore enables rapid acquisition with high reproducibility, but overestimates the aggregate size as CNC content increases. Multi-channel spectral confocal laser scanning microscopy is presented as an alternative technique that combines the accuracy of Raman imaging with the speed and reproducibility of conventional confocal laser scanning microscopy, enabling the rapid determination of CNC aggregate distribution within composites.

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample's refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Optical Characterization of Zinc Pyrithione.

Zinc pyrithione is ubiquitous in commercial products particularly antidandruff shampoos. For the efficacy of zinc pyrithione therapeutic cleansers to be assessed accurately, the distribution of particles on the scalp needs to be visualized. Currently, no technique is available which provides the chemical specificity and sensitivity required. Here, we report application of fluorescence-lifetime imaging microscopy (FLIM) for high-contrast mapping of zinc pyrithione distribution on the scalp. Characterization of the zinc pyrithione emission by using both one-photon excitation at five specific wavelengths and two-photon excitation in the range of 740-820 nm revealed its FLIM fingerprint-a characteristic short average time-weighted emission lifetime of ΤZnPT = 250 ps. Bandpass-filtering FLIM signals at ΤZnPT enabled an efficient discrimination between the zinc pyrithione and major endogenous skin species in comparison with that of the conventional reflectance confocal microscopy. Our findings provide means for in vivo high-sensitivity assaying and high-contrast imaging of zinc pyrithione in biological systems.

Whole-brain block-face serial microscopy tomography at subcellular resolution using FAST.

Here, we describe an optimized and detailed protocol for block-face serial microscopy tomography (FAST). FAST enables high-speed serial section fluorescence imaging of fixed brains at an axonal spatial resolution and subsequent image data processing. It renders brain-wide anatomical and functional analyses, including structural profiling of nuclear-stained brain at the single-cell level, cell-type-specific mapping with reporter animal brains and neuronal tracing with anterograde/retrograde labeling. Light-sheet fluorescence microscopy of cleared brains is advantageous in regard to imaging speed, but its spatial resolution is generally limited, whereas the opposite is true for conventional confocal microscopy. FAST offers a solution to overcome these technical limitations. This protocol describes detailed procedures for assembling the FAST hardware, sample preparation, imaging and image processing. A single imaging session takes as little as 2.4 h per mouse brain, and sample preparation requires 1 to several days, depending on pretreatments; however, multiple samples can be prepared simultaneously. We anticipate that FAST will contribute to unbiased and hypothesis-free approaches for a better understanding of brain systems.

Measuring Hindered Diffusion Dynamics in Live Cell Plasma Membranes with Confocal and Super-Resolution Imaging

The cellular plasma membrane is a highly heterogeneous structure organised on nano-scales and displays a crucial interaction platform for proteins, lipids and soluble ligands. Investigating the molecular membrane organisation by measuring diffusion dynamics offers a better understanding of its biological function. Fluorescence correlation spectroscopy (FCS) is one of the prominent tools to elucidate these dynamics in living cells but can only report on the dynamics at one given spatial position at a time. Using scanning fluorescence correlation spectroscopy (sFCS), we obtain a multitude of FCS measurements at different spatial locations. Here, we present a statistical analysis pipeline for sFCS data which allows for the accurate determination of the diffusion dynamics and the differentiation of free (Brownian) from hindered (non-Brownian) diffusion modes. We show free diffusion for phospholipids in model membranes and cells but reveal hindered diffusion of sphingolipids and GPI-anchored proteins in cells. Notably, these measurements can be performed using standard fluorescent dyes or proteins on a conventional confocal laser scanning microscope. To further investigate the dynamics on single cell level, we combine sFCS with stimulated emission depletion (STED) microscopy and by alternating conventional and super-resolved excitation we introduce line interleaved excitation scanning STED-FCS (LIESS-FCS). With LIESS-FCS the diffusion modes can be directly determined at multiple spots within the cellular plasma membrane providing detailed insights into organisation and function. Overall we are presenting a novel toolkit to investigate nano-scale molecular diffusion dynamics for shedding a new light on membrane organisation and heterogeneity.

