Autofluorescence Imaging to Evaluate Cellular Metabolism

Summary

This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.

Abstract

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.

Introduction

Metabolism is the cellular process of producing energy. Cellular metabolism encompasses multiple pathways, including glycolysis, oxidative phosphorylation, and glutaminolysis. Healthy cells use these metabolic pathways to generate energy for proliferation and function, such as the production of cytokines by immune cells. Many diseases, including metabolic disorders, cancer, and neurodegeneration, are characterized by altered cellular metabolism¹. For example, some cancer cell types have elevated rates of glycolysis, even in the presence of oxygen, to generate molecules for the synthesis of nucleic acids, proteins, and lipids^2<....

Protocol

1. Cell plating for imaging

1. Aspirate the medium from an 80-90% confluent T-75 flask of MCF-7 cells, rinse the cells with 10 mL of sterile phosphate-buffered saline (PBS), and add 2 mL of 0.25% trypsin (1x) to detach the cells from the flask bottom.
2. Incubate the flask at 37 °C for ~4 min. Check the cells under the microscope to confirm detachment.
3. Immediately add 8 mL of culture medium to de-activate the trypsin.
4. Collect the cells in a conical tube (15 mL or 50 mL). Count the cells using a hemocytometer.
5. Centrifuge the cells 200 × g for 5 min.
6. Once centrifuged, aspirate th....

Results

The epithelial breast cancer cell line, MCF-7, was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. For fluorescence imaging, the cells were seeded at a density of 4 × 10⁵ cells per 35 mm glass-bottom imaging dish 48 h before imaging. The cells were imaged before and after cyanide treatment using the protocols stated above. The goal of the cyanide experiment is to confirm spectral isolation of NAD(P)H and FAD fluorescence and validate the imaging system and a.......

Discussion

Autofluorescence intensity and lifetime imaging have been widely used to assess metabolism in cells^21,55. FLIM is high resolution and therefore resolves single cells, which is important for cancer studies because cellular heterogeneity contributes to tumor aggression and drug resistance^{7,39,41,44,45}

Materials

Name	Company	Catalog Number	Comments
2-deoxy-d-glucose (2-DG)	Sigma	AC111980000; AC111980010; AC111980050; AC111980250
Antibiotic Antimicrobial (pen-strep)	Gibco	15240096
Cell Samples	American Type Culture Collection	N/A	MCF-7 cancer line
CellProfiler	Broad Institute	N/A	Image analysis software
Conical Tube	VWR	89039-664	15 mL conical tube
DMEM	ThermoFisher	11965092	Culture media
FAD dichroic mirror	Semrock	FF495-Di03-25x36	495 nm
FAD emission filter	Semrock	FF01-550/88-25	550/88 nm
FAD excitation filter	Semrock	FF01-458/64-25	458/64 nm
FBS	ThermoFisher	16000036
Fluorescence Lifetime Microscope	3i	N/A
Glass bottom dish	MatTek Corp	P35G-1.0-14-C
Multiphoton Laser	Coherent	N/A	2P Coherent Laser, Tunable 680 nm-1080 nm
NAD(P)H dichroic mirror	Semrock	FF409-Di03-25x36	409 nm
NAD(P)H emission filter	Semrock	FF02-447/60-25	447/60 nm
NAD(P)H excitation filter	Semrock	FF01-357/44-25	357/44 nm
PBS	ThermoFisher	70011044
Potassium Cyanide	Sigma-Aldrich	380970
SlideBooks 6	3i	N/A	Image acquisition software
SPCImage	Becker & Hickl GmbH	N/A	Fluorescence lifetime analysis software
Stage Top Incubator	okoLab	N/A
Trypsin	Biosciences	786-262
Urea	Sigma-Aldrich	U5128
YG beads	Polysciences	19096-2	Yg microspheres (20.0 µm)