Optical Sectioning Capacity Research Articles

Single nephron glomerular filtration rate measured by linescan multiphoton microscopy compared to conventional micropuncture

Renal micropuncture, which requires the direct access to the renal tubules, has for long time been the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models. This approach is challenging by virtue of complex animal preparation and numerous technically difficult steps. The introduction of intravital multiphoton microscopy (MPM) offers another approach to the measure of the SNGFR by mean of the high laser-tissue penetration and the optical sectioning capacity. Previous MPM studies measuring SNGFR in vivo relied on fast full-frame acquisition during the filtration process obtainable with high performance resonant scanners. In this study, we describe an innovative linescan–based MPM method. The new method can discriminate SNGFR variations both in conditions of low and high glomerular filtration, and shows results comparable to conventional micropuncture both for rats and mice. Moreover, this novel approach has improved spatial and time resolution and is faster than previous methods, thus enabling the investigation of SNGFR from more tubules and improving options for data-analysis.

MO429A NOVEL METHOD LINESCAN-DRIVEN FOR SNGFR MEASUREMENTS AS ALTERNATIVE TO HIGH FULL FRAME MULTIPHOTON MICROSCOPY ACQUISITION

Abstract Background and Aims Renal micropuncture, which requires the direct access to the renal tubules, has been for long time the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models, but this approach is challenging by virtue of complex animal preparation and numerous careful steps. The introduction of intravital multiphoton microscopy (MPM) permitted to improve the study of renal functions exploiting the high laser penetration and the optical sectioning capacity. Previous MPM studies measuring in vivo the SNGFR relied on fast full frame acquisition during the filtration process obtainable with microscope resonant scanners, which represent optional expensive equipment able to reach very high acquisition speed. In this work we propose an innovative linescan-based MPM method to calculate SNGFR in rodents doable without using the fast acquisition rate offered by resonant scanners. Method An in vivo MPM approach was used to measure the SNGFR in control Munich Wistar Frömter rats (MWF) and to test the feasibility of the innovative linescan approach. In order to validate this method in conditions known for reduced and increased SNGFR, it was applied to ischemia reperfusion injury (IRI) and low-dose dopamine treated conditions, respectively. Results The glomeruli connected to S1 proximal tubules extending at least 100 μm from the exit of the Bowman’s space were chosen for the measurement. A linescan path starting from the urinary pole and crossing many times the tubular lumen orthogonally to the cellular wall was hand drawn. The linescan was acquired soon after a i.v. bolus of low-molecular weight fluorescent marker was injected. The tubular length, the mean diameter and the transit time of the fluorescent marker within two lines of interest (called cross1 and cross2) were measured to obtain the SNGFR. SNGFR measured in control rats was comparable with previous reported data both at MPM and micropuncture. Significantly higher values compared to control were obtained in 3 μg/kg/min dopamine-treated rats. In IRI-treated rats the SNGFR was reduced about 35% compared to the controls. Conclusion The results achieved with our linescan method were quite similar to those obtained with conventional micropuncture, suggesting that the two methods overlap for the normal, dopamine and IRI treatment. Our results show that linescan approach is a promising and cheap alternative to the fast full frame acquisition for the investigation of SNGFR in health and disease, offering results comparable to conventional micropuncture with unprecedent temporal resolution.

Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that—in many aspects—recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that—unlike two- or three-lens light-sheet microscopes—it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Low-coherent optical diffraction tomography by angle-scanning illumination.

Temporally low-coherent optical diffraction tomography (ODT) is proposed and demonstrated based on angle-scanning Mach-Zehnder interferometry. Using a digital micromirror device based on diffractive tilting, the full-field interference of incoherent light is successfully maintained during every angle-scanning sequences. Further, current ODT reconstruction principles for temporally incoherent illuminations are thoroughly reviewed and developed. Several limitations of incoherent illumination are also discussed, such as the nondispersive assumption, optical sectioning capacity and illumination angle limitation. Using the proposed setup and reconstruction algorithms, low-coherent ODT imaging of plastic microspheres, human red blood cells and rat pheochromocytoma cells is experimentally demonstrated.

