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.
