scholarly journals An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

2020 ◽  
Author(s):  
Jana K. Schniete ◽  
Peter W. Tinning ◽  
Ross C. Scrimgeour ◽  
Gillian Robb ◽  
Lisa S. Kölln ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Fluorescence Microscopy ◽  
Standing Wave ◽  
Optical Axis ◽  
Cell Structure ◽  
Lateral Resolution ◽  
Excitation Wavelength ◽  
Sampling Density ◽  
Notable Change

AbstractConventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50% to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

scholarly journals An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jana K. Schniete ◽  
Peter W. Tinning ◽  
Ross C. Scrimgeour ◽  
Gillian Robb ◽  
Lisa S. Kölln ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Fluorescence Microscopy ◽  
Standing Wave ◽  
Optical Axis ◽  
Cell Structure ◽  
Lateral Resolution ◽  
Excitation Wavelength ◽  
Sampling Density ◽  
Notable Change

AbstractConventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells

2007 ◽  
Vol 293 (4) ◽  
pp. C1420-C1425 ◽  
Author(s):  
Ross Corriden ◽  
Paul A. Insel ◽  
Wolfgang G. Junger
Keyword(s):  
Cell Membrane ◽  
Fluorescence Microscopy ◽  
Dynamic Range ◽  
Atp Release ◽  
Cell Types ◽  
New Method ◽  
Excitation Wavelength ◽  
Novel Method ◽  
Human Polymorphonuclear Leukocytes ◽  
Different Cell Types

Many cell types release ATP in response to mechanical or biochemical stimulation. The mechanisms responsible for this release, however, are not well understood and may differ among different cell types. In addition, there are numerous difficulties associated with studying the dynamics of ATP release immediately outside the cell membrane. Here, we report a new method that allows the visualization and quantification of ATP release by fluorescence microscopy. Our method utilizes a two-enzyme system that generates NADPH when ATP is present. NADPH is a fluorescent molecule that can be visualized by fluorescence microscopy using an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The method is capable of detecting ATP concentrations <1 μM and has a dynamic range of up to 100 μM. Using this method, we visualized and quantified ATP release from human polymorphonuclear leukocytes and Jurkat T cells. We show that upon cell stimulation, the concentrations of ATP can reach levels of up to 80 μM immediately outside of the cell membrane. This new method should prove useful for the study of the mechanisms of release and functional role of ATP in various cell systems, including individual cells.

scholarly journals Heterodyne Standing-Wave Interferometer with Improved Phase Stability

2021 ◽  
Author(s):  
Ingo Ortlepp ◽  
Jens-Peter Zöllner ◽  
Ivo W. Rangelow ◽  
Eberhard Manske
Keyword(s):  
Laser Beam ◽  
Frequency Shift ◽  
Standing Wave ◽  
Phase Difference ◽  
Phase Stability ◽  
Constant Velocity ◽  
Optical Axis ◽  
Fixed Position ◽  
Length Measurements ◽  
Laser Sources

AbstractThis paper describes a standing-wave interferometer with two laser sources of different wavelengths, diametrically opposed and emitting towards each other. The resulting standing wave has an intensity profile which is moving with a constant velocity, and is directly detected inside the laser beam by two thin and transparent photo sensors. The first sensor is at a fixed position, serving as a phase reference for the second one which is moved along the optical axis, resulting in a frequency shift, proportional to the velocity. The phase difference between both sensors is evaluated for the purpose of interferometric length measurements.

Effect of Composite Nanoparticle CeO2 on Myocardial Ischemic Re-Infusion of Cardio Myocyte Apoptosis in Mouse

2021 ◽  
Vol 21 (2) ◽  
pp. 1397-1402
Author(s):  
Chengbin Wang ◽  
Lin Ding ◽  
Jiamei Zhao ◽  
Beibei Cao ◽  
Mingwei He
Keyword(s):  
Free Radicals ◽  
Cell Membrane ◽  
Cardiac Muscle ◽  
Unsaturated Fatty Acids ◽  
Ischemia Reperfusion Injury ◽  
Cell Structure ◽  
Ischemia Reperfusion ◽  
Myocardial Tissue ◽  
R Process ◽  
Organelle Membrane

The myocardial I/R damage is very complicated. Apoptosis is considered to its a critical mechanism. During the cardiac muscle I/R process, oxygen-free radicals play a pivotal role. Arrhythmias, as well as enlargement of the area of myocardial infarction after cardiac muscle I/R process, are caused by adequate blast generated O2- ion free radicals. During the ischemia-reperfusion process, a large amount of O2- ion free radicals destroyed the cell structure, and it undergoes lipid peroxidation with unsaturated fatty acids that contain a large number of phospholipids in the cell membrane, causing membrane proteins such as ion channels and enzymes on the cell membrane. The activity of cell is reduced, which affects the function of cell membrane and organelle membrane, destroys its integrity and reduces fluidity.We observed the effects of cerium dioxide nanoparticles on glutathione peroxidase as well as superoxide dismutase, also propionate in myocardial tissue of I/R injury in the mouse. Its effects of malondialdehyde and apoptosis were explored to see its protective effect and to provide more preventive measures for ischemia-reperfusion injury.

