Advancements over the years has helped scientist to trace molecules in live cells and understand the. A spectrofluorometer is extremely flexible, providing continuous ranges of excitation and emission wavelengths. Caymans jc1 mitochondrial membrane potential assay kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis. Tplsm images show the overlay of red and green jc1 fluorescence, plotted traces of jc1 fluorescence were extracted from ccdcameradualview timelapse recordings. Decreasing the pinhole will increase x,y resolution 1.
The assay is applicable to fluorescence microscopy and fluorescent plate reader. Principle using fluorescence microscopy and image analysis, the nucleocounter nc3000 system automates detection of cells with collapsed mitochondrial membrane potential. Fluorescence is widely used in biological microscopy. Optical microscopy conventional light microscopy, fluorescence microscopy, confocalmultiphoton microscopy and stimulated emission depletion microscopy 2.
Mitochondria staining kit cs0390 technical bulletin. Investigation of fluorescence of jc 1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm. Mitochondrial behavior during oogenesis in zebrafish. A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescing agent. Fluorescence microscopy of energized and deenergized rat cardiac muscle mito chondria stained with jci.
The values of fluorescence intensity are indicated as ratio between aggregate form of jc 1 dye on monomer form of jc 1 dye. Omug jan2015 fundamentals of fluorescence microscopy. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a. Nov 22, 2012 investigation of fluorescence of jc 1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard. In apoptotic cells the mitochondrial potential collapses and jc1 localizes to the cytosol in its monomeric green fluorescent form. Some mitochondria remained, however, partly depolarized. Jc1 exhibits potentialdependent accumulation in mitochondria, indicated by a fluorescence emission shift from green 529 nm to red 590 nm. Sep 01, 2011 in cultured astrocytes, occasional fluctuations of m occurred and they were most obvious in the red jc1 fluorescence channel of the ccd cameradualview recordings. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. When cells were prestained with jc1 and placed in nigericin fig. Jc1 mitochondrial membrane potential detection kit abp. Jc1 cas 3520432 adipogen life sciences mitochondrial. Jc 1 and jc 9 mitochondrial potential sensors 3 optical filters a number of different optical filter configurations can be used for analysis of jc 1 or jc 9 by fluorescence microscopy table 3. Fluorescence microscopy is a basic requirement in cell biology, molecular biology and biotechnology.
Therefore, our data do not support the interpretation of a role for mitochondrial k atp channelinduced depolarization of the inner membrane as a key. Changes in m reflected by different forms of jc1 as either green or red fluorescence can be determined as a ratio of green. A fluorescence microscope or plate reader equipped with laserfluorescent filters capable of detecting the jaggregate form of jc 1 using an excitation of 535 nm and an emission at 590 nm as well as the monomeric form of jc 1 at excitation and emission wavelengths of 485 and 535 nm, respectively 4. If cloning is impossible for instance in histologic samples it is required to use other techniques like immunofluorescence staining to. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Jun 10, 2012 a basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescing agent. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted stage 3 to the number of photons absorbed stage 1, is a measure of the rela. The values of fluorescence intensity are indicated as ratio between aggregate form of jc1 dye on monomer form of jc1 dye. Optimization of a microplate reader method for the analysis. Mug 11th annual comprehensive course on fluorescence microscopy june 8. Optimization of a microplate reader method for the. The accumulation of jc1 in these organelles leads to the formation jaggregates with a specific red fluorescence emission maximum at 590 nm, which is in addition. This lecture describes the principles of fluorescence and fluorescence microscopy.
M, 5 min markedly decreased the jc1 fluorescence ratio, indicating a pronounced mitochondrial depolarization figures 7a and 7d. Mitochondrial fluorescence of tmre, nao, and the monomeric form of jc. Using either confocal microscopy with tmre, tmrm and jc 1, or very low light fluorescence with tmre, we did not detect any effect of diazoxide on the fluorescence of potential dependent dyes. He observed blue light emitted from the surface of a solution of quinine. Many published protocols examining jc1 in mammalian cell types, or isolated mitochondria, are currently available reers et al. By detecting and calculating greenred fluorescence ratios of cluster. Familiarity with fluorescence is a prerequisite for taking. Fluorescence microscopy requires intense, nearmonochromatic, illumination which some widespread light sources, like halogen lamps cannot provide. Changes in m reflected by different forms of jc1 as either green or red fluorescence can be determined as a. Illustrated in figure 1 is a cutaway diagram of a modern epi fluorescence microscope equipped for both transmitted and reflected fluorescence microscopy. Light of the excitation wavelength is focused on the specimen through the objective lens.
A isolated mitochondria energized with succinaterotenone were incubated with jc1 1 z for 10 min at 37 and visualized with a zeiss axiophot fluorescent microscope. For many years, the use of jc1 for flow cytometry, fluorescence microscopy, and fluorescence spectrophotometry has been limited to mammalian cells. Epifluorescence microscopy the majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the diagram. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscopy. The vertical illuminator in the center of the diagram has the light source positioned at one end labeled the episcopic lamphouse and the filter cube turret at the other. Jc1, a cationic fluorescent dye when added to living cells, is known to be localized exclusively in mitochondria, particularly in good physiological conditions characterized by sufficient mitochondrial membrane potential. For fluo 3am recordings a 505 nm beamsplitter, a 53535 nm bandpass filter emitter and 485.
