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I am interested in metabolic imaging FLIM. Where should I start reading?

Metabolic imaging of cells and tissues by FLIM is a powerful tool in biology and medical research. There are many applications ranging from cancer detection, cell metabolic activity classification, embryo classification for fertilisation and many more. Metabolic imaging is mainly based on detection of autofluorescence of endogenous fluorophores such as NADH/NAD(P)H FAD. Fluorescence lifetime measured by FLIM contributes valuable information for quantitative metabolic imaging.

Here, we have compiled a few starting points for people interested in metabolic imaging.

bh TCSPC handbook 9th Ed.

The bh TCSPC handbook 9th Ed. contains multiple sections about metabolic imaging and related topics. The handbook is available for download. Hear a few starting points:
p. 361: Metabolic Imaging by Simultaneous FLIM of NADH and FAD
p. 493: Autofluorescence FLIM of Cells and Tissues
p. 500: Parameters Characterising the Cell Metabolism
p. 507: Review of NAD(P)H / FAD FLIM Literature
p. 518: Oxygen Sensing by Phosphorescence Lifetime Measurement

Application Note

The application note “Metabolic Imaging with the DCS-120 Confocal FLIM System: Simultaneous FLIM of NAD(P)H and FAD” briefly describes the basics of metabolic imaging (Redox ratio, OMI index, FLIRR index) and how it is performed on the DCS-120 Confocal Scanning FLIM System.


Becker, W. (Ed.), 2015. Advanced Time-Correlated Single Photon Counting Applications, Springer Series in Chemical Physics. Springer International Publishing, Cham. Link

Dmitriev, R.I. (Ed.), 2017. Multi-Parametric Live Cell Microscopy of 3D Tissue Models, Advances in Experimental Medicine and Biology. Springer International Publishing, Cham.

Scientific papers

There are numerous scientific papers related to metabolic imaging and NADH/FAD detection. The list below is by far incomplete, yet a good starting point for reading.

Review: Schaefer, P.M., Kalinina, S., Rueck, A., Arnim, C.A.F. von, Einem, B. von, 2019. NADH Autofluorescence—A Marker on its Way to Boost Bioenergetic Research. Cytometry Part A 95, 34–46.

Shcheslavskiy, V.I., Shirmanova, M.V., Dudenkova, V.V., Lukyanov, K.A., Gavrina, A.I., Shumilova, A.V., Zagaynova, E., Becker, W., 2018. Fluorescence time-resolved macroimaging. Opt. Lett., OL 43, 3152–3155.

Cao, R., Wallrabe, H., Siller, K., Rehman Alam, S., Periasamy, A., 2019. Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H. Cytometry A 95, 110–121.

Gómez, C.A., Sutin, J., Wu, W., Fu, B., Uhlirova, H., Devor, A., Boas, D.A., Sakadžić, S., Yaseen, M.A., 2018. Phasor analysis of NADH FLIM identifies pharmacological disruptions to mitochondrial metabolic processes in the rodent cerebral cortex. PLOS ONE 13, e0194578.

Blacker, T.S., Mann, Z.F., Gale, J.E., Ziegler, M., Bain, A.J., Szabadkai, G., Duchen, M.R., 2014. Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat Commun 5.

Pastore, M.N., Studier, H., Bonder, C.S., Roberts, M.S., 2017. Non-invasive metabolic imaging of melanoma progression. Experimental Dermatology 26, 607–614.

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