<p class="Body">The anal secretory apparatus of Eriophyoidea and description of Phyllocoptes bilobospinosus n. sp. (Acariformes: Eriophyidae) from Tamarix (Tamaricaceae) from Ukraine, Crimea and USA

A new vagrant phyllocoptine species, Phyllocoptes bilobospinosus n. sp. (Eriophyidae, Phyllocoptinae), found on tamarisks (Tamarix tetrandra Pallas, T. smyrnensis Bunge, T. ramossisima Ledeb) in Donbass (Ukraine), Crimea, and USA is described based on conventional light microscopy and confocal laser scanning microscopy. Apart from two distinct areas of ventral cuticle bearing large, spike-like microtubercles, the new species possesses a thin translucent supracapitular plate (situated below the frontal lobe of the prodorsal shield), a short longitudinal ventral ridge anterior to the anal lobes, and unusual internal tube-like structures associated with the rectum. Careful examination of purposefully made slide mounts of partially cleared specimens revealed that adults of P. bilobospinosus possess a complex of structures associated with the rectum, including a hypertrophied, four-lobed putative anal gland and four thin tubes connected with a rectal sac. Similar tubular structures previously described in aberoptine mites of the genus Aberoptus from Brazilian Cesalpiniaceae are discussed. The synonymy of genera Aberoptus Keifer and Aceria Keifer is rejected and a new combination, Aberoptus inusitatus (Britto & Navia (in Britto et al. 2008)) n. comb., is proposed. A brief review of the anal glands of Eriophyoidea is given, including a discussion on homology and the variety of forms of the anal secretory apparatus among eriophyoid genera. Further research is needed on the anatomy of anal glands in Eriophyoidea, including transmission electron microscopy based histological analyses and additional studies of eriophyoids with well-developed secretory structures associated with the rectum. These methods will lead to a much better understanding of the evolution and homology of the anal secretory apparatus, which may render it useful for future phylogenetic studies.

Intravital Confocal Microscopy of Dermal Innate Immune Responses to Flea-Transmitted Yersinia pestis.

The technique known as intravital microscopy (IVM), when used in conjunction with transgenic mice expressing fluorescent proteins in various cell populations, is a powerful tool with the potential to provide new insights into host-pathogen interactions in infectious disease pathogenesis in vivo. Yersinia pestis, the causative agent of plague, is typically deposited in a host's skin during feeding of an infected flea. IVM has been used to characterize the innate immune response to Y. pestis in the skin and identify differences between the responses to needle-inoculated and flea-transmitted bacteria that would have been difficult, if not impossible, to detect by other means. Here we describe techniques used to image the neutrophil response to flea-transmitted Y. pestis in the dermis of live mice using conventional confocal microscopy.

Cell-Specific Markers for the Identification of Retinal Cells and Subcellular Organelles by Immunofluorescence Microscopy.

Identification of specific cells and subcellular structures in the retina is fundamental for understanding the visual process, retinal development, disease progression, and therapeutic intervention. The increased use of knockout, transgenic, and naturally occurring mutant mice has further underlined the need for retinal cell-specific imaging. Immunofluorescence microscopy of retinal cryosections and whole-mount tissue labeled with cell-specific markers has emerged as the method of choice for identifying and quantifying specific cell populations and mapping their distribution within the retina. Immunofluorescence microscopy has also been important in localizing proteins to specific compartments of retinal cells. In most cases indirect labeling methods are employed in which lightly fixed retinal samples are first labeled with a primary antibody targeted against a cell-specific protein of interest and then labeled with a fluorescent dye-tagged secondary antibody that recognizes the primary antibody. The localization and relative abundance of the protein can be readily imaged under a conventional fluorescent or confocal scanning microscope. Immunofluorescence labeling can be adapted for imaging more than one antigen through the use of multiple antibodies and different, non-overlapping fluorescent dyes. A number of well-characterized immunochemical markers are now available for detecting photoreceptors, bipolar cells, amacrine cells, horizontal cells, Müller cells, and retinal pigment epithelial cells in the retina of mice and other mammals. Immunochemical markers are also available for visualizing the distribution of specific proteins within cells with most studies directed toward photoreceptor cells.

Inline integration of offset MMF-capillary-MMF structure as a portable and compact fiber-optic surface-enhanced Raman scattering microfluidic chip.

A novel fiber-optic surface-enhanced Raman scattering (SERS) microfluidic chip integrated with an embedded Raman probe is presented and demonstrated. The Raman probe consists of the offset-multimode-fiber (MMF)-capillary-MMF (OMCM) structure and SERS substrate. The probe is embedded in the microfluidic channel to form a compact and portable chip. The chip is employed with a fiber coupler in an all-fiber detection system, which has a good stability compared to the free space focusing by the conventional confocal Raman microscope. The excitation light transmits from both ends of the OMCM probe into the capillary, and the generated Raman scattered signals are collected by two MMFs simultaneously. Experimental results of the Rhodamine 6G (R6G) detection show that the Raman signal intensity increases in a linear pattern at ∼1509 cm-1 with the increase of R6G concentrations. This kind of chip is compact, integrated, and miniaturized for the Raman signal detection. Furthermore, it can be fabricated easily in large quantities at cost, rendering promising applications.

