Funding sources include the Cancer Prevention and Research Institute of Texas (CPRIT RP200668) and Texas A&#38;M University. Figure 1&#160;was created with BioRender.com.

1. Cell plating for imaging
<ol>
	<li>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.</li>
	<li>Incubate the flask at 37 &#176;C for ~4 min. Check the cells under the microscope to confirm detachment.</li>
	<li>Immediately add 8 mL of culture medium to de-activate the trypsin.</li>
	<li>Collect the cells in a conical tube (15 mL or ...

Autofluorescence intensity and lifetime imaging have been widely used to assess metabolism in cells21,55. FLIM is high resolution and therefore resolves single cells, which is important for cancer studies because cellular heterogeneity contributes to tumor aggression and drug resistance7,39,41,44,45<su...

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 lipids2,3. This phenomenon, known as the Warburg effect, is a hallmark of many cancer types, including breast cancer, lung cancer, and glioblastomas4. Because of the alterations of cellular metabolism associated with cancer progression, cellular metabolism can be a surrogate biomarker for drug response5,6. Moreover, understanding drug efficacy at a cellular level is crucial as cell heterogeneity can lead to differing drug responses in individuals7,8.
Technologies that identify and quantify changes in cellular metabolism are essential for studies of cancer and drug response. Chemical and protein analyses are used to evaluate the metabolism of cells or tissues but lack single-cell resolution and spatial information. Metabolic plate reader-based assays can measure pH and oxygen consumption in the sample over time and the subsequent metabolic perturbation by chemicals. The pH can be used to calculate the extracellular acidification rate (ECAR), which provides an insight into the glycolytic activity of the cells9. Whole-body imaging methods, including 2-[fluorine-18] fluoro-D-glucose positron emission tomography (FDG PET) and magnetic resonance spectroscopy (MRS), are noninvasive imaging modalities used clinically to identify tumor recurrence and drug efficacy through metabolic measurements10,11,12,13,14.
FDG-PET images the tissue uptake of FDG, a radiolabeled glucose analog. Increased uptake of FDG-PET by tumors relative to surrounding tissue is due to the Warburg effect12,13. MRS images common nuclei of molecules used for metabolism, such as 13C and 31P, and can obtain dynamic information about how metabolism changes in response to stimuli, such as exercise or eating14. Although FDG-PET and MRS can be used clinically, these technologies lack the spatial resolution to resolve intratumoral heterogeneity. Likewise, oxygen consumption measurements are made on a bulk population of cells. Autofluorescence imaging overcomes the spatial resolution obstacle of these technologies and provides a noninvasive method of quantifying cellular metabolism.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63282/63282fig01v2.jpg" /> 
Figure 1: NADH and FAD in common metabolic pathways. NADH and FAD are coenzymes used in glycolysis, the Krebs cycle, and the electron transport chain. Autofluorescence imaging of these molecules provides information about cellular metabolism. <a href="https://www.jove.com/files/ftp_upload/63282/63282fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions, including glycolysis, oxidative phosphorylation, and glutaminolysis (Figure 1). Both NAD(P)H and FAD are autofluorescent and provide endogenous contrast for fluorescence imaging1,15. NADPH has similar fluorescent properties to NADH. Because of this, NAD(P)H is often used to represent the combined signal of NADH and NADPH2,16.
