FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy

Santos, Carla De Los; Chang, Ching Wei; Mycek, Mary Ann; Cardullo, Richard A.

doi:10.1002/mrd.22501

Cited by 53 publications

(25 citation statements)

References 124 publications

(211 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Fluorescence-based microscopy techniques have become important tools for tissue imaging and open new possibilities for investigating the tissue behavior in response to different influences at a molecular level. 2 One of many fluorescencebased microscopic imaging modalities is the fluorescence lifetime imaging microscopy (FLIM), which has successfully been used to investigate biological processes in human tissue, as well as on the cellular level. 3 The principle of time-correlated single photon counting (TCSPC) is used for FLIM in this context.…”

Section: In Vitro Fluorescence Detection: Fluorescence Lifetime Imagimentioning

confidence: 99%

Review of clinical approaches in fluorescence lifetime imaging ophthalmoscopy

et al. 2018

View full text Add to dashboard Cite

Autofluorescence-based imaging techniques have become very important in the ophthalmological field. Being noninvasive and very sensitive, they are broadly used in clinical routines. Conventional autofluorescence intensity imaging is largely influenced by the strong fluorescence of lipofuscin, a fluorophore that can be found at the level of the retinal pigment epithelium. However, different endogenous retinal fluorophores can be altered in various diseases. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an imaging modality to investigate the autofluorescence of the human fundus in vivo. It expands the level of information, as an addition to investigating the fluorescence intensity, and autofluorescence lifetimes are captured. The Heidelberg Engineering Spectralis-based fluorescence lifetime imaging ophthalmoscope is used to investigate a 30-deg retinal field centered at the fovea. It detects FAF decays in short [498 to 560 nm, short spectral channel (SSC) and long (560 to 720 nm, long spectral channel (LSC)] spectral channels, the mean fluorescence lifetimes (τm) are calculated using bi- or triexponential approaches. These are meant to be relatively independent of the fluorophore's intensity; therefore, fluorophores with less intense fluorescence can be detected. As an example, FLIO detects the fluorescence of macular pigment, retinal carotenoids that help protect the human fundus from light damages. Furthermore, FLIO is able to detect changes related to various retinal diseases, such as age-related macular degeneration, albinism, Alzheimer's disease, diabetic retinopathy, macular telangiectasia type 2, retinitis pigmentosa, and Stargardt disease. Some of these changes can already be found in healthy eyes and may indicate a risk to developing such diseases. Other changes in already affected eyes seem to indicate disease progression. This review article focuses on providing detailed information on the clinical findings of FLIO. This technique detects not only structural changes at very early stages but also metabolic and disease-related alterations. Therefore, it is a very promising tool that might soon be used for early diagnostics.

show abstract

Section: In Vitro Fluorescence Detection: Fluorescence Lifetime Imagimentioning

confidence: 99%

Review of clinical approaches in fluorescence lifetime imaging ophthalmoscopy

et al. 2018

View full text Add to dashboard Cite

show abstract

“…FLIM-FRET reflects the spatial distance between two molecules inside the cell based on the FRET between two fluorophores, which can only occur when they are less than 10 nm apart. This energy transfer results in a decrease in the fluorescence lifetime of the donor, measured by FLIM ( 25 , 26 ) (Figure 6B and Supplementary Figure S6 ). We first determined the fluorescence lifetime of the donor alone (CDK8-GFP), either in treated or non-treated cells, and either followed or not by CSK washes.…”

Section: Resultsmentioning

confidence: 99%

Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal

Lebraud

Pinna

Siberchicot

et al. 2020

Nucleic Acids Research

View full text Add to dashboard Cite

One of the most abundant DNA lesions induced by oxidative stress is the highly mutagenic 8-oxoguanine (8-oxoG), which is specifically recognized by 8-oxoguanine DNA glycosylase 1 (OGG1) to initiate its repair. How DNA glycosylases find small non-helix-distorting DNA lesions amongst millions of bases packaged in the chromatin-based architecture of the genome remains an open question. Here, we used a high-throughput siRNA screening to identify factors involved in the recognition of 8-oxoG by OGG1. We show that cohesin and mediator subunits are required for re-localization of OGG1 and other base excision repair factors to chromatin upon oxidative stress. The association of OGG1 with euchromatin is necessary for the removal of 8-oxoG. Mediator subunits CDK8 and MED12 bind to chromatin and interact with OGG1 in response to oxidative stress, suggesting they participate in the recruitment of the DNA glycosylase. The oxidative stress-induced association between the cohesin and mediator complexes and OGG1 reveals an unsuspected function of those complexes in the maintenance of genomic stability.

show abstract

“…Fluorescence lifetime is the time of electron transition to the excited state and its return to the ground state. Changes in the fluorescence lifetimes of NADH and FAD indicate changes in metabolic pathways such as glycolysis, oxidative phosphorylation, the pentose phosphate pathway, and the complex of biosynthetic processes in the cell [120][121][122]. Several reports showed the applicability of FLIM for assessing metabolic changes in primary sclerosing cholangitis and biliary tract fibrosis, chronic fibrosis, steatosis, hepatocellular carcinoma, and liver ischemia-reperfusion syndrome [95, 96,100].…”

Section: Promising Methods For Assessing the Structure Function Andmentioning

confidence: 99%

Modern Methods for Assessing the Regenerative Potential of the Liver after Partial Hepatectomy (Review)

Rodimova¹,

Kuznetsova²,

Bobrov³

et al. 2019

Sovrem Tehnol Med

View full text Add to dashboard Cite

The review addresses the main methods for assessing the function and regenerative potential of the liver. They include both the traditional methods, commonly used in clinical practice, and the latest promising techniques suitable for the analysis of cellular and tissue pathology and having a proven diagnostic value.It is known that the dynamics of liver regeneration is reflected in the metabolic status of liver cells, their morphology, and the molecular rearrangement. Therefore, by looking at these parameters we will be able to assess the regenerative potential of the liver as a whole.At present, the most promising method is represented by multiphoton microscopy able to generate the second harmonic; there are also such techniques as coherent anti-stokes Raman spectroscopy (CARS), stimulated Raman scattering (SRS) microscopy, and fluorescence lifetime imaging microscopy (FLIM). In addition, a number of options for analyzing metabolic and structural changes are provided by mass spectrometry, in particular time-of-flight secondary ion mass spectrometry (ToF-SIMS). Studies using these methods with in vivo models and with human biopsy samples demonstrate their relevance in biomedical research and in clinical practice alike.

show abstract

FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy

Cited by 53 publications

References 124 publications

Review of clinical approaches in fluorescence lifetime imaging ophthalmoscopy

Review of clinical approaches in fluorescence lifetime imaging ophthalmoscopy

Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal

Modern Methods for Assessing the Regenerative Potential of the Liver after Partial Hepatectomy (Review)

Contact Info

Product

Resources

About