Mammalian Apoptosis in Whole Neonatal Ovaries, Embryos and Fetal Limbs Using Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 598-599
Author(s):  
Robert M Zucker
Keyword(s):  
Limb Development ◽  
Laser Scanning ◽  
Optical Resolution ◽  
Confocal Microscope ◽  
Laser Scanning Microscopy ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Depth Of Penetration ◽  
Technical Problems ◽  
Sample Technique

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the depth of penetration of the laser light into the tissue. We have optimized the confocal microscope performance and developed a sample technique that increases the optical resolution of the system.CLSM has been used to study cellular death (apoptosis) during GD 8-12 normal rat/mouse embryonic development, neonatal ovarian development (PD10-45) and fetal limb development (GD 11-15). LysoTracker Red (LT) was incorporated into the tissue prior to laser excitation. It is aldehyde fixable stain that concentrates into acidic structures or into cells that have high lysosomal activity.

scholarly journals Peeking under the Iron Curtain: Development of a Microcosm for Imaging the Colonization of Steel Surfaces by Mariprofundus sp. Strain DIS-1, an Oxygen-Tolerant Fe-Oxidizing Bacterium

2016 ◽  
Vol 82 (22) ◽  
pp. 6799-6807 ◽  
Author(s):  
Adam C. Mumford ◽  
Irini J. Adaktylou ◽  
David Emerson
Keyword(s):  
Laser Scanning ◽  
Three Dimensional ◽  
Experimental System ◽  
Laser Scanning Microscopy ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Sterile Seawater ◽  
Microbially Influenced Corrosion ◽  
Steel Surfaces

ABSTRACTMicrobially influenced corrosion (MIC) is a major cause of damage to steel infrastructure in the marine environment. Despite their ability to grow directly on Fe(II) released from steel, comparatively little is known about the role played by neutrophilic iron-oxidizing bacteria (FeOB). Recent work has shown that FeOB grow readily on mild steel (1018 MS) incubatedin situor as a substrate for pure culturesin vitro; however, details of how they colonize steel surfaces are unknown yet are important for understanding their effects. In this study, we combine a novel continuously upwelling microcosm with confocal laser scanning microscopy (CLSM) to determine the degree of colonization of 1018 MS by the marine FeOB strain DIS-1. 1018 MS coupons were incubated with sterile seawater (pH 8) inoculated with strain DIS-1. Incubations were performed both under oxic conditions and in an anoxic-to-oxic gradient. Following incubations of 1 to 10 days, the slides were removed from the microcosms and stained to visualize both cells and stalk structures. Stained coupons were visualized by CLSM after being mounted in a custom frame to preserve the three-dimensional structure of the biofilm. The incubation of 1018 MS coupons with strain DIS-1 under oxic conditions resulted in initial attachment of cells within 2 days and nearly total coverage of the coupon with an ochre film within 5 days. CLSM imaging revealed a nonadherent biofilm composed primarily of the Fe-oxide stalks characteristic of strain DIS-1. When incubated with elevated concentrations of Fe(II), DIS-1 colonization of 1018 MS was inhibited.IMPORTANCEThese experiments describe the growth of a marine FeOB in a continuous culture system and represent direct visualizations of steel colonization by FeOB. We anticipate that these experiments will lay the groundwork for studying the mechanisms by which FeOB colonize steel and help to elucidate the role played by marine FeOB in MIC. These observations of the interaction between an FeOB, strain DIS-1, and steel suggest that this experimental system will provide a useful model for studying the interactions between microbes and solid substrates.

scholarly journals Evaluation of a Novel Prototype for Pressurized Intraperitoneal Aerosol Chemotherapy

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 633
Author(s):  
Hee Su Lee ◽  
Junsik Kim ◽  
Eun Ji Lee ◽  
Soo Jin Park ◽  
Jaehee Mun ◽  
...  
Keyword(s):  
Penetration Depth ◽  
Laser Scanning ◽  
Performance Test ◽  
Ex Vivo ◽  
Drug Distribution ◽  
Laser Scanning Microscopy ◽  
Distribution Area ◽  
Confocal Laser ◽  
The Novel ◽  
Depth Of Penetration