Dual-color quantum dot detection of a heterotetrameric potassium channel (hKCa3.1)

2011 ◽  
Vol 300 (4) ◽  
pp. C843-C849 ◽  
Author(s):  
Daniel E. J. Waschk ◽  
Anke Fabian ◽  
Thomas Budde ◽  
Albrecht Schwab
Keyword(s):  
Quantum Dots ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Quantum Dot ◽  
Fluorescence Microscopy ◽  
Potassium Channel ◽  
Binomial Distribution ◽  
Hek293 Cells ◽  
F Cells

Potassium channels play a key role in establishing the cell membrane potential and are expressed ubiquitously. Today, more than 70 mammalian K+ channel genes are known. The diversity of K+ channels is further increased by the fact that different K+ channel family members may assemble to form heterotetramers. We present a method based on fluorescence microscopy to determine the subunit composition of a tetrameric K+ channel. We generated artificial “heteromers” of the K+ channel hKCa3.1 by coexpressing two differently tagged hKCa3.1 constructs containing either an extracellular hemagglutinin (HA) or an intracellular V5 epitope. hKCa3.1 channel subunits were detected in the plasma membrane of MDCK-F cells or HEK293 cells by labeling the extra- and intracellular epitopes with differently colored quantum dots (QDs). As previously shown for the extracellular part of hKCa3.1 channels, its intracellular domain can also bind only one QD label at a time. When both channel subunits were coexpressed, 27.5 ± 1.8% and 24.9 ± 2.1% were homotetramers consisting of HA- and V5-tagged subunits, respectively. 47.6 ± 3.2% of the channels were heteromeric and composed of both subunits. The frequency distribution of HA- and V5-tagged homo- and heteromeric hKCa3.1 channels is reminiscent of the binomial distribution ( a + b)2 = a2 + 2 ab + b2. Along these lines, our findings are consistent with the notion that hKCa3.1 channels are assembled from two homomeric dimers and not randomly from four independent subunits. We anticipate that our technique will be applicable to other heteromeric membrane proteins, too.

Visualization of the Microtubules of Glutaraldehyde-Fixed Cells by Reflection-Enhanced Backscatter Confocal Microscopy

2005 ◽  
Vol 12 (2) ◽  
pp. 113-123
Author(s):  
Charles H. Keith ◽  
Mark A. Farmer
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Standing Wave ◽  
Reflection Mode ◽  
Intact Cells ◽  
Fluorescence Techniques ◽  
Number Of Factors

Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size—and refractility—of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.

Enhanced axial resolution of the cytoskeleton in fluorescence microscopy by standing-wave excitation

1993 ◽  
Vol 51 ◽  
pp. 88-89
Author(s):  
Frederick Lanni ◽  
Brent Bailey ◽  
Daniel L. Farkas ◽  
D. Lansing Taylor
Keyword(s):  
Fluorescence Microscopy ◽  
Standing Wave ◽  
Focal Plane ◽  
Information Transfer ◽  
High Speed ◽  
Depth Resolution ◽  
Depth Of Field ◽  
Imaging Method ◽  
Axial Resolution ◽  
Direct Imaging

When the depth-of-field of a microscope is less than the axial dimension of the specimen, 3d information can be derived from a set of images recorded as the specimen is stepped through the object focal plane of the microscope. This procedure, known as optical sectioning microscopy (OSM), is the same in direct imaging and confocal scanning. For both of these cases in fluorescence microscopy, axial (depth) resolution is more limited than transverse resolution, for fundamental reasons. Our research aim has been to enhance axial resolution in fluorescence OSM (FOSM) while retaining the high-speed information transfer characteristics of direct imaging that are necessary for 3d studies of living cells in culture.Standing-wave fluorescence microscopy (SWFM) is a direct imaging method in which the object is illuminated by a three-dimensional field of planar interference fringes (standing waves) oriented parallel to the focal plane of the microscope. This field is produced in the specimen by crossing two coherent, collimated, s-polarized beams of equal amplitude directed through the specimen at complementary angles (θ, π -θ) relative to the axis of the microscope.

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