Fluorescence Lifetime-Resolved Imaging Microscopy: A General Description of Lifetime-Resolved Imaging Measurements

Clegg, Robert M.; Schneider, P. C.

doi:10.1007/978-1-4899-1866-6_2

Cited by 46 publications

(45 citation statements)

References 20 publications

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“…While E can be determined by measurements of the fluorescence lifetime of the donor fluorophore [8][9][10] or of the polarization of light emitted from the sample, 11,12 the most commonly used techniques depend upon accurate measurements of the intensities of light emitted by one or both of the donor and acceptor fluorophores. A requirement for FRET is that the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, with a greater degree of overlap yielding larger transfer efficiencies.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

et al. 2013

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Abstract. Accurate quantification of Förster resonance energy transfer (FRET) using intensity-based methods is difficult due to the overlap of fluorophore excitation and emission spectra. Consequently, mechanisms are required to remove bleedthrough of the donor emission into the acceptor channel and direct excitation of the acceptor when aiming to excite only the donor fluorophores. Methods to circumvent donor bleedthrough using the unmixing of emission spectra have been reported, but these require additional corrections to account for direct excitation of the acceptor. Here we present an alternative method for robust quantification of FRET efficiencies based upon the simultaneous spectral unmixing of both excitation and emission spectra. This has the benefit over existing methodologies in circumventing the issue of donor bleedthrough and acceptor cross excitation without the need for additional corrections. Furthermore, we show that it is applicable with as few as two excitation wavelengths and so can be used for quantifying FRET efficiency in microscope images as easily as for data collected on a spectrofluorometer. We demonstrate the accuracy of the approach by reproducing efficiency values in well characterized FRET standards: HEK cells expressing a variety of linked cerulean and venus fluorescent proteins. Finally we describe simple ImageJ plugins that can be used to calculate and create images of FRET efficiencies from microscope images.

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Section: Introductionmentioning

confidence: 99%

Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

et al. 2013

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“…Fluorescence lifetime imaging is a powerful technique used for in vitro, live cell or live animal (in vivo) measurements [24][25][26]. In contrast to other fluorescence-based microscopy ap- proaches, FLIM is not merely concerned with fluorescence intensity or spectrum, but also in the fluorescence decay time scale after excitation (fluorescence lifetime).…”

Section: Application 2: Flim Measurementsmentioning

confidence: 99%

“…It can also report on the different environments in which a known fluorophore is located, if its fluorescence lifetime is affected by the chemical species surrounding it. In particular, distance-dependent fluorescence resonant energy transfer (FRET) between fluorophores bound to different molecular species, can be detected by a reduced lifetime of the "donor" species compared to its lifetime in the absence of nearby "acceptor" molecules [24][25][26].…”

Section: Application 2: Flim Measurementsmentioning

confidence: 99%

Architecture and applications of a high resolution gated SPAD image sensor

Burri¹,

Maruyama²,

Michalet³

et al. 2014

Opt. Express

105

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Abstract:We present the architecture and three applications of the largest resolution image sensor based on single-photon avalanche diodes (SPADs) published to date. The sensor, fabricated in a high-voltage CMOS process, has a resolution of 512 x 128 pixels and a pitch of 24 μm. The fill-factor of 5% can be increased to 30% with the use of microlenses. For precise control of the exposure and for time-resolved imaging, we use fast global gating signals to define exposure windows as small as 4 ns. The uniformity of the gate edges location is~140 ps (FWHM) over the whole array, while in-pixel digital counting enables frame rates as high as 156 kfps. Currently, our camera is used as a highly sensitive sensor with high temporal resolution, for applications ranging from fluorescence lifetime measurements to fluorescence correlation spectroscopy and generation of true random numbers.

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“…Fluorescence resonance energy transfer (FRET) is widely used for studying biological systems on the nanometer scale (i.e., Ͻ10 nm; ref. 3). FRET occurs over distances similar to the Förster radius, which is Ϸ5 nm for common fluorophores (4).…”

mentioning

confidence: 99%

Nanometer-localized multiple single-molecule fluorescence microscopy

Wu²,

Mets³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

292

231

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Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale ''rulers'' to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate singlemolecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated. It is well known that the spatial resolution of an optical system is limited by the Rayleigh criterion (1),where is the wavelength of the collected photons and N.A. is the numerical aperture of the system. Using visible light (i.e., Ϸ500 nm) and a high N.A. objective (i.e. Ͼ1) allows achieving Ϸ250-nm lateral resolution. However, most biological macromolecules (DNA, RNA, proteins, etc.) and molecular machines (e.g., ribosome and spliceosome) are much smaller in two or all three dimensions, with important features or subassemblies lying within 10 nm of each other (2). Fluorescence resonance energy transfer (FRET) is widely used for studying biological systems on the nanometer scale (i.e., Ͻ10 nm; ref. 3). FRET occurs over distances similar to the Förster radius, which is Ϸ5 nm for common fluorophores (4). However, FRET only gives the relative distance between two probes, whereas their absolute positions remain unknown. Centroid localization, which has been used for single-particle tracking, allows determination of the centroid position of the particle to a much better precision than the length scale defined by the Rayleigh criterion (5-7). Recently, this approach to localizing fluorophores has been applied to single dye molecules (Cy3 and Rhodamine dyes) bound to molecular motors, and 2-nm precision in localization has been achieved when an oxygen scavenging buffer was used to suppress photobleaching (8, 9). However, centroid localization can only be used for isolated single molecules and it does not improve the resolution of the imaging system. It has been shown that two probes with different fluorescent wavelengths can be resolved within 10 nm because they are spectrally distinguishable (10). This method does not help when two or more objects with the same emission spectrum are within a diffraction-limited spot. Imaging applications beyond even the capabilities of ''su...

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Fluorescence Lifetime-Resolved Imaging Microscopy: A General Description of Lifetime-Resolved Imaging Measurements

Cited by 46 publications

References 20 publications

Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

Architecture and applications of a high resolution gated SPAD image sensor

Nanometer-localized multiple single-molecule fluorescence microscopy

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