A typical response of mitochondria exposed to such stress is the socalled threadgrain transition, that is, a disruption of longer mitochondrial. Mitochondrial potential, jc1, apoptosis using the nc3000. Using either confocal microscopy with tmre, tmrm and jc1, or very low light fluorescence with tmre, we did not detect any effect of diazoxide on the fluorescence of potential dependent dyes. Investigation of fluorescence of jc1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm. The field of fluorescence microscopy is experiencing a renaissance with the introduction of new techniques such as confocal, multiphoton, deconvolution, and total internal reflection, especially when coupled to advances in chromophore and fluorophore technology. Feb 15, 2017 epifluorescence microscopy the majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the diagram. Mar 21, 2002 mitochondrial fluorescence of tmre, nao, and the monomeric form of jc. Jc1 mitochondrial membrane potential assay kit ab1850. The ratio of red to green fluorescence of jc1 is dependent only on membrane potential, and not influenced by mitochondrial size, shape, or density. Nov 22, 2012 investigation of fluorescence of jc 1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm. Many published protocols examining jc 1 in mammalian cell types, or isolated mitochondria, are currently available reers et al. Jc 1 exhibits potentialdependent accumulation in mitochondria, indicated by a fluorescence emission shift from green 529 nm to red 590 nm. Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical for example, absorption of light, mechanical friction, or chemical mechanism.
In each case the photomultiplier voltage was set so that the signal peak from nonstained. Jul 10, 2019 for many years, the use of jc 1 for flow cytometry, fluorescence microscopy, and fluorescence spectrophotometry has been limited to mammalian cells. In cultured astrocytes, occasional fluctuations of m occurred and they were most obvious in the red jc1 fluorescence channel of the ccd cameradualview recordings. Ratiometric highresolution imaging of jc1 fluorescence. Optical microscopy davidson and abramowitz optical. Increased mitochondrial mass and cytosolic redox imbalance in. Detection of the mitochondrial membrane potential by the. Investigation of fluorescence of jc1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard. The fluorescence emitted by the specimen is focused to the detector by the objective. The membranepermeant dualemission potentialsensitive jc 1 dye is widely used in apoptosis studies to monitor mitochondrial health by flow cytometry, fluorescence microscopy and in microplatebased fluorescent assays. The principal advantage of jc1 relative to other commonly employed fluorescent probes of mitochondrial membrane potential is that it allows qualitative visualization, considering the shift from orange to green fluorescence emission, and quantitative. Introduction to fluorescence microscopy microscopyu enus. Combs1 and hari shroff2 1nhlbi light microscopy facility, national institutes of health, bethesda, maryland 2nibib section on high resolution optical imaging, national institutes of health, bethesda, maryland the.
This can be a fluorescing protein for example gfp genetically linked to the protein of interest. This is basically a reflected light microscopy mode in which the wavelength of the reflected light is longer than that of the excitation. Dr otto schott formulated glass lenses that colorcorrected objectives and. Rapid development of new fluorescent labels has accelerated the expansion of fluorescence microscopy in laboratory applications and research 68.
Fluorescence is a process in which matter absorbs light and reemits at a different wavelength. Caymans jc1 mitochondrial membrane potential assay kit provides all the necessary reagents, as well as complete instructions, for analysis of mitochondrial integrity in whole cells. The fluorescence of the cells stained with this kit may be observed by fluorescence microscopy or measured. Four main types of light source are used, including xenon arc lamps or mercuryvapor lamps with an excitation filter, lasers, supercontinuum sources, and highpower leds. D, e jc1 green d and orangered e fluorescence histograms of control filled tracings and mclccptreated samples open tracings. Jc 1 was excited at 490 nm and an optical image splitter device dualview tm, optical insights was attached to the microscope to separate spectrally the green and red components of jc 1 fluorescence see fig. Increased mitochondrial mass and cytosolic redox imbalance. Mitochondrial and nonmitochondrial reduction of mtt. Apr 01, 2000 photomultiplier settings were adjusted to detect rho123 and dioc 6 3 fluorescence on the fl1 detector, cmxros and pi fluorescence on the fl3 detector, and the jc 1 monomer and aggregate fluorescence signals on the fl1 and fl2 detectors, respectively. A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. Consequently, mitochondrial depolarization is indicated by a decrease in the redgreen fluorescence intensity ratio.
Fundamentals of light microscopy and electronic imaging douglas b. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design. For confocal laser scanning microscopy, the monomer and jaggregate forms can be excited simultaneously by 488 nm argonion laser sources. This guide is a compilation of the principles and knowhow that the engineers at chroma technology corp use to design filters for a variety of fluorescence. Scanning probe microscopy scanning tunneling microscopy stm, atomic force microscopy afm, nearfield scanning optical microscopy and others 3. Fluorescence microscope refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence. Basic concepts of microscopy confocal aperture decreasing the pinhole size rejects more out of focus light, therefore improving contrast and effective z resolution. An introduction to fluorescence microscopy 2 excitation and emission spectra. Red jc1 fluorescence clearly increased up to threefold, whereas green jc1 fluorescence showed less intense and opposite changes see fig. Investigation of fluorescence of jc 1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard. Fundamentals of light microscopy and electronic imaging.
1380 278 690 1096 1444 209 1301 130 986 33 1394 1265 1137 1169 260 1310 1154 498 781 329 187 1004 1305 114 660 1402 391 1227 182 299 636 655 81 1366 503 202 103 400 929 1275 1153 752 357 612 705 1359