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Multimicroscopic study of curcumin effect on fixed nonmalignant and cancerous mammalian epithelial cells

The morphology changes, in particular the organization of microtubules in mammalian nonmalignant HMEC 184A1 and cancerous MCF-7 cells during curcumin treatment have been investigated by utilizing multiphoton, fluorescence, and atomic force (AFM) microscopies. Fluorescence microscopy reveals formation of ring-like structures of microtubules circumscribing the nuclear area in HMEC 184A1 cells after treatment, while in MCF-7 cells, no important changes were observed. Topography analyses of fixed HMEC 184A1 and MCF-7 before and after treatment with curcumin were performed using AFM and the effect of the employed cells' fixation method was investigated on MCF-7 cells. Due to its indepth optical sectioning capacity multiphoton microscopy provided valuable complementary information on curcumin's effect on both cells' types. Combining information provided by AFM and optical fluorescence and biphoton microscopes allows us to gain a better understanding of the cells and their curcumin-induced changes, especially for microtubules which are the main target of antitumor chemotherapy treatments. Our multimicroscopic study demonstrates that 6 h incubation with curcumin does not induce significant modifications in the interphase microtubules in the malignant MCF7cell, whereas it has measurable effects on those of the nonmalignant HMEC 184A1 cells, revealing also morphology modifications over the nuclear area of these cells.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Digital slicing of 3D scenes by Fourier filtering of integral images

We present a novel technique to extract depth information from 3D scenes recorded using an Integral Imaging system. The technique exploits the periodic structure of the recorded integral image to implement a Fourier-domain filtering algorithm. A proper projection of the filtered integral image permits reconstruction of different planes that constitute the 3D scene. The main feature of our method is that the Fourier-domain filtering allows the reduction of out-of-focus information, providing the InI system with real optical sectioning capacity.

Quasi-spherical focal spot in two-photon scanning microscopy by three-ring apodization

We present a beam-shaping technique for two-photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three-ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi-spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot-fluorescence efficiency.

Optical-sectioning improvement in two-color excitation scanning microscopy.

We present a new beam-shaping technique for two-color excitation fluorescence microscopy. We show that by simply inserting a properly designed shaded-ring filter in the illumination beam of smaller wavelength, it is possible to improve the effective optical sectioning capacity of such microscopes by 23%. Such an improvement is obtained at the expense of only a very small increasing of the overall energy in the point-spread-function sidelobes. The performance of this technique is illustrated by a numerical imaging simulation.

Axial gain resolution in optical sectioning fluorescence microscopy by shaded-ring filters

We present a new family of pupil masks to control the axial component of the intensity distribution in the focal region of tightly focused light fields. The filters, which consist of a circular clear pupil with a single shaded ring, allow to control the width of the central lobe of the axial spot together with the residual sidelobes energy. The filters can be applied to improve the optical sectioning capacity of different scanning microscopes.

Optical sectioning by two-pinhole confocal fluorescence microscopy

A two-pinhole axially superresolving confocal fluorescence imaging system is presented. Based on the concept of subtractive incoherent imaging, the system described here is equipped with a zero-focus complex-transmittance pupil filter in one of the collector paths. The optical sectioning capacity of the system is 25% superior to that of a free-pupil one-pinhole instrument.

Tunable optical sectioning in confocal microscopy by use of symmetrical defocusing and apodization

We present two novel optical methods to achieve a significative improvement in the optical-sectioning capacity of confocal scanning microscopes. The techniques, whose real power is the simplicity with which they can be implemented, consist of a suitable combination of symmetrical defocusing with two different manners of apodizing both parts of the confocal architecture. It is shown that the proposed techniques are useful in both the bright-field and the fluorescence modes and for reflection and transmission geometries.

From two dimensional (2D) to three dimensional (3D) analysis by confocal microscopy.

The confocal microscope is becoming increasingly important as an apparatus to analyze the 3-D topography of the cell. Main reasons are the high resolution optical sectioning capacity, the non-invasiveness which leaves the object intact, and the imaging capabilities. This chapter introduces a description of the confocal principle, the basic concepts of confocal fluorescence microscopy and some criteria for cell preservation. Optimization of in situ immunofluorescence, hybridization and detection procedures in combination with new digital microscope techniques can fully express their capacities only if the preparation of biological specimens is accurate for 3-D analysis. Some applications of confocal microscopy to the study of intranucleolar antigens, enzyme translocations and fluorescence in situ hybridization, are described in association with 3-D software image processing, as a useful framework for the study of the 3-D visualization of proteins and chromatin domains.