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) has been suggested as an alternative option for treating peritoneal carcinomatosis (PC). Even with its clinical advantages, the current PIPAC system still suffers from limitations regarding drug distribution area and penetration depth. Thus, we evaluated the new PIPAC system using a novel prototype, and compared its performance to the results from previous studies related with the current MIP® indirectly because the system is currently not available for purchase in the market. The developed prototype includes a syringe pump, a nozzle, and controllers. Drug distribution was conducted using a methylene blue solution for performance test. For penetration depth evaluation, an ex-vivo experiment was performed with porcine tissues in a 3.5 L plastic box. Doxorubicin was sprayed using the novel prototype, and its penetration depth was investigated by confocal laser scanning microscopy. The experiment was repeated with varying nozzle levels from the bottom. The novel prototype sprays approximately 30 μm drug droplets at a flow rate of 30 mL/min with 7 bars of pressure. The average diameter of sprayed region with concentrated dye was 18.5 ± 1.2 cm, which was comparable to that of the current MIP® (about 10 cm). The depth of concentrated diffusion (DCD) did not differ among varying nozzle levels, whereas the depth of maximal diffusion (DMD) decreased with increasing distance between the prototype and the bottom (mean values, 515.3 μm at 2 cm; 437.6 μm at 4 cm; 363.2 μm at 8 cm), which was comparable to those of the current MIP® (about 350–500 μm). We developed a novel prototype that generate small droplets for drug aerosolization and that have a comparably wide sprayed area and depth of penetration to the current MIP® at a lower pressure.

Three-Dimensional Reconstruction of the Rat Brain Cortical Microcirculation in vivo

1991 ◽  
Vol 11 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Ulrich Dirnagl ◽  
Arno Villringer ◽  
Roland Gebhardt ◽  
Roman L. Haberl ◽  
Peter Schmiedek ◽  
...  
Keyword(s):  
Rat Brain ◽  
Laser Scanning ◽  
Brain Cortex ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Depth Of Penetration ◽  
Cranial Window ◽  
The Brain

We used confocal laser scanning microscopy (CLSM) to investigate the morphology and three-dimensional relationships of the microcirculation of the superficial layers of the rat brain cortex in vivo. In anesthetized rats equipped with a closed cranial window (dura mater removed), after i.v. injection of 3 mg/100 g of body weight of fluorescein in 0.5 ml of saline, serial optical sections of the brain cortex intraparenchymal microcirculation were taken. Excitation was at a wavelength of 488 nm (argon laser), and emission was collected above 515 nm. CLSM provided images of brain vessels with sufficient signal-to-noise ratio for three-dimensional reconstructions down to a depth of 250 μm beneath the surface of the brain. Compared to conventional fluorescence microscopy, CLSM has a much higher axial resolution and higher depth of penetration. Laser light-induced intravascular aggregates, irregularities of erythrocyte flow, or microvascular occlusions (“light and dye injury”) were not apparent in the current experimental paradigm. CLSM is a promising new tool for in vivo visualization of the cerebral microcirculation. Future studies have to characterize the potential damage to the tissue caused by the cranial window preparation and light and dye mechanisms.

Phase modulation nanoscopy: a simple approach to enhanced optical resolution

2015 ◽  
Vol 177 ◽  
pp. 507-515 ◽  
Author(s):  
Robert Pal
Keyword(s):  
Phase Modulation ◽  
Laser Scanning ◽  
Optical Resolution ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Optical Diffraction ◽  
Optical Modulator ◽  
Confocal Laser ◽  
Electro Optical Modulator ◽  
Experimental Resolution

A new modular super-resolution technique called Phase Modulation Nanoscopy (PhMoNa) has been developed in order to break the optical diffraction barrier in Confocal Laser Scanning Microscopy (LSCM). This technique is based on using spatially modulated illumination intensity, whilst harnessing the fluorophore's non-linear emission response. It allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2. The work is in its initial phase, but by using a custom built Electro Optical Modulator (EOM) in conjunction with functionalised Ln(iii) complexes as probes, a sub-diffraction resolution of ∼60 nm was achieved of selected cellular organelles in long term live cell imaging experiments.

