To begin, place the prewarmed imaging medium and sterile microscopy dishes with coated or non-coated cover glass surfaces on a working platform. After removing the medium surrounding the agarose mold, carefully resuspend the content of the molds in 190 microliters media before transferring floating spheroids into a two milliliter vial. For spheroids in a low-attachment plate, transfer spheroids one by one from individual wells to a vial.
Allow the spheroids to settle down at the bottom of the vial for five minutes, forming a visible pellet. Remove the media from the vial, leaving spheroids undisturbed, and gently resuspend them in a sufficient amount of fresh McCoy's 5A medium. Now, transfer an equal volume of spheroid suspension per well of the microscopy dish.
Incubate spheroid for 1 to 2 hours at 37 degrees Celsius in a carbon dioxide incubator. Add a fluorescence probe to a known volume of spheroid suspension and continue incubation for 1 hour at 37 degrees Celsius. After incubation, exchange the media containing fluorescent probes with a necessary volume of imaging media.
Start the microscope control software and initialize the stage calibration. Then, choose the required objective. Choose the Open project window and click Create a New Project.
Then, right click on the newly created project and select the Rename option in the dropdown menu. Give a standard name to the research project file. Open the Acquisition window.
Set the pulse white light laser excitation wavelength and the required range of hybrid or resonance scanning detectors. Then, choose line or frame types of scan. In the Acquisition window, select FLIM mode to perform imaging combined with photon counting.
Then, choose the white light laser pulse repetition rate. Start the preview imaging using Fast Live mode and adjust the fine focus of the imaging object on a section of interest. By looking at the pixel intensity histogram, adjust the appropriate laser intensity pinhole size and resolution.
While in Fast Live, set the coordinates and scan direction and attribute them to begin and end in the Z-Stack window. Choose a z-step size or number of steps. Once all necessary settings are applied, start imaging.
Give the image an appropriate name and save the imaging project. Different formation methods resulted in varying spheroid sizes. HCT116 spheroids in low attachment plates were significantly larger after five days compared to high throughput methods.
Low attachment methods led to the evident development of a necrotic core detected by the propidium iodide staining. In contrast, spheroids generated with other methods demonstrated the diffused distribution of dead cells across the spheroid body. Oxygenation in HCT116 spheroids produced by different methods showed more oxygenated spheroid with the microtissue and lab-made micromolds than the Sphericalplate 5D.
Spheroid oxygenation of Sphericalplate 5D spheroids showed lower oxygenation compared to microtissue and lab-made micromolds. Phasor analysis of HCT116 spheroids made using low-attachment plates reveals the presence of glycolytic and non-glycolytic cores via two-photon NADPH-FLIM.
