“Grid-mapped” freeze-fracture: Three-Dimensional confocal laser scanning microscopy for directed fracturing and histological mapping of neurons in spinal cord and brain

1993 ◽  
Vol 51 ◽  
pp. 64-65
Author(s):  
J. E. Rash ◽  
L. R. Whalen ◽  
P. B. Guthrie ◽  
M. Morita ◽  
R. Dillman ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Male Rats ◽  
Whole Body ◽  
Confocal Laser ◽  
Fracture Plane

A new correlative microscopic technique, “grid-mapped” freeze fracture, is introduced. This technique allows individual cells in histological slices to be freeze fractured, and their ultrastructural details to be correlated with conventional histological and gross anatomical features. Adult male rats were anesthetized, the sciatic nerve was exposed and crushed, and rhodaminefilled latex microspheres (Lumafluor, Inc) were injected at the crush site to label motor neurons. After 3-7 days, rats were fixed by whole-body perfusion. The brains and spinal cords were removed, embedded in 5% gelatin, and 50 or 100 μm thick slices were cut with a Vibratome. Slices were mapped in three-dimensions (Figs. 1-2 and 3-4) using a Molecular Dynamics Multiport 2001 confocal microscope, and the depths of selected cells were measured (±2μm) from the cut surfaces. After freezing on gold specimen supports, the fracture plane was directed through selected neurons using the precise planar microtome of the JEOL JFD-9000-CR freeze-fracture machine.

Confocal laser scanning microscopy and Raman imagery of the late Neoproterozoic Chichkan microbiota of South Kazakhstan

2010 ◽  
Vol 84 (3) ◽  
pp. 402-416 ◽  
Author(s):  
J. William Schopf ◽  
Anatoliy B. Kudryavtsev ◽  
Vladimir N. Sergeev
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Depth Study ◽  
Late Neoproterozoic

Precambrian microbiotas, such as that permineralized in bedded and stromatolitic cherts of the late Neoproterozoic, 750- to 800-Ma-old, Chichkan Formation of South Kazakhstan, have traditionally been studied by optical microscopy only. Such studies, however, are incapable of documenting accurately either the three-dimensional morphology of such fossils or their chemical composition and that of their embedding minerals. As shown here by analyses of fossils of the Chichkan Lagerstätte, the solution to these long-standing problems is provided by two techniques recently introduced to paleontology: confocal laser scanning microscopy (CLSM) and Raman imagery. The two techniques are used together to characterize, in situ and at micron-scale resolution, the cellular and organismal morphology of the thin section-embedded organic-walled Chichkan fossils. In addition, Raman imagery is used to analyze the molecular-structural composition of the carbonaceous fossils and of their embedding mineral matrix, identify the composition of intracellular inclusions, and quantitatively assess the geochemical maturity of the Chichkan organic matter.CLSM and Raman imagery are both broadly applicable to the study of fossils, whether megascopic or microscopic and regardless of mode of preservation, and both are non-intrusive and non-destructive, factors that permit their use for analyses of archived specimens. They are especially useful for the study of microscopic fossils, as is demonstrated in this first in-depth study of diverse taxa of a single Precambrian microbiota for which they provide information in three dimensions at high spatial resolution about their organismal morphology, cellular anatomy, kerogenous composition, mode of preservation, and taphonomy and fidelity of preservation.

scholarly journals Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms

1998 ◽  
Vol 64 (11) ◽  
pp. 4115-4127 ◽  
Author(s):  
Martin Kuehn ◽  
Martina Hausner ◽  
Hans-Joachim Bungartz ◽  
Michael Wagner ◽  
Peter A. Wilderer ◽  
...  
Keyword(s):  
Image Analysis ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Image Acquisition ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Confocal Laser ◽  
Cross Sectional ◽  
In Situ Conditions

ABSTRACT The purpose of this study was to develop and apply a quantitative optical method suitable for routine measurements of biofilm structures under in situ conditions. A computer program was designed to perform automated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing biofilm, a nondestructive procedure was created to study and quantify undisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dimensional (2D) cross-sectional images from a preset 3D domain of interest followed by the off-line analysis of these 2D images. With the quantitative extraction of information contained in each image, a three-dimensional reconstruction of the principal biological events can be achieved. The program is convenient to handle and was generated to determine biovolumes and thus facilitate the examination of dynamic processes within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, was inoculated into glass flow cells and the respective monoculture biofilms were analyzed in three dimensions. In this paper we describe a method for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis.