Fluorescence lifetime imaging (FLIM) quantifies the fluorescence lifetime or the time for which a fluorophore is in the excited state. Fluorescence lifetimes are responsive to the microenvironment of the fluorophores and provide information about cellular metabolism17. NAD(P)H and FAD can exist within cells in either protein-bound or free conformations, each of which has a different lifetime. Free NAD(P)H has a shorter lifetime than protein-bound NAD(P)H; conversely, free FAD has a longer lifetime than bound FAD18,19. The lifetimes and lifetime component weights can be quantified from fluorescence lifetime decay data through Eq. (1)20:
I(t) = &#945;1e-t/&#964;1&#160;+ &#945;2e-t/&#964;2&#160;+ C&#160; (1)
Eq (1) represents the normalized fluorescence intensity as a function of time. The &#945;1 and &#945;2 in this equation represent the proportional components of short and long lifetimes (&#945;1+ &#945;2=1), respectively, &#964;1 and &#964;2 represent the short and long lifetimes, respectively, and C accounts for background light7,20. The amplitude-weighted lifetime, represented here as &#964;m, is calculated using Eq. (2).
&#964;m= &#945;1&#964;1+ &#945;2&#964;2&#160; (2)
A mean lifetime can be computed by averaging &#34;t&#34; over the intensity decay of the fluorophore, which for a two-exponential decay is shown by Eq. (3)17,21.
&#964;*m= (&#945;1&#964;12+ &#945;2&#964;22)/ (&#945;1&#964;1+ &#945;2&#964;2)&#160; (3)
A fluorescence intensity image can be computed from the lifetime image by integrating the fluorescence lifetime decay. Autofluorescence imaging is a nondestructive and label-free method that can be used to characterize the metabolism of live cells at a subcellular resolution. The optical redox ratio provides an optical analog metric of the chemical redox state of the cell and is calculated as the ratio of NAD(P)H and FAD intensities. Although the formula for calculating the optical redox ratio is not standardized22,23,24,25, it is defined here as the intensity of FAD over the combined intensities of NAD(P)H and FAD. This definition is used because the summed intensity in the denominator normalizes the metric between 0 and 1, and the expected result of the cyanide inhibition is a decrease in the redox ratio. The fluorescence lifetimes of free NAD(P)H and FAD provide insight into changes in the metabolic solvent microenvironment, including pH, temperature, proximity to oxygen, and osmolarity17.
Changes in the fluorescence lifetime of the bound fractions of NAD(P)H and FAD can indicate metabolic pathway utilization and substrate-specific metabolism26. Component weights can be interpreted for changes in the free to the bound fraction of the coenzymes18,19. Altogether, these quantitative autofluorescence lifetime metrics allow the analysis of cellular metabolism, and autofluorescence imaging has been used for identifying neoplasms from normal tissues27,28, characterizing stem cells29,30, evaluating immune cell function31,32,33,34,35, gauging neurological activity36,37,38, and understanding drug efficacy in cancer types such as breast cancer and head and neck cancer21,39,40,41,42. High-resolution autofluorescence imaging can be combined with image segmentation for single-cell analysis and quantification of intrapopulation heterogeneity43,44,45,46,47.
