Fluorescence Spectroscopy and Microscopy: Methods and Protocols


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In the majority of experiments, particularly when cells have been transiently transfected with fluorescent proteins, a large amount of morphological variability of the cells can be present. Sometimes cells are not transfected or show poor localization of the probe. Some of the transfected cells might overexpress the fluorescent protein, which can lead to toxicity.

Cells can show abnormal morphology, such as swollen mitochondria, blebbing, and detachment from the substrate. Cells that do not show normal morphology should not be used for imaging experiments. The system used should make maximum use of the light and use the fewest optical elements in the light path.

Advances in detector technology have enabled reduction in illumination levels. Photomultiplier tube cathodes for confocal microscopy have become increasingly sensitive. Sensitivity of the camera is also an important consideration. With regard to speed of acquisition, particularly with simultaneous imaging of multiple fluorophores or radiometric analysis of a single probe, switching between filters or output from a monochromator will reduce the acquisition time. Researchers must therefore compromise by determining the optimal parameters while minimizing cell damage or death.

Researchers also need to understand the pros and cons of different microscopes. Most cellular processes occur in three dimensions over time. Therefore, cells need to be imaged in four dimensions to obtain a complete picture. This technique enables repeated imaging of a cell culture at particular time points Fig. However, dynamic imaging data are complex and interpretation can be difficult. Therefore, specialized image processing methods for estimation of motion, object detection, visualization, and quantitation can be used Gerlic et al.

Some of the more common systems used for imaging live cells are discussed below; the more widely used systems are discussed first and then the less commonly used systems are described. A basic overview of these types of fluorescent microscopy has been published in a previous issue of The Anatomical Record. Currently, the most common fluorescent protein used in imaging of cells is green fluorescent protein GFP; Shimomura et al. Table 1 shows some examples of the fluorescent proteins used by researchers, and use and advantages of these proteins.

Fast acquisition can be achieved with flexible excitation and a low cost Stephens and Allan, However, deconvolution should be carefully and accurately applied to avoid generation of artifacts Wallace et al. The plasma membrane undergoes ruffling activity around the fiber as it becomes progressively internalized until it is completely contained within the cytoplasm of the cell.

Labeling with DiI demonstrates that during phagocytosis the fiber becomes encased within a portion of the plasma membrane. The speed of incorporation is approximately 0.

Fluorescence microscopy - fluorescence microscope principle

Time in hr:min:sec is shown in the lower right corner. The images were kindly provided by Dr. Cynthia Jensen, the University of Auckland. Part of a time sequence of neonatal rat ventricular myocytes plated onto a dish and paced at 1 Hz.

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The white areas are free calcium ions. The scale is 0.


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Immunofluorescence microscopy was used. The image was kindly provided by Gregory Bass, the University of Auckland. This is important for investigating cells that undergo changes in shape, such as mitosis and apoptosis. Differential interference contrast and phase contrast are the most common types of microscopy used. Imaging live tissues using confocal microscopy is more difficult than imaging fixed specimens, and is not always practical because the specimen may not tolerate the particular conditions.

Choice of a cell type that is amenable to the conditions of imaging in confocal microscopy is important to avoid experimental problems. Images should be captured as rapidly as possible, using the minimal amount of laser power that enables imaging. Two images are shown from the same experiment. B The summary of fluorescent intensity of 42 slides over a period of 84 sec is shown.

Using another nm laser, the fluorescein was excited and imaged. The image was kindly provided by Dr. Nancy Liu, the University of Auckland. Confocal microscopy has high irradiance, which may cause photobleaching of specimens Conchello and Lichtman, Photobleaching from the laser beam is cumulative over multiple scans and, therefore, exposure to the beam should be reduced as much as possible for image acquisition.

Another limitation of confocal microscopy is its inefficient use of light. This loss can be reduced, but it will never be eliminated. Therefore, high intensity of excitation light is required for confocal microscopy, which increases the possibility of photobleaching to the specimen Khodjakov and Rieder, Confocal microscopy provides control of the area of illumination and, therefore, it is useful for photobleaching techniques, such as fluorescence loss in photobleaching and fluorescence recovery after photobleaching FRAP; see below.

Fast frame rates are advantageous because CCD cameras can be used for direct acquisition of confocal images rather than photomultiplier tubes, which are still the most commonly used light detectors in laser scanning confocal microscopy. Multiphoton microscopy has many advantages for imaging living cells. Examples of multiphoton fluorescence microscopy include imaging of intracellular calcium dynamics in various tissues, probing functional integration of donor cells after intracardiac transplantation, cellular redox state, and developmental studies, which require viability to be maintained for detecting sequential events in the same specimen over long time periods.

