In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM)

1988 ◽  
Vol 46 ◽  
pp. 56-57
Author(s):  
J. V. Jester ◽  
H. D. Cavanagh ◽  
M. A. Lemp
Keyword(s):  
Confocal Imaging ◽  
Optical Sectioning ◽  
Scanning Confocal Microscopy ◽  
Reflected Light ◽  
Interesting Possibility ◽  
Superficial Epithelium ◽  
Confocal Scanning ◽  
Time Image

New developments in optical microscopy involving confocal imaging are now becoming available which dramatically increase resolution, contrast and depth of focus by optically sectioning through structures. The transparency of the anterior ocular structures, cornea and lens, make microscopic visualization and optical sectioning of the living intact eye an interesting possibility. Of the confocal microscopes available, the Tandem Scanning Reflected Light Microscope (referred to here as the Tandem Scanning Confocal Microscope), developed by Professors Petran and Hadravsky at Charles University in Pilzen, Czechoslovakia, permits real-time image acquisition and analysis facilitating in vivo studies of ocular structures.Currently, TSCM imaging is most successful for the cornea. The corneal epithelium, stroma, and endothelium have been studied in vivo and photographed in situ. Confocal scanning images of the superficial epithelium, similar to those obtained by scanning electron microscopy, show both light and dark surface epithelial cells.

scholarly journals Phagocytosis and Efferocytosis by Resident Macrophages in the Mouse Pancreas

2021 ◽  
Vol 12 ◽  
Author(s):  
Kristel Parv ◽  
Nestori Westerlund ◽  
Kevin Merchant ◽  
Milad Komijani ◽  
Robin S. Lindsay ◽  
...  
Keyword(s):  
Confocal Imaging ◽  
Mouse Pancreas ◽  
Tissue Microenvironment ◽  
Inflammatory State ◽  
Islet Dysfunction ◽  
Diabetes Development ◽  
The Impact

The tissue microenvironment in the mouse pancreas has been shown to promote very different polarizations of resident macrophages with islet-resident macrophages displaying an inflammatory “M1” profile and macrophages in the exocrine tissue mostly displaying an alternatively activated “M2” profile. The impact of this polarization on tissue homeostasis and diabetes development is unclear. In this study, the ability of pancreas-resident macrophages to phagocyte bacterial and endogenous debris was investigated. Mouse endocrine and exocrine tissues were separated, and tissue-resident macrophages were isolated by magnetic immunolabeling. Isolated macrophages were subjected to flow cytometry for polarization markers and qPCR for phagocytosis-related genes. Functional in vitro investigations included phagocytosis and efferocytosis assays using pH-sensitive fluorescent bacterial particles and dead fluorescent neutrophils, respectively. Intravital confocal imaging of in situ phagocytosis and efferocytosis in the pancreas was used to confirm findings in vivo. Gene expression analysis revealed no significant overall difference in expression of most phagocytosis-related genes in islet-resident vs. exocrine-resident macrophages included in the analysis. In this study, pancreas-resident macrophages were shown to differ in their ability to phagocyte bacterial and endogenous debris depending on their microenvironment. This difference in abilities may be one of the factors polarizing islet-resident macrophages to an inflammatory state since phagocytosis has been found to imprint macrophage heterogeneity. It remains unclear if this difference has any implications in the development of islet dysfunction or autoimmunity.

scholarly journals Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry

2004 ◽  
Vol 70 (6) ◽  
pp. 3745-3750 ◽  
Author(s):  
M. Rold�n ◽  
F. Thomas ◽  
S. Castel ◽  
A. Quesada ◽  
M. Hern�ndez-Marin�
Keyword(s):  
Single Cells ◽  
Three Dimensional ◽  
Confocal Imaging ◽  
Confocal Scanning Laser Microscopy ◽  
Pigment Analysis ◽  
Phototrophic Biofilms ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

ABSTRACT A new imaging technique for the analysis of fluorescent pigments from a single cell is reported. It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods. The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages.

