Fluorescence Recovery After Photobleaching (FRAP) with simultaneous Fluorescence Lifetime and time-resolved Fluorescence Anisotropy Imaging (FLIM and tr-FAIM)

Teijeiro-Gonzalez, Yurema; Marois, Alix Le; Economou, A. M.; Hirvonen, L; Levitt, James A.; Beavil, Andrew J.; Beavil, Rebecca L.; Crnjar, Alessandro; Molteni, Carla; Ortiz-Zapater, Elena; Parsons, Maddy; Suhling, Klaus

doi:10.1117/12.2508692

Cited by 3 publications

(6 citation statements)

References 63 publications

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“…A simultaneous combination of FRAP-FLIM and time-resolved anisotropy has been reported, using a similar temporal mosaic recording method. 67,68 A way to resolve even faster dynamic lifetime e®ects is to combine the TCSPC recording with line scanning, which is also called°uorescence lifetimetransient scanning (FLITS). 18,39,69 The fastest line times for galvanometer scanners are in the range of 1 ms, consequently transient e®ects can be resolved down to about 1 ms.…”

Section: Temporal Mosaic Recording With Triggered Accumulationmentioning

confidence: 99%

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Liu

Lin

Becker

et al. 2019

J. Innov. Opt. Health Sci.

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Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

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Section: Temporal Mosaic Recording With Triggered Accumulationmentioning

confidence: 99%

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Liu

Lin

Becker

et al. 2019

J. Innov. Opt. Health Sci.

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“…[11][12][13] Yet, 2-dimensional (2D) time resolved fluorescence anisotropy imaging (TR-FAIM) has been reported only a handful of times. [14][15][16][17][18][19][20][21][22] Despite the lack of progression of TR-FAIM methods, 2D images of the rotational diffusion of rigid dyes using TR-FAIM has great potential for viscosity studies of biological systems. Partly because the investigator can choose a probe whose fluorescence lifetime is sensitive to another cellular environment parameter, and simultaneously capture FLIM and TR-FAIM images of the biological sample.…”

Section: Introductionmentioning

confidence: 99%

“…[23][24][25][26] However, only few demonstrations of TCSPC TR-FAIM of biological samples have been reported. [16][17][18] One reason for this could be due to the lack of available photo-detectors practical for TCSPC TR-FAIM. TCSPC FLIM images typically require hundreds of photons in a pixel to accurately resolve a mono-exponential fluorescence decay, 27 whereas TR-FAIM images require tens of thousands of photons in a pixel to accurately resolve a mono-exponential anisotropy decay.…”

Section: Introductionmentioning

confidence: 99%

Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array

Obeid Mogridge,

Nedbal,

Awale

et al. 2024

Multiphoton Microscopy in the Biomedical Sciences XXIV

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The time-resolved fluorescence anisotropy of a biological sample can reveal rotational dynamics and structure of a fluorescent probe's local environment. Here, methods are presented towards developing widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array (QuantICAM). Such detector allows for simultaneous time-correlated single photon counting (TCSPC) measurements in each pixel with singlephoton sensitivity and picosecond time resolution. Our method integrates the SPAD array with a widefield microscope, and automated rotating polarizers in the excitation and emission pathways to demonstrate TR-FAIM. We have shown the robustness of the method through spectroscopic measurements of the fluorophore PM546, and we have demonstrated the usefulness of simultaneous FLIM and TR-FAIM for studying properties of plasma membranes in live yeast cells.

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“…These include studying the dynamic properties of proteins, [18][19][20] estimating the internal viscosities of membranes, 21 performing fluorescencepolarization immunoassays, 22,23 probing molecular proximity manifested by hetero-or homo-FRET assays, 20,24,25 and monitoring cellular activities. 14,26 Nevertheless, since the pioneering work in rotational correlation time imaging of Siegel et al in the time domain (TD) (2003) 4 and Clayton et al in the frequency domain (FD) (2002), 27 only few reports described their extension into two-dimensional (2D) TR-FA imaging (TR-FAIM) in the TD 26,[28][29][30] and almost none in the FD. 31 Nonetheless, there are a variety of works that signify the use of FD measurements for the resolution of fluorescence intensity (FI) and FA decays thus probing cell behavior and monitoring changes in its microenvironment.…”

Section: Introductionmentioning

confidence: 99%

“…in the time domain (TD) (2003) 4 and Clayton et al. in the frequency domain (FD) (2002), 27 only few reports described their extension into two-dimensional (2D) TR-FA imaging (TR-FAIM) in the TD 26 , 28 – 30 and almost none in the FD 31 . Nonetheless, there are a variety of works that signify the use of FD measurements for the resolution of fluorescence intensity (FI) and FA decays thus probing cell behavior and monitoring changes in its microenvironment 16 , 32 …”

Section: Introductionmentioning

confidence: 99%

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

et al. 2023

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Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore's diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, θ imaging is still rarely investigated in general and in carbon dots (CDs) in particular.Aim: To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and θ, together with the steady-state images of fluorescence intensity (FI) and FA (r ). Approach:The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. Results:The FLT of fluorescein samples was found to decrease from 4.01 AE 0.01 to 3.56 AE 0.02 ns, whereas both r and θ were significantly increased from 0.053 AE 0.012 to 0.252 AE 0.003 and 0.15 AE 0.05 to 11.25 AE 1.87 ns, respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of r from 0.100 AE 0.011 to 0.150 AE 0.013 and θ from 0.98 AE 0.13 to 1.65 AE 0.20 ns for the first CDs and from 0.280 AE 0.008 to 0.310 AE 0.004 and 5.55 AE 1.08 to 7.95 AE 0.97 ns for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs.Conclusions: Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, r , and θ). Nevertheless, θ was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

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Fluorescence Recovery After Photobleaching (FRAP) with simultaneous Fluorescence Lifetime and time-resolved Fluorescence Anisotropy Imaging (FLIM and tr-FAIM)

Cited by 3 publications

References 63 publications

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

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