2-D fluorescence lifetime imaging using a time-gated image intensifier

Dowling, K.; Hyde, S.C.W.; Dainty, J. C.; French, Paul M. W.; Hares, J.D.

doi:10.1016/s0030-4018(96)00618-9

Cited by 75 publications

(41 citation statements)

References 7 publications

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“…Moreover, the use of time-gating [137] or frequencymodulation [138] with intensifier-equipped cameras used in fluorescence lifetime imaging microscopy (FLIM) significantly reduces the effective sensitivity of these detectors, making those approaches inappropriate for single-molecule detection [139].…”

Section: (D) Detectors Used For Wide Field Single-molecule Imagingmentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

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Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

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Section: (D) Detectors Used For Wide Field Single-molecule Imagingmentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

View full text Add to dashboard Cite

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“…Fluorescence Lifetime Imaging (FLIM) techniques can be divided into time-domain and frequency-domain techniques, 6-9 photon counting 10,11 and analog techniques, 6,[12][13][14][15] and point-scanning 7,16 and widefield imaging techniques. 6,12,15,17 It also matters whether a technique acquires the signal waveform in a few time gates 10 or in a large number of time channels 11,18 and whether this happens simultaneously 10,11,18 or sequentially. 12,15,17 Virtually all combinations are in use.…”

Section: Introductionmentioning

confidence: 99%

“…6,12,15,17 It also matters whether a technique acquires the signal waveform in a few time gates 10 or in a large number of time channels 11,18 and whether this happens simultaneously 10,11,18 or sequentially. 12,15,17 Virtually all combinations are in use. This leads to a wide variety of instrumental principles.…”

Section: Introductionmentioning

confidence: 99%

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

Becker

Hirvonen

Milnes

et al. 2016

Review of Scientific Instruments

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We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the “start” input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the “stop” input. The time between start and stop is directly converted into position, with a resolution of 200–250 μm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.

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“…Several FLI schemes have been invented and successfully applied for extraction of temporal information [13][14][15]. These techniques are commonly divided into two subgroups depending on whether the fluorescence lifetime is extracted through analysis in the frequency or in the time domain.…”

Section: Introductionmentioning

confidence: 99%