Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

Hanley, Quentin S.; Subramaniam, Vinod; Arndt‐Jovin, Donna J.; Jovin, Thomas M.

doi:10.1002/1097-0320(20010401)43:4<248::aid-cyto1057>3.0.co;2-y

Cited by 111 publications

(103 citation statements)

References 52 publications

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“…In a previous publication on FLIM from our laboratory, we noted that detectable intra-image variations in lifetime are about an order of magnitude smaller than detectable inter-image variations (43). Thus, methods exploiting the high sensitivity of intra-image lifetime variations should provide improved discrimination of processes leading to FRET.…”

Section: Discussionmentioning

confidence: 88%

“…viously (15,18,43). The PAM system consisted of a DMD mounted in the primary image plane of a Nikon E-600 microscope, where it served to define patterns of illumination and detection (Fig.…”

Section: Programmable Array Microscopementioning

confidence: 99%

“…The light from the output phosphor plate of the intensifier was relayed with a pair of f/1.8 (Nikon AF Nikkor) camera lenses to a charge-coupled device camera (Apogee Instruments KX-2). Lifetime images were calibrated relative to rhodamine 6G in water (4.1 ns, [43]). …”

Section: Programmable Array Microscopementioning

confidence: 99%

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Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

et al. 2005

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Background: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. Methods and Results: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simu-

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Section: Discussionmentioning

confidence: 88%

Section: Programmable Array Microscopementioning

confidence: 99%

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Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

et al. 2005

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“…As an example we have simulated the correlation time and lifetime distributions for AB values that deviate by 1% of the average value for a rotator with correlation time of 2 ns and lifetime of 4 ns (initial anisotropy 0.4, limiting anisotropy 0 (Hanley et al, 2001) whereas the minimum detectable intra image difference is 4-5 ps (<1%). We would expect that the AB analysis will lead to large errors if used indiscriminately on a pixel-by-pixel basis.…”

Section: Noise and Aperture Depolarization Effectsmentioning

confidence: 99%

The polarized AB plot for the frequency‐domain analysis and representation of fluorophore rotation and resonance energy homotransfer

Clayton

2008

Journal of Microscopy

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SummaryThe graphical representation of single-frequency phasemodulation fluorescence lifetime imaging data, referred to as the AB plot, is extended to take into account measurements of the polarized components of the fluorescence. For a hindered rotator model (characterized with a single excited-state lifetime, a single rotational correlation time and limiting initial and final anisotropies) the rotational correlation time and the excited lifetime can be determined from the AB plot of any two of the following emission components: parallel, perpendicular, total emission or combinations thereof. A strategy for resolving the component hindered rotations and lifetimes for mixtures of two hindered rotators from measurements of the total, parallel and perpendicular components of the emission is developed. The analysis does not require prior knowledge of the initial limiting anisotropy or of the steady-state anisotropy or of the excited state lifetime. Plots in polarized AB space derived for heterogeneous systems are constructed to aid interpretation of frequency-domain dynamic depolarization imaging microscopy experiments. These plots can be used to distinguish spatially dependent rotational correlation time heterogeneity from heterogeneity in limiting anisotropies. The effects of noise and aperture depolarization are discussed. It is anticipated that the polarized AB plot will provide a useful adjunct to existing methods for visualizing and analysing dynamic polarization phenomena arising from molecular dynamics and homo-energy transfer in singlefrequency microscopy applications.

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“…This group of coral proteins, which have a GFP-like structural motif, have a number of interesting properties. They show complex flickering (22), color change from green to red during ''maturation'' (23), photoinduced color changes (24), and tetramer formation (26). While much is known about the properties of these proteins in isolation, the photophysical properties of intact corals are not well understood.…”

Section: Imaging Spectroscopic Photobleachingmentioning

confidence: 99%

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

2006

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Background: Fluorescence imaging spectroscopy is a powerful but under-utilized tool. This article gives perspective on the use of imaging spectroscopy, and provides two examples of imaging spectroscopy done with a prism-based system. The intent is to give insight into the power of imaging spectroscopy when used in combination with other imaging techniques. In particular, studies of intact coral photobleaching and beads designed to show energy transfer are reported. In the bead study, spectroscopic lifetime imaging was performed at each photobleaching step. Results: Spectroscopic photobleaching of the hard coral, Montastrea annularis, revealed two spectral regions. A region in the red portion of the spectrum bleached rapidly while progressively increasing fluorescence was observed over a wide portion of the spectrum. This behavior is consistent with current theories for the role of fluorescent proteins in corals.

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Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

Cited by 111 publications

References 52 publications

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

The polarized AB plot for the frequency‐domain analysis and representation of fluorophore rotation and resonance energy homotransfer

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

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