Three-dimensional imaging of the living eye

1991 ◽  
Vol 49 ◽  
pp. 170-171 ◽  
Author(s):  
Barry R. Masters
Keyword(s):  
Laser Scanning ◽  
Water Immersion ◽  
Physiological State ◽  
Three Dimensional ◽  
Confocal Microscope ◽  
Image Stack ◽  
Rabbit Eye ◽  
Reflected Light

The structure of the in situ rabbit cornea can be observed at high resolution and contrast with reflected light confocal microscopy. In vivo confocal images of the living cornea have been made at lower resolution and lower contrast using a SIT video camera together with a real-time Nipkow disk confocal microscope adapted for in vivo observations. This paper describes the three dimensional reconstruction of the in situ cornea from an enucleated rabbit eye with confocal reflected light microscopy and volume rendering computer techniques.A laser scanning confocal microscope (BioRad MRC 600) was used in the reflected light mode to obtain the two-dimensional image stack from the cornea of a freshly enucleated rabbit eye. The eye was maintained in a physiological state with aerated Ringer's solution. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The optical sectioning was performed perpendicular to the optical axis of the eye globe.

Three-dimensional visualization of the eye

1992 ◽  
Vol 50 (1) ◽  
pp. 586-587
Author(s):  
Barry R. Masters
Keyword(s):  
High Resolution ◽  
Confocal Microscopy ◽  
Volume Rendering ◽  
Three Dimensional ◽  
Full Thickness ◽  
Rabbit Cornea ◽  
Rabbit Eye ◽  
Reflected Light ◽  
Dimensional Reconstruction

The structure of the in situ rabbit cornea can be observed at high resolution and contrast with reflected light confocal microscopy. This paper describes the three dimensional reconstruction of the in situ cornea from an enucleated rabbit eye with confocal reflected light microscopy and volume rendering computer techniques. This paper shows the 3-D reconstruction of the full thickness of the cornea based on cubic voxels.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

scholarly journals High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  
Keyword(s):  
Blood Flow ◽  
High Speed ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Fluorescence Signal ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

scholarly journals Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Robert J. Francis ◽  
Gillian Robb ◽  
Lee McCann ◽  
Bhagwati Khatri ◽  
James Keeble ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
Large Field ◽  
Staining Procedure ◽  
3D Analysis ◽  
Mycobacterium Tuberculosis Infection ◽  
In Vivo Models ◽  
Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

scholarly journals In Situ Vitrification of Lung Cancer Organoids on a Microwell Array

Micromachines ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 624
Author(s):  
Qiang Liu ◽  
Tian Zhao ◽  
Xianning Wang ◽  
Zhongyao Chen ◽  
Yawei Hu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Drug Sensitivity ◽  
Simple Procedure ◽  
Three Dimensional ◽  
Cancer Drug ◽  
Microwell Array ◽  
Sensitivity Tests ◽  
Anti Cancer

Three-dimensional cultured patient-derived cancer organoids (PDOs) represent a powerful tool for anti-cancer drug development due to their similarity to the in vivo tumor tissues. However, the culture and manipulation of PDOs is more difficult than 2D cultured cell lines due to the presence of the culture matrix and the 3D feature of the organoids. In our other study, we established a method for lung cancer organoid (LCO)-based drug sensitivity tests on the superhydrophobic microwell array chip (SMAR-chip). Here, we describe a novel in situ cryopreservation technology on the SMAR-chip to preserve the viability of the organoids for future drug sensitivity tests. We compared two cryopreservation approaches (slow freezing and vitrification) and demonstrated that vitrification performed better at preserving the viability of LCOs. Next, we developed a simple procedure for in situ cryopreservation and thawing of the LCOs on the SMAR-chip. We proved that the on-chip cryopreserved organoids can be recovered successfully and, more importantly, showing similar responses to anti-cancer drugs as the unfrozen controls. This in situ vitrification technology eliminated the harvesting and centrifugation steps in conventional cryopreservation, making the whole freeze–thaw process easier to perform and the preserved LCOs ready to be used for the subsequent drug sensitivity test.

Three-Dimensional Laser-Scanning Confocal Microscopy of In Situ Hybridization in the Skin

1994 ◽  
Vol 16 (1) ◽  
pp. 44-51 ◽  
Author(s):  
Stephen E. Mahoney ◽  
Stephen W. Paddock ◽  
Louis C. Smith ◽  
Dorothy E. Lewis ◽  
Madeleine Duvic
Keyword(s):  
In Situ Hybridization ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

Blind Deconvolution of Low and High Signal-to-noise 3-D Images of Fluorescent Subcellular Structures

1998 ◽  
Vol 4 (S2) ◽  
pp. 882-883
Author(s):  
B. Rajwa ◽  
J. Czyz ◽  
J. Dobrucki
Keyword(s):  
Biological Samples ◽  
Software Package ◽  
Blind Deconvolution ◽  
Water Immersion ◽  
Plant Cells ◽  
Confocal Microscope ◽  
High Signal ◽  
Signal To Noise ◽  
Reflected Light ◽  
Fluorescence Images

This report summarizes our experiences gained with deconvolving 3-d images of various types of biological samples - animal and plant cells; live and fixed. We compared raw, unmodified images with images processed with standard filtering methods and images improved by using deconvolution. The blind decovolution software package from AutoQuant, Inc. was used. All images were acquired using a BioRad MRC 1024 confocal microscope (attached to a Nikon Diaphot microscope), using fluorescence or reflected light. For thin (up to 25 μm) flat specimens a 60x PlanApo NA1.4 lens was used while for thick (25-200 μm) water containing samples a 40x Fluor NA1.15 water immersion lens was used.In an attempt to test the usefulness and validity of deconvolution for various types of biological samples we adopted the following approaches:1. collect images of structures that are known to be both fluorescent and reflective - compare the results of deconvolution of fluorescence images with reflected light images that are not deconvolved.

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