Phasor method to fluorescence lifetime microscopy to discriminate metabolic state of cells in living tissue
A label-free imaging method to monitor stem cell metabolism discriminates different states of stem cell as they differentiate in a living tissues. We use intrinsic fluorescence biomarkers and the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM). We identify and map intrinsic fluorophores such as collagen, retinol, retinoic acid, flavins, nicotinamide adenine dinucleotide (NADH) and porphyrin. We measure the phasor values of germ cells in C. Elegans germ line. Their metabolic fingerprint cluster according to their differentiation state, reflecting changes in FAD concentration and NADH binding during the differentiation pathway. The phasor approach to lifetime imaging provides a label-free, fit-free and sensitive method to identify different metabolic state of cells during differentiation, to sense small changes in the redox state of cells and may identify symmetric and asymmetric divisions and predict cell fate.
1. A method for discriminating the metabolic state of cells with in vivo measurements in a metabolically active in vivo tissue which is part of a living mammalian individual, comprising
using a fluorescence lifetime imaging microscope apparatus that acquires fluorescence lifetime imaging microscopy (FLIM) data from fluorescence signals of the in vivo tissue; and
using an electronic circuit configured to apply fluorescence lifetime imaging to an autofluorescence signal of the tissue and thereby acquire autofluorescence FLIM data,
wherein the electronic circuit is configured to
i) receive information identifying at least two selected cells of the tissue,
ii) calculate an average autofluorescence phasor value for a region of each selected cell based on phasor values determined from autofluorescence FLIM data for each selected cell, and
iii) generate a scatterplot representing redox states of the selected cells based on the average autofluorescence phasor values.
2. The method of claim 1 , wherein the electronic circuit is configured to calculate a relative concentration of a tissue or cell component based on the fluorescence lifetime imaging microscopy data.
3. The method of claim 1 , wherein the electronic circuit is configured to monitor metabolic changes of cells that occur after (a) drug interaction, (b) oxidative stress, (c) differentiation, or (d) carcinogenesis.
4. The method of claim 1 , wherein the electronic circuit is configured to detect differences in redox ratios of cells in the tissue.
5. The method of claim 1 , wherein the system is able to perform high throughput screening to detect precancer stages and perform early diagnosis in an individual.
6. The method of claim 1 , wherein the electronic circuit is configured to perform multi-harmonic analysis of the fluorescence lifetime imaging data using higher harmonics of laser repetition rate.
7. The method of claim 1 , wherein the autofluorescence signal is from NADH.
8. The method of claim 1 , wherein the autofluorescence signal is from NADH, FAD and/or another flavin.