Two-Photon Laser Scanning Confocal Microscopy

1997 ◽  
Vol 3 (S2) ◽  
pp. 847-848
Author(s):  
P.C. Cheng ◽  
S.J. Pan ◽  
A. Shih ◽  
W.S. Liou ◽  
M.S. Park ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Linear Absorption ◽  
Excitation Light ◽  
Excitation Beam ◽  
Scanning Confocal Microscopy ◽  
Two Photon ◽  
Photobleaching Recovery

Two-photon fluorescence microscopy has become an important research tool in both biological and material sciences. The technique uses long wavelength, typically in the near IR, as the excitation light to obtain shorter wavelength fluorescence (e.g. visible light). Because of the low linear absorption coefficient of most biological and polymeric specimens, this technique allows deeper penetration of the excitation beam, achieving optical sectioning to a depth of 250μm or more into the specimen. As a result of the quadratic dependency of the two-photon induced fluorescence to the excitation intensity, the fluorescent emission and photobleaching are limited to the vicinity of focal spot. This capability of addressing a specimen’s 3D space allows exciting possibilities in biological researches, such as 3D photobleaching recovery experiment.Two-photon confocal fluorescence microscopy is ideal for the study of thick biological and material specimen in 3D. For example, Figure 1 shows a three-dimensional isosurface rendered image of a vascular bundle from a maize stem.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

1993 ◽  
Vol 51 ◽  
pp. 562-563
Author(s):  
Hakan Ancin
Keyword(s):  
Laser Scanning ◽  
Population Analysis ◽  
Three Dimensional ◽  
Large Data ◽  
Laser Scanning Confocal Microscopy ◽  
Tissue Slices ◽  
Scanning Confocal Microscopy ◽  
Tissue Sections ◽  
Spatially Adaptive ◽  
Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

scholarly journals Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Drug Delivery Systems ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Confocal Microscopy ◽  
Delivery Systems ◽  
Scanning Confocal Microscopy ◽  
Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

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

scholarly journals Three-Dimensional Nano-Morphology of Carbon Nanotube/Epoxy Filled Poly(methyl methacrylate) Microcapsules

Materials ◽  
2019 ◽  
Vol 12 (9) ◽  
pp. 1387 ◽  
Author(s):  
M. Galip Icduygu ◽  
Meltem Asilturk ◽  
M. Akif Yalcinkaya ◽  
Youssef K. Hamidi ◽  
M. Cengiz Altan
Keyword(s):  
Confocal Microscopy ◽  
Methyl Methacrylate ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Poly Methyl Methacrylate ◽  
Scanning Confocal Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Average Size

The three-dimensional nano-morphology of poly(methyl methacrylate; PMMA) microcapsules filled with carbon nanotubes (CNTs) and epoxy resin were investigated by various microscopy methods, including a novel, laser scanning confocal microscopy (LSCM) method. Initially, PMMA microcapsules containing various amounts of CNTs were synthesized by a solvent evaporation method. Scanning electron microscopy analysis showed that pore-free, smooth-surface microcapsules formed with various types of core-shell morphologies. The average size of CNT/epoxy/PMMA microcapsules was shown to decrease from ~52 μm to ~15 μm when mixing speed during synthesis increased from 300 rpm to 1000 rpm. In general, the presence of CNTs resulted in slightly larger microcapsules and higher variations in size. Moreover, three-dimensional scans obtained from confocal microscopy revealed that higher CNT content increased the occurrence and size of CNT aggregates inside the microcapsules. Entrapped submicron air bubbles were also observed inside most microcapsules, particularly within those with higher CNT content.

Some possibilities of representing microcirculation in human spleen

Biologia ◽  
2009 ◽  
Vol 64 (6) ◽  
Author(s):  
Paulína Gálfiová ◽  
Ivan Varga ◽  
Martin Kopáni ◽  
Peter Michalka ◽  
Jana Michalková ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
The Other ◽  
Corrosion Casts ◽  
Histochemical Analysis ◽  
Human Spleen ◽  
Scanning Confocal Microscopy ◽  
Scanning Electron

AbstractThe representation of microcirculation can be approached in several ways. One of the possibilities is to represent the endothelium (endothelial or sinus lining cells) and their basement membrane on the basis of detecting the known components and the expression of the surface antigenes by the methods of immuno-, enzyme- or lectino-histochemical analysis, or by staining or impregnation histological methods. The other possibility is the examination of samples by transmission and scanning electron microscopy. For three-dimensional demonstration corrosion casts techniques or laser scanning confocal microscopy can be used. In this paper we describe the survey of immuno-, enzyme- and lectino-histochemical characteristics of selected components of microcirculation and our own results of its demonstration in human spleen.

Characterization Of Micropipes And Other Defect Structures In 6H-SiC Through Fluorescence Microscopy

1995 ◽  
Vol 406 ◽  
Author(s):  
W. D. Vetter ◽  
M. Dudley ◽  
T.- F. Wong ◽  
J. T. Frderich
Keyword(s):  
Silicon Carbide ◽  
Fluorescence Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Defect Structures ◽  
X Ray ◽  
Scanning Confocal Microscopy ◽  
Low Viscosity ◽  
Dye Staining

AbstractCrystals of silicon carbide, and other polytypic materials often have micropipes associated with screw dislocations of large Burgers vectors running along their axial dimensions. These defects are considered the most deleterious to the performance of SiC semiconductor devices.Optical micrographs of micropipes in silicon carbide crystals are ordinarily faint. To obtain micrographs showing higher contrast and detail, laser scanning confocal microscopy (LSCM) and simple fluorescence microscopy were used on 6H-SiC single crystals after infiltrating them with a low-viscosity epoxy containing a fluorescent dye. “Staining” the micropipes rendered them much more visible both in fluorescence and conventional optical microscopies. Details of their structures and shapes were revealed, and their dimensions were measured accurately, using LSCM and other, less sophisticated, fluorescence microscopies. Other voids present, such as microcracks, were also visualized. Observations by this optical technique were related to information obtained by synchrotron white beam x-ray topography.

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