Measurement of volume injected into individual cells by quantitative fluorescence microscopy

1989 ◽  
Vol 94 (3) ◽  
pp. 443-447
Author(s):  
G.M. Lee
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Intensity ◽  
Mammalian Cells ◽  
Lucifer Yellow ◽  
Fluorescence Microscope ◽  
Standard Curve ◽  
Cellular Processes ◽  
Reliable Procedure ◽  
Dextran Solution ◽  
Quantitative Fluorescence

Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

scholarly journals Quantitative Fluorescence Microscopy on SARS-CoV-2

2021 ◽  
Vol 120 (3) ◽  
pp. 360a
Author(s):  
Rayna M. Addabbo ◽  
John Kohler ◽  
Isaac Angert ◽  
Yan Chen ◽  
Heather Hanson ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Quantitative Fluorescence

scholarly journals Quantitative Fluorescence Microscopy Reveals Higher Order Oligomerization of FGFR5

2017 ◽  
Vol 112 (3) ◽  
pp. 88a
Author(s):  
Romario Regeenes ◽  
Pamuditha Silva ◽  
Dawn M. Kilkenny ◽  
Jonathan V. Rocheleau
Keyword(s):  
Fluorescence Microscopy ◽  
Higher Order ◽  
Quantitative Fluorescence

scholarly journals Every laboratory with a fluorescence microscope should consider counting molecules

2014 ◽  
Vol 25 (10) ◽  
pp. 1545-1548 ◽  
Author(s):  
Valerie C. Coffman ◽  
Jian-Qiu Wu
Keyword(s):  
Mathematical Modeling ◽  
Fluorescence Microscopy ◽  
Protein Complexes ◽  
The State ◽  
Fluorescence Microscope ◽  
Cellular Structures ◽  
Biological Processes ◽  
Protein Molecules ◽  
Parameter Values

Protein numbers in cells determine rates of biological processes, influence the architecture of cellular structures, reveal the stoichiometries of protein complexes, guide in vitro biochemical reconstitutions, and provide parameter values for mathematical modeling. The purpose of this essay is to increase awareness of methods for counting protein molecules using fluorescence microscopy and encourage more cell biologists to report these numbers. We address the state of the field in terms of utility and accuracy of the numbers reported and point readers to references for details of specific techniques and applications.

scholarly journals Miga-mediated endoplasmic reticulum–mitochondria contact sites regulate neuronal homeostasis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lingna Xu ◽  
Xi Wang ◽  
Jia Zhou ◽  
Yunyi Qiu ◽  
Weina Shang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Mitochondrial Dynamics ◽  
Molecular Organization ◽  
Lipid Transport ◽  
Cellular Processes ◽  
Contact Sites ◽  
Neuronal Homeostasis ◽  
Higher Eukaryotes ◽  
Lateral Sclerosis

Endoplasmic reticulum (ER)–mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. We found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.

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