Microspectroscopic fluorescence analysis with prism‐based imaging spectrometers: Review and current studies

Hanley, Quentin S.; Murray, Patricia I; Forde, Toni S

doi:10.1002/cyto.a.20265

Cited by 15 publications

(8 citation statements)

References 35 publications

(34 reference statements)

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“…The optimized E and a values can be achieved in a few (3)(4)(5) iterations. In both Method 1 and Method 2, the e D /e A ratio at the donor excitation wavelength can be determined either by spectrophotometry, or by the method described in (27).…”

Section: The Intensity-based Ratiometric Fret Methodsmentioning

confidence: 99%

“…The efficiency of FRET (E) depends on the inverse sixth power of the distance between the donor and the acceptor, so monitoring E can serve as a spectroscopic ruler (3). Several spectroscopic parameters (e.g., intensity, lifetime, anisotropy) can be used to determine E (1,(4)(5)(6)(7)(8). In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations.…”

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confidence: 99%

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Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

et al. 2009

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The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results. ' 2009 International Society for Advancement of Cytometry Key terms fluorescence resonance energy transfer; ratiometric FRET; intensity-based FRET; FRET calibration; confocal laser scanning microscopy; ImageJ; 3D image processing IN biological systems, fluorescence resonance energy transfer (FRET) is a widely used method for studying molecular interactions and processes (1). FRET is a nonradiative transfer of energy from an excited fluorophore, the donor, to another fluorophore, the acceptor, that are at a distance of 1-10 nm (2). The efficiency of FRET (E) depends on the inverse sixth power of the distance between the donor and the acceptor, so monitoring E can serve as a spectroscopic ruler (3). Several spectroscopic parameters (e.g., intensity, lifetime, anisotropy) can be used to determine E (1,4-8). In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations. On the other hand, when subcellular details are needed, various microscopic FRET methods can be used (9-16). The ratiometric method (10), which was adapted from flow cytometry to microscopy (17), has the advantage of preserving the donor and acceptor dyes, which is necessary for following the time course of molecular interactions in living cells. Although the ratiometric approach is similar to the 3-cube FRET method (14), it uses less complicated calculations.The achievable signal to noise ratio is usually limited by the autofluorescence of the cells. Since autofluorescence is less pronounced toward the red spectral region, the preferred donor acceptor dye pair should be red-shifted as well, such as the AlexaFluor546 -AlexaFluor647 pair with 543 nm and 633 nm laser excitations, respectively. In a...

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Section: The Intensity-based Ratiometric Fret Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

et al. 2009

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“…Although several studies have already employed imaging tools to study cnidarian photophysiology, most efforts have investigated microscopic and environmental scales, through the use of microscopy (e.g. Salih et al 1995;Hanley et al 2006) and satellite imaging (e.g. Kutser et al 2006;Ziskin et al 2011), respectively.…”

Section: Introductionmentioning

confidence: 99%

Concurrent imaging of chlorophyll fluorescence, Chlorophyll a content and green fluorescent proteins‐like proteins of symbiotic cnidarians

et al. 2014

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Research on photosynthetic cnidarians has been mainly focused on the symbiosis established between the cnidarian host and its dinoflagellates endosymbionts from genus Symbiodinium. Despite the potential of imaging techniques for assessing the spatial distribution of key parameters of cnidarian photobiology, such as photochemical activity, chlorophyll a content or green fluorescent proteins (GFPs), to our best knowledge, no study has ever attempted to simultaneous map these three features. In this study, we developed a modified imaging pulse amplitude fluorometer by applying excitation light of different wavelengths and selectively detecting short spectral bands through bandpass filters. The imaging system was used to sequentially excite and quantify chlorophyll variable fluorescence (maximum quantum yield of photosystem II, F v /F m ), Chl a content (normalized difference vegetation index) and relative content of GFPs. The spatial distribution of these photophysiological parameters was mapped both horizontally, across the surface of the soft corals Sarcophyton cf. glaucum and Sinularia flexibilis and the zoanthid Protopalythoa sp., and vertically, throughout a vertical section of S. cf. glaucum. Results showed bleached areas within each individual coral colony and registered photophysiological changes with S. cf. glaucum tissue depth. Analysis of Protopalythoa sp. polyps' expansion revealed differential surface patterns of NDVI and GFP concentration, and a negative relation between these latter parameters within each polyp. This novel non-invasive approach allowed a high-resolution characterization of the spatial relationship between these key parameters through the analysis of image information on a pixel-by-pixel basis, which has great potential for investigating the physiological state of symbiotic associations.

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“…This equipment has the capacity to obtain unique fluorescence spectra of specific dye molecules that will increase our understanding of biological fluorescence emission process (9)(10)(11)(12)(13)(14)(15)(16). The popularity of this new field in biology has been described in a special issue of Cytometry that includes both review articles and specific application articles (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To insure that this equipment is functioning properly, new and improved tests have been developed to evaluate spectral performance on these instruments (1,2,5,6).…”

Section: Discussionmentioning

confidence: 99%

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Zucker¹,

Rigby²,

Clements³

et al. 2007

Cytometry Pt A

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Microspectroscopic fluorescence analysis with prism‐based imaging spectrometers: Review and current studies

Cited by 15 publications

References 35 publications

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Concurrent imaging of chlorophyll fluorescence, Chlorophyll a content and green fluorescent proteins‐like proteins of symbiotic cnidarians

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

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