TIRF microscopy is used for direct imaging of living cells that are within close proximity to the coverslip. TIRF can be used to follow biochemical reactions, structural changes, and movements of biomolecules at the level of single molecules in real time. To achieve this, a selected pool of fluorescent proteins needs to be distinguished from other fluorescent proteins in cells.

This method can be used to determine the mobility and dynamics of proteins that are fluorescently labeled in or associated with cells.

A quantitative protocol for intensity-based live cell FRET imaging.

The bleached molecules diffuse out of this region and nonphotobleached fluorescently labeled molecules simultaneously move into the bleached region, which results in the fluorescence signal being recovered. The rate and the amount of recovery at the photobleached region is analyzed, revealing information on the dynamic properties of proteins or cytoskeletal structures e.

However, this usually means that spatial resolution is traded for temporal resolution. Excimer formation by pyrene in ethanol. Spectra are normalized to the All spectra are essentially identical below nm after normalization.

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Spectra are as follows: 1 2 mM pyrene, purged with argon to remove oxygen; 2 2 mM pyrene, air-equilibrated; 3 0. The monomer-to-excimer ratio Many other environmental factors exert influences on fluorescence properties.


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The three most common are:. Fluorescence spectra may be strongly dependent on solvent. This characteristic is most often observed with fluorophores that have large excited-state dipole moments, resulting in fluorescence spectral shifts to longer wavelengths in polar solvents.

Measurement of Cell Membrane Fluidity by Laurdan GP: Fluorescence Spectroscopy and Microscopy

Representative fluorophores include the aminonaphthalenes such as prodan, badan Figure 14 and dansyl, which are effective probes of environmental polarity in, for example, a protein's interior. Binding of a probe to its target can dramatically affect its fluorescence quantum yield Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.

Probes that have a high fluorescence quantum yield when bound to a particular target but are otherwise effectively nonfluorescent yield extremely low reagent background signals. Similarly, fluorogenic enzyme substrates, which are nonfluorescent or have only short-wavelength emission until they are converted to fluorescent products by enzymatic cleavage, allow sensitive detection of enzymatic activity Enzyme Substrates and Assays—Chapter If quenching is caused by collisional interactions, as is usually the case, information on the proximity of the fluorophore and quencher and their mutual diffusion rate can be derived.

This quenching effect has been used productively to measure chloride-ion flux in cells Detecting Chloride, Phosphate, Nitrite and Other Anions—Section Many fluorophores are also quenched by proteins. Examples are Alexa Fluor, fluorescein and BODIPY dyes, in which the effect is apparently due to charge-transfer interactions with aromatic amino acid residues. Fluorescein and hydroxycoumarins umbelliferones are further examples of this type of fluorophore. Fluorescence emission spectra of the 2-mercaptoethanol adduct of badan B in: 1 toluene, 2 chloroform, 3 acetonitrile, 4 ethanol, 5 methanol and 6 water.

Each solution contains the same concentration of the adduct. Excitation of all samples is at nm. The preceding discussion has introduced some general principles to consider when selecting a fluorescent probe. Application-specific details are addressed in subsequent chapters of the Molecular Probes Handbook. For in-depth treatments of fluorescence techniques and their biological applications, the reader is referred to the many excellent books and review articles listed below.

Note: You clicked on an external link, which has been disabled in order to keep your shopping session open. Search Thermo Fisher Scientific. Search All. Fluorescence Fundamentals.

A quantitative protocol for intensity-based live cell FRET imaging.

See Navigation. The Fluorescence Process. Stage 2: Excited-State Lifetime The excited state exists for a finite time typically 1—10 nanoseconds. Fluorescence Spectra The entire fluorescence process is cyclical. Table 1. Spectroscopic properties of fluorescent dyes. Optimum instrument setup should deliver excitation light as close to the peak of the excitation spectrum of the fluorophore as possible. Absorption spectrum An X,Y plot of wavelength versus absorbance of a chromophore or fluorophore.

To a first approximation, the absorption spectrum of a fluorophore is equivalent to the fluorescence excitation spectrum.

Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols
Fluorescence Spectroscopy and Microscopy: Methods and Protocols Fluorescence Spectroscopy and Microscopy: Methods and Protocols

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