Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy

Abstract

Monitoring of cell metabolism represents an important application area for fluorescence lifetime imaging microscopy (FLIM). In particular, assessment of mitochondrial membrane potential (MMP) in complex three-dimensional multicellular in vitro, ex vivo, and in vivo models would enable improved segmentation and functional discrimination of cell types, directly report on the mitochondrial function and complement the quenched-phosphorescence detection of cellular O 2 and two-photon excited FLIM of endogenous NAD(P)H. Here, we report the green and orange-emitting fluorescent dyes SYTO and tetramethylrhodamine methyl ester (TMRM) as potential FLIM probes for MMP. In addition to nuclear, SYTO 16 and 24 dyes also display mitochondrial accumulation. FLIM with the culture of human colon cancer HCT116 cells allowed observation of the heterogeneity of mitochondrial polarization during the cell cycle progression. The dyes also demonstrated good performance with 3D cultures of Lgr5-GFP mouse intestinal organoids, providing efficient and quick cell staining and compatibility with two-photon excitation. Multiplexed imaging of Lgr5-GFP, proliferating cells (Hoechst 33342-aided FLIM), and TMRM-FLIM allowed us to identify the population of metabolically active cells in stem cell niche. TMRM-FLIM enabled to visualize the differences in membrane potential between Lgr5-positive and other proliferating and differentiated cell types. Altogether, SYTO 24 and TMRM dyes represent promising markers for advanced FLIM-based studies of cell bioenergetics with complex 3D and in vivo models.Key terms FLIM; intestinal organoids; Lgr5-GFP; mitochondrial membrane potential; SYTO; tetramethylrhodamine methyl ester Energy production and regulation of cell metabolism are essential for the living cell. Corresponding biomarkers are important for cancer, stem and basic cell biology, drug screening and discovery, cell death and toxicity, and tissue engineering (1-4). Quantitative assessment of cell metabolism and mitochondrial function is possible via a number of techniques, which include analyses of oxygenation and oxygen consumption rate (OCR), mitochondrial membrane potential (MMP), glycolytic flux, ATP and redox status, analysis of metabolome and other approaches (5-8). Photoluminescence (i.e., fluorescence and phosphorescence)-based methods enable quantification of these parameters in formats of a plate reader, fluorescence microscope, and other readouts compatible with various cells and tissues. For in vivo and advanced microscopy applications, quantitative analysis of mitochondrial function is possible only by limited number of probes and approaches, such as measurement of cell oxygenation by phosphorescence quenching method (9,10), analysis of redox status by imaging of NAD(P)H, FAD + , and tryptophan autofluorescence by FLIM or by using various genetically-encoded fluorescent biosensors (11-15). The quantitative assessment of the direct indicator of mitochondrial function, MMP, remains a challenging task (16). This situation creates a gap in the...

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses

“…The heterogeneity of daughter cells cell-cycle within the stem cell niche is another factor potentially affecting measurements of O2: it is known that mitochondrial membrane polarization changes during the cell cycle and reaches a maximum during early S phase 49 suggesting that stem cell niche-residing cells can have enhanced utilization of OxPhos cyclically. Such mitochondrial heterogeneity within GFP + cells in intestinal organoids was recently reported 18 . Understanding the functional heterogeneity of differentiated epithelial cells within organoids is also important for an accurate interpretation of results.…”

“…Microscopy-based FLIM and PLIM methods, while rarely reported together, provided direct readouts of cell metabolism both within and outside Lgr5-GFP-labeled stem cell niches. Potentially, NAD(P)H-FLIM and O2-PLIM methods could be multiplexed with each other, and with additional measurement assays, such as labeling of other cell types (genetically or via fluorescent tracers), labeling cell proliferation 29 and measurement of mitochondrial membrane potential 18 . Curiously, the majority of commercially available FLIM-PLIM microscopes have limited microsecond PLIM measurement range 33 , many high-performance O2 probes are not suitable for twophoton excitation 37 , cannot stain organoids or possess other drawbacks 45,46 .…”

“…Lgr5-GFP (Lgr5-EGFP-ires-CreERT2) mouse intestinal organoid culture 35 were kindly provided by Prof. H. Clevers Lab (Hubrecht Institute, The Netherlands). Organoids were handled as described previously 18 and typically were grown in ENRVC media, consisting of Dulbecco modified Eagle's medium nutrient mixture F-12 [HAM] supplemented with 1% penicillin-streptomycin, 1% GlutaMax, 1% N2 supplement, 2% B-27 supplement, 1.25 mM NAC, 50 ng/ml EGF, 1 µg/ml R-spondin-1, 100 ng/ml Noggin, 1 mM sodium valproate and 3 µM CHIR99021. Intact organoids were seeded onto 20 µl of Matrigel in 35 mm tissue culture treated dishes (Sarstedt, Dublin, Ireland) in ENRVC medium, 1-2 days prior to imaging.…”

“…Collectively, organoids are amenable for studies of stem cell niche function by highthroughput next generation sequencing, metabolomics and high content microscopy screening, and by 'traditional' methods such as quantitative PCR, western blotting or fluorescence microscopy [13][14][15] . Until recently, only few works dealt with assessment of mitochondrial function of the stem cell niche in intestinal organoids 5,6,[16][17][18] . The standard approach is to analyze the extracellular flux, i.e.…”

Extracellular Ca2+-sensitive fluorescent protein biosensor based on a collagen-binding domain

“…We chose mouse Lgr5-GFP small intestinal organoid model [60], which enables live tracing of stem (Lgr5-GFP + ) and differentiated enterocyte cells, grows in Matrigel matrix and recapitulates most of the functions of the native epithelium [61,62]. Mouse intestinal organoids have been already extensively evaluated in a number of FLIM and PLIM assays, informing on cell oxygenation, proliferation, mitochondrial polarization, redox status and others [33,[63][64][65][66][67]. Although in previous work we have demonstrated pH-FLIM imaging of organoids combined with O2 imaging, we were not able to measure acidification directly in Matrigel and not assessed extracellular Ca 2+ [33].…”