In vivo label-free confocal imaging of the deep mouse brain with long-wavelength illumination.

Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1-micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.

Unilateral-shift-subtracting confocal microscopy with nanoscale axial focusing precision.

A novel unilateral-shift-subtracting confocal microscopy (USSCM) method with nanoscale axial focusing precision is proposed based on the optical arrangement of conventional confocal microscopy (CM). As the two segments of data on both sides of the confocal axial response curve are very sensitive to variations of the axial position, USSCM introduces an axial shift of S for one segment, to intersect it with the other segment. It then separately interpolates the two segments of intersecting data, subtracts the corresponding interpolated data, and selects the data that exhibit a good linearity from all of the subtracted data to fit a straight line. It calculates the zero position of the fitting line and offsets it by S/2, to precisely reveal the focus position of the confocal system, thereby achieving high-precision imaging of the three-dimensional sample's structure. Theoretical analyses and preliminary experiments indicate that, for excitation wavelength of λ=405 nm, numerical aperture of NA=0.95, and normalized axial shift of S=5.21, USSCM achieves an axial resolution of 3nm and a repetitive focusing precision of 1.5nm, while it does not change the lateral resolution of CM. Furthermore, compared with conventional CM, under the same noise condition, USSCM is less affected by system aberration, which leads to higher focusing precision. These findings demonstrate that USSCM is a very efficient method for imaging.

Loss of Tight Junction Protein Claudin 18 Promotes Progressive Neoplasia Development in Mouse Stomach

Loss of claudin 18 (CLDN18), a membrane-spanning tight junction protein, occurs during early stages of development of gastric cancer and associates with shorter survival times of patients. We investigated whether loss of CLDN18 occurs in mice that develop intraepithelial neoplasia with invasive glands due to infection with Helicobacter pylori, and whether loss is sufficient to promote the development of similar lesions in mice with or without H pylori infection. We performed immunohistochemical analyses in levels of CLDN18 in archived tissues from B6:129 mice infected with H pylori for 6 to 15 months. We analyzed gastric tissues from B6:129S5-Cldn18tm1Lex/Mmucd mice, in which the CLDN18 gene was disrupted in gastric tissues (CLDN18-knockout mice), or from control mice with a full-length CLDN18 gene (CLDN18+/+; B6:129S5/SvEvBrd) or heterozygous disruption of CLDN18 (CLDN18+/-; B6:129S5/SvEvBrd) that were infected with H pylori SS1 or PMSS1 at 6 weeks of age and tissues collected for analysis at 20 and 30 weeks after infection. Tissues from CLDN18-knockout mice and control mice with full-length CLDN18 gene expression were also analyzed without infection at 7 weeks and 2 years after birth. Tissues from control and CLDN18-knockout mice were analyzed by electron microscopy, stained by conventional methods and analyzed for histopathology, prepared by laser capture microdissection and analyzed by RNAseq, and immunostained for lineage markers, proliferation markers, and stem cell markers and analyzed by super-resolution or conventional confocal microscopy. CLDN18 had a basolateral rather than apical tight junction localization in gastric epithelial cells. B6:129 mice infected with H pylori, which developed intraepithelial neoplasia with invasive glands, had increasing levels of CLDN18 loss over time compared with uninfected mice. In B6:129 mice infected with H pylori compared with uninfected mice, CLDN18 was first lost from most gastric glands followed by disrupted and reduced expression in the gastric neck and in surface cells. Gastric tissues from CLDN18-knockout mice had low levels of inflammation but increased cell proliferation, expressed markers of intestinalized proliferative spasmolytic polypeptide-expressing metaplasia, and had defects in signaltransduction pathways including p53 and STAT signaling by 7 weeks after birth compared with full-length CLDN18 gene control mice. By 20 to 30 weeks after birth, gastric tissues from uninfected CLDN18-knockout mice developed intraepithelial neoplasia that invaded the submucosa; by 2 years, gastric tissues contained large and focally dysplastic polypoid tumors with invasive glands that invaded the serosa. H pylori infection of B6:129 mice reduced the expression of CLDN18 early in gastric cancer progression, similar to previous observations from human gastric tissues. CLDN18 regulates cell lineage differentiation and cellular signaling in mouse stomach; CLDN18-knockout mice develop intraepithelial neoplasia and then large and focally dysplastic polypoid tumors in the absence of H pylori infection.