NAD(P)H and FAD can be imaged on single-photon or multiphoton fluorescence microscopes configured for intensity or lifetime imaging. For single-photon microscopes, NAD(P)H and FAD are typically excited at wavelengths of 375-405 nm and 488 nm, respectively, due to common laser sources at these wavelengths48. In two-photon fluorescence excitation, NAD(P)H and FAD will excite at wavelengths of approximately 700 to 750 nm and 700 to 900 nm, respectively15,49. Once the fluorophores are excited, NAD(P)H and FAD emit photons at wavelengths between ~410 nm to ~490 nm and ~510 nm to ~640 nm, respectively15. The NAD(P)H and FAD maxima emission wavelengths are approximately 450 nm and 535 nm, respectively48.
Because of their different excitation and emission wavelengths, the fluorescence of the two metabolic coenzymes can be spectrally isolated. An understanding of the spectral characteristics of NAD(P)H and FAD is necessary for the design and optimization of autofluorescence imaging protocols. Cyanide is an electron transport chain (ETC) complex IV inhibitor. The effects of cyanide on cellular metabolism and the autofluorescence intensities and lifetimes of NAD(P)H and FAD within cells are well characterized27,40. Therefore, a cyanide perturbation experiment is an effective means of validating NAD(P)H and FAD imaging protocols. A successful cyanide experiment provides confidence that the NAD(P)H and FAD imaging protocol can be used to assess the metabolism of unknown groups or perturbations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-deoxy-d-glucose (2-DG)</td>
			<td>Sigma</td>
			<td>AC111980000; AC111980010; AC111980050; AC111980250</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Antimicrobial (pen-strep)</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Samples</td>
			<td>American Type Culture Collection</td>
			<td>N/A</td>
			<td>MCF-7 cancer line</td>
		</tr>
		<tr>
			<td>CellProfiler</td>
			<td>Broad Institute</td>
			<td>N/A</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Conical Tube</td>
			<td>VWR</td>
			<td>89039-664</td>
			<td>15 mL conical tube</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>11965092</td>
			<td>Culture media</td>
		</tr>
		<tr>
			<td>FAD dichroic mirror</td>
			<td>Semrock</td>
			<td>FF495-Di03-25x36</td>
			<td>495 nm</td>
		</tr>
		<tr>
			<td>FAD emission filter</td>
			<td>Semrock</td>
			<td>FF01-550/88-25</td>
			<td>550/88 nm</td>
		</tr>
		<tr>
			<td>FAD excitation filter</td>
			<td>Semrock</td>
			<td>FF01-458/64-25</td>
			<td>458/64 nm</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16000036</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Lifetime Microscope</td>
			<td>3i</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>MatTek Corp</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiphoton Laser</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td>2P Coherent Laser, Tunable 680 nm-1080 nm</td>
		</tr>
		<tr>
			<td>NAD(P)H dichroic mirror</td>
			<td>Semrock</td>
			<td>FF409-Di03-25x36</td>
			<td>409 nm</td>
		</tr>
		<tr>
			<td>NAD(P)H emission filter</td>
			<td>Semrock</td>
			<td>FF02-447/60-25</td>
			<td>447/60 nm</td>
		</tr>
		<tr>
			<td>NAD(P)H excitation filter</td>
			<td>Semrock</td>
			<td>FF01-357/44-25</td>
			<td>357/44 nm</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Cyanide</td>
			<td>Sigma-Aldrich</td>
			<td>380970</td>
			<td></td>
		</tr>
		<tr>
			<td>SlideBooks 6</td>
			<td>3i</td>
			<td>N/A</td>
			<td>Image acquisition software</td>
		</tr>
		<tr>
			<td>SPCImage</td>
			<td>Becker &#38; Hickl GmbH</td>
			<td>N/A</td>
			<td>Fluorescence lifetime analysis software</td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>okoLab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Biosciences</td>
			<td>786-262</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>YG beads</td>
			<td>Polysciences</td>
			<td>19096-2</td>
			<td>Yg microspheres (20.0 &#181;m)</td>
		</tr>
	</tbody>
</table>

