EUS-guided needle-based Confocal Laser induced Endomicroscopy (nCLE): a correlation study of “through the needle' imaging with normal histology in a porcine model

Background: Microcirculatory alterations have been observed at the early phase of sepsis, although macrocirculation seems preserved. The aim of this study was to analyze the effect of crystalloid fluid therapy on mesenteric microcirculation, assessed by using the confocal laser endomicroscope Cellvizio®, in an endotoxic porcine model. Methods: It is a prospective endotoxic shock (lipopolysaccharide infusion) experimental trial. Piglets were divided into 3 groups: 6 in the sham group (no LPS injection, no fluid), 9 in the control group (LPS infusion, no fluid), and 6 in the crystalloids group (LPS infusion and fluid resuscitation with crystalloids). Fluid resuscitation consisted in a fluid bolus of 20 mL/kg 0.9% saline over 30 min followed by a 10 mL/kg/h fluid rate over 4 h. Mesenteric microcirculation was assessed using a confocal laser endomicroscope (Cellvizio®). Blood flow within capillaries was visually assessed according to the point of care microcirculation (POEM) score. Results: At baseline, the 3 groups were similar regarding hemodynamic, biological, and microcirculatory parameters. At T360, the POEM score significantly decreased in the control and crystalloids groups, whereas it remained unchanged in the sham group (respectively, 1.62 ± 1.06, 1.2 ± 0.45, and 5.0 ± 0, p = 0.011). There was no significant difference in cardiac output at T360 between the sham and crystalloids groups (3.1 ± 0.8 vs. 2.3 ± 0.6, p = 0.132) or between the control and crystalloids groups (2.0 ± 0.6 vs. 2.3 ± 0.6, p = 0.90). Conclusion: There was no significant improvement of microcirculatory alterations after crystalloids resuscitation despite improvement in macrocirculatory parameters in early experimental sepsis.

Download Full-text

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102560 ◽

1988 ◽

Vol 46 ◽

pp. 96-97

Author(s):

Thomas M. Jovin ◽

Michel Robert-Nicoud ◽

Donna J. Arndt-Jovin ◽

Thorsten Schormann

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Scanning Microscope ◽

Laser Scanning Microscope ◽

Biochemical Processes ◽

Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Download Full-text

Confocal laser microscopy of impregnated central nervous system tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102559 ◽

1988 ◽

Vol 46 ◽

pp. 94-95

Author(s):

J.N. Turner ◽

M. Siemens ◽

D. Szarowski ◽

D.N. Collins

Keyword(s):

Electron Microscopy ◽

Central Nervous System ◽

Nervous System ◽

Three Dimensional ◽

Dimensional Structure ◽

Confocal Laser ◽

High Contrast ◽

Central Nervous System Tissue ◽

Tissue Samples ◽

Reflected Light

A classic preparation of central nervous system tissue (CNS) is the Golgi procedure popularized by Cajal. The method is partially specific as only a few cells are impregnated with silver chromate usualy after osmium post fixation. Samples are observable by light (LM) or electron microscopy (EM). However, the impregnation is often so dense that structures are masked in EM, and the osmium background may be undesirable in LM. Gold toning is used for a subtle but high contrast EM preparation, and osmium can be omitted for LM. We are investigating these preparations as part of a study to develop correlative LM and EM (particularly HVEM) methodologies in neurobiology. Confocal light microscopy is particularly useful as the impregnated cells have extensive three-dimensional structure in tissue samples from one to several hundred micrometers thick. Boyde has observed similar preparations in the tandem scanning reflected light microscope (TSRLM).

Download Full-text

Some quantitative applications of vascular corrosion casting

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124094 ◽

1992 ◽

Vol 50 (1) ◽

pp. 738-739

Author(s):

Fred E. Hossler

Keyword(s):

Blood Vessels ◽

Nuclear Orientation ◽

Three Dimensional ◽

Surrounding Tissue ◽

Confocal Laser ◽

Corrosion Casting ◽

Venous Valve ◽

Casting Technique ◽

Low Viscosity ◽

Quantitative Measurements

Preparation of replicas of the complex arrangement of blood vessels in various organs and tissues has been accomplished by infusing low viscosity resins into the vasculature. Subsequent removal of the surrounding tissue by maceration leaves a model of the intricate three-dimensional anatomy of the blood vessels of the tissue not obtainable by any other procedure. When applied with care, the vascular corrosion casting technique can reveal fine details of the microvasculature including endothelial nuclear orientation and distribution (Fig. 1), locations of arteriolar sphincters (Fig. 2), venous valve anatomy (Fig. 3), and vessel size, density, and branching patterns. Because casts faithfully replicate tissue vasculature, they can be used for quantitative measurements of that vasculature. The purpose of this report is to summarize and highlight some quantitative applications of vascular corrosion casting. In each example, casts were prepared by infusing Mercox, a methyl-methacrylate resin, and macerating the tissue with 20% KOH. Casts were either mounted for conventional scanning electron microscopy, or sliced for viewing with a confocal laser microscope.

Download Full-text

3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168554 ◽

1994 ◽

Vol 52 ◽

pp. 164-165

Author(s):

Thomas J. Deerinck ◽

Maryann E. Martone ◽

Varda Lev-Ram ◽

David P. L. Green ◽

Roger Y. Tsien ◽

...

Keyword(s):

Laser Scanning ◽

Reactive Oxygen ◽

Confocal Laser Scanning Microscope ◽

Fluorescent Dye ◽

Confocal Laser ◽

3 Dimensional ◽

Voltage Electron ◽

Dimensional Distribution ◽

Electron Microscopes ◽

New Generation

The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

Download Full-text