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.

scholarly journals Chronic Dysfunction of Astrocytic Inwardly Rectifying K+ Channels Specific to the Neocortical Epileptic Focus After Fluid Percussion Injury in the Rat

2010 ◽  
Vol 104 (6) ◽  
pp. 3345-3360 ◽  
Author(s):  
Tessandra H. Stewart ◽  
Clifford L. Eastman ◽  
Peter A. Groblewski ◽  
Jason S. Fender ◽  
Derek R. Verley ◽  
...  
Keyword(s):  
Neuronal Excitability ◽  
Confocal Imaging ◽  
Epileptic Focus ◽  
Whole Cell ◽  
Fluid Percussion ◽  
Fluid Percussion Injury ◽  
Spontaneous Seizures ◽  
Inwardly Rectifying

Astrocytic inwardly rectifying K+ currents ( IKIR) have an important role in extracellular K+ homeostasis, which influences neuronal excitability, and serum extravasation has been linked to impaired KIR-mediated K+ buffering and chronic hyperexcitability. Head injury induces acute impairment in astroglial membrane IKIR and impaired K+ buffering in the rat hippocampus, but chronic spontaneous seizures appear in the perilesional neocortex—not the hippocampus—in the early weeks to months after injury. Thus we examined astrocytic KIR channel pathophysiology in both neocortex and hippocampus after rostral parasaggital fluid percussion injury (rpFPI). rpFPI induced greater acute serum extravasation and metabolic impairment in the perilesional neocortex than in the underlying hippocampus, and in situ whole cell recordings showed a greater acute loss of astrocytic IKIR in neocortex than hippocampus. IKIR loss persisted through 1 mo after injury only in the neocortical epileptic focus, but fully recovered in the hippocampus that did not generate chronic seizures. Neocortical cell-attached recordings showed no loss or an increase of IKIR in astrocytic somata. Confocal imaging showed depletion of KIR4.1 immunoreactivity especially in processes—not somata—of neocortical astrocytes, whereas hippocampal astrocytes appeared normal. In naïve animals, intracortical infusion of serum, devoid of coagulation-mediating thrombin activity, reproduces the effects of rpFPI both in vivo and at the cellular level. In vivo serum infusion induces partial seizures similar to those induced by rpFPI, whereas bath-applied serum, but not dialyzed albumin, rapidly silenced astrocytic KIR membrane currents in whole cell and cell-attached patch-clamp recordings in situ. Thus both acute impairment in astrocytic IKIR and chronic spontaneous seizures typical of rpFPI are reproduced by serum extravasation, whereas the chronic impairment in astroglial IKIR is specific to the neocortex that develops the epileptic focus.

scholarly journals Resistance of Synthetic and Biologic Surgical Meshes to Methicillin-ResistantStaphylococcus aureusBiofilm: AnIn VitroInvestigation

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Ethan Kallick ◽  
Laura Nistico ◽  
Mark Longwell ◽  
Barbara Byers ◽  
Frank Cartieri ◽  
...  
Keyword(s):  
Standard Procedure ◽  
Single Species ◽  
Infection Model ◽  
Attached Bacteria ◽  
Surgical Meshes ◽  
Confocal Scanning ◽  
Confocal Scanning Microscopy

Surgical meshes have become the standard procedure for a variety of surgical applications with 20 million meshes being implanted each year. The popularity of mesh usage among surgeons is backed by the multiple studies that support its functionality as a tool for improving surgical outcomes. However, their use has also been associated with infectious surgical complications and many surgeons have turned to biologic meshes. While there have been several studies investigating synthetic meshes, there is limited data comparing synthetic and biologic meshesin vitroin an infection model. This study evaluates thein vitrosusceptibility of both synthetic and biologic meshes to single-species methicillin-resistantStaphylococcus aureus(MRSA) biofilms. This research compares biofilm biomass, average thickness, and coverage between the three meshes throughflorescent in situ hybridization(FISH), confocal scanning microscopy (CSLM), and image analysis. We also report the varying levels of planktonic and attached bacteria through sonication and cfu counts. While the data illustrates increased biofilm formation on biologic meshin vitro, the study must further be investigatedin vivoto confirm the study observations.

scholarly journals Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: System design and calibration

Scanning ◽  
2006 ◽  
Vol 18 (1) ◽  
pp. 45-49 ◽  
Author(s):  
W. Matthew Petroll ◽  
James V. Jester ◽  
H. Dwight Cavanagh
Keyword(s):  
System Design ◽  
Three Dimensional ◽  
Confocal Imaging

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.

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