autofluorescence imaging to evaluate cellular metabolism

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.

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 &#215; 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...

Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Cellular Metabolism at JoVE.com

Autofluorescence Imaging to Evaluate Cellular Metabolism

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.

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced ...

ADH and FAD are endogenous molecules that exhibit autofluorescence at different excitation and emission wavelengths. Because of their use as co-enzymes of metabolic reactions, autofluorescence microscopy can assess cellular metabolism. Autofluorescence imaging of NADH and FAD does not require exogenous labels and is a non-destructive method with subcellular resolution.Autofluorescence microscopy can be used on live samples and for time course studies. Autofluorescent imaging can be broadly applied to any living cells or tissue. Autofluorescent imaging has been used on neuron, cancer cell, tumor, in-vivo, stem cell, and immune cells.Demonstrating the procedure will be Linghao Hu, a graduate student from the Quantitative Optical Imaging Laboratory at Texas A&M University. Start the experiment by turning on all the components of the multi-photon fluorescence lifetime microscope, including the laser source and the detectors. Before sample placement, turn on the bright field lamp and ensure that the light goes into the eyepiece.Then, choose an objective for the cell imaging. If not using an air objective, apply one drop of the appropriate immersion medium on top of the objective. Place the glass-bottom dish onto the sample holder on the microscope stage.Then, center the specimen with the objective using the X/Y stage control Ensure that the specimen is secured and will not move during imaging. Once done, look into the eyepiece and move the objective up to focus on the cells. If the microscope is within an enclosure, close the light box door.Next, open the image acquisition software and click on the Multi-photon Imaging tab to set the multi photon imaging parameters as described in the manuscript. Adjust the gain of the detector to the optimal value for the fluorescence lifetime imaging, or FILM. Then, change the dwell-time of the laser spending at each pixel of the specimen to the desired value.For nicotinamide adenine phosphate dinucleotide, or NADPH, set the multi-photon laser to 750 nanometers. Then, confirm that the power control for the laser is set at zero so that the cells are not damaged upon opening the shutter on the laser. Set an emission filter at 400 to 500 nanometers.When the parameters are set, begin imaging in a focusing or live-view manner to optimize the image settings. Slowly increase the laser power from three to eight milliwatts while ensuring that the cells are in focus. Once adjusted, record the maximum power used.Use the recorded maximum power setting for the imaging on other segments of the Petri dish. Collect a NADPH-FILM image with an image integration time of 60 seconds and check that the image has a sufficient number of photons, such as a peak of 100 photons for a cytoplasm pixel within the fluorescence lifetime decay curve. If the number of photons is too low, increase the laser power or duration of image acquisition.For the flavin adenine dinucleotide, or FAD, imaging, set the multi-photon laser to 890 nanometers and wait for the laser to mode-lock at the new wavelength. Ensure that the power control for the laser is set at zero initially. Set an emission filter at 500 to 600 nanometers.To optimize the image settings, begin imaging in a focusing or live-view manner by increasing the laser power to 5 to 10 milliwatts and recording the maximum power used. Use the same power setting for the FAD imaging of subsequent fields-of-view. Collect the FAD-FILM image with an image integration time of 60 seconds and check that the image has sufficient photons within the fluorescence lifetime decay curve.If the number of photons is too low, increase the laser power or duration of image acquisition and repeat the imaging for an additional four to five fields-of-view, or FOVs, with each image spaced at two FOVs away. To make an 80-millimolar sodium cyanide solution, dissolve 130.24 milligrams of sodium cyanide in 25 milliliters of PBS. From the culture dish, aspirate 100 microliters of culture medium to replace with 100 microliters of sodium cyanide solution to obtain a four-millimolar concentration of cyanide in the dish.Put the cells in an incubator for five minutes to allow the cells to react with the cyanide solution. After the cyanide exposure, acquire NADPH and FAD images of the cells as described earlier. The study calculated the fluorescence lifetime parameters using the measured instrument response function, or IRF, from urea.The parameters were averaged across the pixels of the cytoplasm of each cell used for segmentation. Individual cells were identified and masked to eliminate background noise. Then, the nucleus was identified and projected onto the cell mask.Further, the cells were filtered to remove the masked areas that do not fit the size of typical cells. The multi-photon fluorescence lifetime imaging of NADPH and FAD allowed visualization of cell morphology and metabolism before and after cyanide treatment. It was observed that NADPH lifetime decreases with cyanide treatment, whereas the FAD lifetime increases after cyanide treatment.The representative box plot indicated that the optical redox ratio of MCF7 cells decreased after cyanide treatment. The cyanide exposure decreased the amplitude-weighted NADPH lifetime of MCF7 cells. The short and long lifetimes decreased for NADPH, but increased for NADPH alpha one.For FAD, the amplitude-weighted lifetime of MCF7 cells increased after cyanide exposure. Both the short and long lifetimes increased for FAD, but decreased for FAD alpha one. It is important to have enough laser power going to the samples, but remember, too much power can cause damage to the cells.Because autofluorescence imaging is not damaging to the cells, subsequent biochemical assays can be used on the same samples.

Autofluorescence Imaging to Evaluate Cellular Metabolism

Abstract