Contribution of Microscopy to a Better Knowledge of the Biology ofGiardia lamblia

2004 ◽  
Vol 10 (5) ◽  
pp. 513-527 ◽  
Author(s):  
Wanderley de Souza ◽  
Adriana Lanfredi-Rangel ◽  
Loraine Campanati
Keyword(s):  
Golgi Complex ◽  
Laser Scanning ◽  
Diarrheal Disease ◽  
Freeze Fracture ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Cytochemistry ◽  
Morphological Studies ◽  
Transmission Electron ◽  
Microscopic Techniques

Giardia lambliais a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown thatG. lambliadoes not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum–Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

Behaviour of nucleolar proteins in nuclei lacking ribosomal genes. A study by confocal laser scanning microscopy

1991 ◽  
Vol 98 (1) ◽  
pp. 99-105
Author(s):  
D. Hernandez-Verdun ◽  
M. Robert-Nicoud ◽  
G. Geraud ◽  
C. Masson
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Specific Marker ◽  
Nucleolar Proteins ◽  
Fibrillar Component

The behaviour of nucleolar proteins in cycling PtK1 cells and in micronuclei with or without NORs was investigated by immunofluorescence using antibodies from autoimmune sera and confocal laser scanning microscopy. These antibodies were shown by electron microscopy to recognize antigens confined to only one of the three basic nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). Serial optical sections allowed us to determine the three-dimensional organization of these components in the nucleolus of cycling cells. Furthermore, clear differences were found in the distribution of the various antigens in micronucleated cells. Three patterns could be observed: (1) the FC antigens were found mainly in the nucleoli, but also in varying amounts in the dots; (2) surprisingly, the DFC antigens were found to accumulate preferentially in the dots; (3) the GC-specific marker stained intensively the nucleoli as well the dots. The results are interpreted with regard to possible mechanisms for targeting nucleolar proteins to the site of nucleolar formation.

scholarly journals Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells

2019 ◽  
Vol 20 (24) ◽  
pp. 6238 ◽  
Author(s):  
Anna L. Gill ◽  
Monica Z. Wang ◽  
Beth Levine ◽  
Alan Premasiri ◽  
Fernando G. Vieira
Keyword(s):  
Motor Neurons ◽  
Laser Scanning ◽  
Neuronal Cell ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Primary Neurons ◽  
Repeat Protein ◽  
Neuronal Cell Lines ◽  
Dipeptide Repeat

A repeat expansion mutation in the C9orf72 gene is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, using multiple cell-based assay systems, we reveal both increased dipeptide repeat protein (DRP) toxicity in primary neurons and in differentiated neuronal cell lines. Using flow cytometry and confocal laser scanning microscopy of cells treated with fluorescein isothiocyanate (FITC)-labeled DRPs, we confirm that poly-glycine-arginine (GR) and poly-proline-arginine (PR) DRPs entered cells more readily than poly-glycine-proline (GP) and poly-proline-alanine (PA) DRPs. Our findings suggest that the toxicity of C9-DRPs may be influenced by properties associated with differentiated and aging motor neurons. Further, our findings provide sensitive cell-based assay systems to test phenotypic rescue ability of potential interventions.

scholarly journals Software-Based Three-Dimensional Deconvolution Microscopy of Cytoskeletal Proteins in Cultured Fibroblast Using Open-Source Software and Open Hardware

2019 ◽  
Vol 5 (12) ◽  
pp. 88
Author(s):  
Kazuo Katoh
Keyword(s):  
Open Source ◽  
Open Source Software ◽  
Laser Scanning ◽  
Low Cost ◽  
Three Dimensional ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Actual Measurement ◽  
Open Hardware

As conventional fluorescence microscopy and confocal laser scanning microscopy generally produce images with blurring at the upper and lower planes along the z-axis due to non-focal plane image information, the observation of biological images requires “deconvolution.” Therefore, a microscope system’s individual blur function (point spread function) is determined theoretically or by actual measurement of microbeads and processed mathematically to reduce noise and eliminate blurring as much as possible. Here the author describes the use of open-source software and open hardware design to build a deconvolution microscope at low cost, using readily available software and hardware. The advantage of this method is its cost-effectiveness and ability to construct a microscope system using commercially available optical components and open-source software. Although this system does not utilize expensive equipment, such as confocal and total internal reflection fluorescence microscopes, decent images can be obtained even without previous experience in electronics and optics.

scholarly journals Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Rachele Tofanelli ◽  
Athul Vijayan ◽  
Sebastian Scholz ◽  
Kay Schneitz
Keyword(s):  
Laser Scanning ◽  
Developmental Stages ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Imaging Data ◽  
Model Species ◽  
Cellular Models ◽  
Cellular Resolution

Abstract Background A salient topic in developmental biology relates to the molecular and genetic mechanisms that underlie tissue morphogenesis. Modern quantitative approaches to this central question frequently involve digital cellular models of the organ or tissue under study. The ovules of the model species Arabidopsis thaliana have long been established as a model system for the study of organogenesis in plants. While ovule development in Arabidopsis can be followed by a variety of different imaging techniques, no experimental strategy presently exists that enables an easy and straightforward investigation of the morphology of internal tissues of the ovule with cellular resolution. Results We developed a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages. The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide. We further improved the microscopy by employing a homogenous immersion system aimed at minimizing refractive index differences. The method allows complete inspection of the cellular architecture even deep within the ovule. Using the new protocol we were able to generate digital three-dimensional models of ovules of various stages. Conclusions The protocol enables the quick and reproducible imaging of fixed Arabidopsis ovules of all developmental stages. From the imaging data three-dimensional digital ovule models with cellular resolution can be rapidly generated using image analysis software, for example MorphographX. Such digital models will provide the foundation for a future quantitative analysis of ovule morphogenesis in a model species.

Sign in / Sign up

Export Citation Format

Share Document