Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera

Krishnan, Ramanujan Venkata; Saitoh, H.; Terada, Hirotoshi; Centonze, Victoria E.; Herman, Brian

doi:10.1063/1.1569410

Cited by 98 publications

(48 citation statements)

References 17 publications

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“…Finally, when using a streak camera, very high temporal resolution at the picosecond level is feasible but this detector is intrinsically limited to line imaging (Krishnan et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Leray

Riquet

Richard

et al. 2008

Microscopy Res & Technique

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Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system.

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“…Finally, when using a streak camera, very high temporal resolution at the picosecond level is feasible but this detector is intrinsically limited to line imaging (Krishnan et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Leray

Riquet

Richard

et al. 2008

Microscopy Res & Technique

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“…Technically, this can be done by using a Time Correlated Single Photon Counting (TCSPC) system (10), or by measuring the fluorescence signal either using a time-gated detector (12), or a streak camera (13). Regardless of the technique applied, the theoretical fluorescence intensity profile I(t) is defined as:…”

Section: The Characterization Of Dynamic Interactions Between Biomolementioning

confidence: 99%

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

et al. 2011

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Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate the local environment of fluorophores in living cells. To correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently long laser exposure times. This is an issue because long exposure times are incompatible with the observation of dynamic molecular events and induce cellular stress. To minimize exposure time, we have developed an original approach that statistically inflates the number of collected photons. Our approach, called Adaptive Monte Carlo Data Inflation (AMDI), combines the well-known bootstrap technique with an adaptive Parzen kernel. We here demonstrate using both Monte Carlo simulations and live cells that our robust method accurately estimate fluorescence lifetimes with exposure time reduced up to 50 times for monoexponential decays (corresponding to a minimum of 20 photons/pixel), and 10 times for biexponential decays (corresponding to a minimum of 5,000 photons/pixel), compared to standard fitting method. Thanks to AMDI, in Förster resonance energy transfer experiments, it is possible to estimate all fitting parameters accurately without constraining any parameters. By reducing the commonly used spatial binning factor, our technique also improves the spatial resolution of FLIM images. ' 2011 International Society for Advancement of Cytometry

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“…Alternative techniques exist to TCSPC--FLIM, such as wide--field time gated FLIM (Webb et al, 2002) and streak--camera based FLIM (Biskup et al, 2004;Krishnan et al, 2003a;Krishnan et al, 2003b). Wide--field time gated FLIM is equivalent to successive acquisitions of the fluorescence emission taken at various nanosecond delays after the excitation.…”

Section: Tcspc--flimmentioning

confidence: 99%

Multifocal multiphoton microscopy with adaptive optical correction

et al. 2013

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Multiphoton microscopy (MPM) is a remarkably versatile technique in biologicalimaging. MPM provides increased depth over confocal imaging and can be combined with other imaging techniques such as fluorescence lifetime imaging microscopy (FLIM), adding functional information. FLIM read--out is relatively straightforward using time--correlated single photon counting (TCSPC). Fluorescence lifetime detection enhances the power of multiphoton imaging to allow three dimensional, concentration independent, measurements of environmental parameters such as pH, Oxygen tension and Ca 2+ in addtion to the interaction or conformational modification of proteins by Förster resonant energy transfer (FRET); the latter is a particular focus of the Dimbleby research groups at King's CollegeLondon. However, there are significant limitations in both FLIM and MM.Limitations of TCSPC--FLIM include prolonged acquisition times along with signal and resolution degradation as a function of depth. This thesis demonstrates advancements multiphoton fluorescence lifetime imaging through improvements in two principal areas: speed and resolution at depth.In order to improve acquistion rates a multifocal multiphoton microscope (MMM) capable of rapid, parallelized TCSPC--FLIM was developed --MegaFLI. Acquisitions demonstrate rapid 3--dimensional, high temporal resolution FLIM in vivo Zebrafish.Performed by massively parallel excitation/detection the speed is signficantly improved by a factor of 64.In parallel to the MegaFLI project, a second microscope employing adaptive optical correction has been developed. The introduction of Adaptive Optics (AO) serves to improve imaging quality by counteracting the refractive index heterogeneities introduced by the sample, limiting the imaging depth. Incorporated with a single beam scanning FLIM system, a pupil--segmentation AO--TCSPC--FLIM demonstrates improved signal--to--noise ratio (SNR) and resolution, permiting a more accurate determination of fluorescent lifetime in turbid media. 3 DECLARATION OF AUTHORSHIPThe work present is my own work with the exception of TRI2, which was developed The introduction (Chapter 1) concentrates on optical methods developed in the context of multiphoton microscopy (MPM) or more specifically two--photon microscopy (TPM) (Denk et al., 1990). TPM enables long--term imaging of in vivo biological specimens, image generation at increased tissue depth, and higher signal--to--noise images compared to wide--field and confocal approaches (Helmchen and In comparison to confocal microscopy, two--photon microscopy (TPM) offers considerable benefits since excitation is confined to a femtolitre volume in the vicinity of the focal plane, reducing overall photobleaching and phototoxicity of thick samples (Centonze and White, 1998;Denk et al., 1990;Helmchen and Denk, 2005;Williams et al., 2001). Out--of--focus excitation is avoided due to the localization of the excitation making the confocal pinhole obsolete (Amos and Where is the numerical aperture of the obj...

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Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera

Cited by 98 publications

References 17 publications

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

Multifocal multiphoton microscopy with adaptive optical correction

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