Use of Confocal Imaging for Arthropod Morphological Studies

2000 ◽  
Vol 6 (S2) ◽  
pp. 822-823
Author(s):  
Angela V. Klaus ◽  
Varuni L. Kulasekera ◽  
Norman Platnick
Keyword(s):  
Light Microscopy ◽  
Laser Scanning ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Morphological Studies ◽  
Critical Point Drying

Morphological studies in spider and insect groups (Phylum Arthropoda) have historically relied upon traditional imaging techniques such as light microscopy (LM) and scanning electron microscopy (SEM). It has often been difficult, however, to examine some structural details of certain specimens due to limitations of the techniques. Light microscopy does not always provide adequate depth of focus for visualizing the true three-dimensional structure of a specimen, and out-of-focus light in the image can obscure detail. SEM is not applicable if the specimen is too delicate to undergo critical point drying. Additionally, SEM is a surface technique and does not provide any information about internal structure.If a specimen is transparent to light, information about the morphology of internal structures, and connections between structures, can easily be obtained using confocal microscopy without the problems associated with traditional LM. In this study, we used confocal laser scanning microscopy (CLSM) to study the morphology of chitinous arthropod structures by taking advantage of their autofluorescent properties.

scholarly journals Confocal Laser Scanning Microscopy By Remote Access

1999 ◽  
Vol 7 (7) ◽  
pp. 32-33
Author(s):  
J.H. Youngblom ◽  
J. Wilkinson ◽  
J.J. Youngblom
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Microscope ◽  
California State University ◽  
Remote Access ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
State University ◽  
Electron Microscopes

In recent years there have been a growing number of facilities interested in developing remote access capabilities to a variety of microscopy systems. While certain types of microscopes, such as electron microscopes and scanning probe microscopes have been well established for telepresence microscopy, no one has yet reported on the development of similar capabilities for the confocal microscope.At California State University, home to the CSUPERB (California State University Program for Education and Research in Biotechnology) Confocal Microscope Core Facility, we have established a remote access confocal laser scanning microscope facility that allows users with virtually any type of computer platform to connect to our system.

scholarly journals Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue

1997 ◽  
Vol 110 (8) ◽  
pp. 975-983 ◽  
Author(s):  
N. Klauke ◽  
H. Plattner
Keyword(s):  
Signal Transduction ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Time Range ◽  
High Time Resolution ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Technical Problems ◽  
Mutant Cells

In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (<1 second) trichocyst exocytosis has been hampered by various technical problems which we now have overcome. While Fura Red was found appropriate for quantitative double wavelength recordings, Fluo-3 allowed to follow, semi-quantitatively but with high time resolution, [Ca2+]i changes by rapid confocal laser scanning microscopy (CLSM). Resting values are between 50 and 70 nM in the strains analysed (7S wild type, as well as a non-discharge and a trichocyst-free mutant, nd9-28 degrees C and tl). In all strains [Ca2+]i first increases at the site of AED application, up to 10-fold above basal values, followed by a spillover into deeper cell regions. This might: (i) allow a vigorous Ca2+ flush during activation, and subsequently (ii) facilitate re-establishment of Ca2+ homeostasis within > or =20 seconds. Because of cell dislocation during vigorous trichocyst exocytosis, 7S cells could be reasonably analysed only by CLSM after Fluo-3 injection. In 7S cells cortical [Ca2+]i transients are strictly parallelled by trichocyst exocytosis, i.e. in the subsecond time range and precisely at the site of AED application. Injection of Ca2+ is a much less efficient trigger for exocytosis. Ca2+-buffer injections suggest a requirement of [Ca2+]i >1 to 10 microM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.

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