Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Levchenko, Svitlana M.; Pliss, Artem; Qu, Junle

doi:10.1142/s1793545817300099

Cited by 21 publications

(14 citation statements)

References 58 publications

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“…[3][4][5][6][7][8][9] It can thus be conveniently used to achieve quantitative measurements and to distinguish di®erent probes with similar°uorescence spectra. Importantly, according to the relationship between°uorescence lifetime and the environment around°uorescent molecules, FLIM technology can favorably be used to quantitatively measure various biochemical parameters in the microenvironment, 6 such as oxygen content, 5,10,11 pH, 12,13 metabolic state, 10,14,15 viscosity, 16,17 solution hydrophobicity, ion concentrations, 18,19 quencher distribution, temperature, 20 and°uorescence resonance energy transfer (FRET) e±ciency. [21][22][23][24] Therefore, FLIM has played an increasingly important role in the biomedical¯eld in the past two decades.…”

Section: Introductionmentioning

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: Introductionmentioning

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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“…The availability of genetically-encoded fluorophores such as fluorescent proteins (FPs) initiated a revolution in biological imaging. 1 Routine use of FPs in assays involving technologies such as Förster resonance energy transfer (FRET), 2 fluorescence lifetime imaging microscopy (FLIM), 3,4 molecular sensing, 5 and nanoscopy, 6 make them indispensible tools for biological research. Current efforts focus on developing FPs with excitation and emission in the far-red/near-infrared wavelengths and with photophysical properties such as photoswitching and fluorescence intermittency optimized for super-resolution imaging modalities.…”

Section: Introductionmentioning

confidence: 99%

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Mukherjee

Hung

Douglas

et al. 2020

Preprint

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The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M -1 cm -1 ), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations -M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using sitedirected mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

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“…Thus, the images of these areas are lack of enough contrast. This could be improved by combining the 3PFM imaging with the°uorescence lifetime imaging microscopy (FLIM), which is suitable for noninvasive study of intracellular processes, 15,16 micro°uidic systems, 17 remote sensing, 18,19 lipid order problems in physical chemistry, 20 temperature sensing 21 and clinical medicine. 22 FLIM can provide a more sensitive and precise image based on the weak signals, compared with the tra-ditional°uorescence intensity imaging.…”

Section: Introductionmentioning

confidence: 99%

Aggregation-induced emission luminogen for in vivo three-photon fluorescence lifetime microscopic imaging

et al. 2019

J. Innov. Opt. Health Sci.

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Compared with visible light, near-infrared (NIR) light has deeper penetration in biological tissues. Three-photon fluorescence microscopy (3PFM) can effectively utilize the NIR excitation to obtain high-contrast images in the deep tissue. However, the weak three-photon fluorescence signals may be not well presented in the traditional fluorescence intensity imaging mode. Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser. Moreover, fluorescence lifetime imaging microscopy (FLIM) can detect weak signals by utilizing time-correlated single photon counting (TCSPC) technique. Thus, it would be an improved strategy to combine the 3PFM imaging with the FLIM together. Herein, DCDPP-2TPA, a novel aggregation-induced emission luminogen (AIEgen), was adopted as the fluorescent probes. The three-photon absorption cross-section of the AIEgen, which has a deep-red fluorescence emission, was proved to be large. DCDPP-2TPA nanoparticles were synthesized, and the three-photon fluorescence lifetime of which was measured in water. Moreover, in vivo three-photon fluorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home-made optical system. High contrast cerebrovascular images of different vertical depths were obtained and the maximum depth was about 600 [Formula: see text]m. Even reaching the depth of 600 [Formula: see text]m, tiny capillary vessels as small as 1.9 [Formula: see text]m could still be distinguished. The three-photon fluorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water. A vivid 3D reconstruction was further organized to present a wealth of lifetime information. In the future, the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.

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Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Cited by 21 publications

References 58 publications

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

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

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Aggregation-induced emission luminogen for in vivo three-photon fluorescence lifetime microscopic imaging

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