Researchers at Imperial College’s photonics department are developing multidimensional fluorescence imaging (MDFI) for cell biology, high-content analysis, and label-free tissue analysis. “The multidimensional part refers to the parameters you can get out of it, such as wavelength and polarization,” explained Sunil Kumar, Ph.D., research associate, photonics group.
Although fluorescence microscopy is a powerful functional imaging tool, in order to obtain quantitative images, one needs to apply spectral or temporal resolution. Since it’s best to avoid labeling live cells with extrinsic fluorophores, the MDFI approach of imaging autofluorescence (certain molecules like collagen and elastin are fluorescent) can distinguish different cell components and provide potential for label-free contrast. This approach is being applied to tissue imaging for research and clinical diagnosis.
Dr. Kumar’s group is also developing high-speed FLIM (fluorescence lifetime imaging), which can be used to image spatio-temporal organization of proteins and their organization. This uses an ultrafast shutter at different time delays after a laser pulse.
“You can build up intensities across your sample at different time delays and then put an exponential delay through them to get a lifetime image. Since you are doing this in a parallel fashion, you can put in a lot more light, acquiring images much faster,” explained Dr. Kumar.
This is an upgrade to time-coordinated single photon counting—considered the gold standard, which uses a single excitation via a pulsed laser to emit single photons. This takes a long time to build up an image. “You have to acquire hundreds to thousands depending on the lifetime decay to actually build up an image,” Dr. Kumar added.
Another technology currently under development is an automated high-speed optically sectioned FLIM multiwell plate reader applicable to fixed and live cells. “This looks for something in the sample that fluoresces and then automatically optimizes acquisition parameters. It will be used for drug discovery, especially if you have a particular biological system that expresses protein.”
A FLIM optical projection tomography system enables imaging of small samples, less than a centimeter in diameter. “It’s optically equivalent to a CT scan—you rotate the sample around its axis and collect data on it—and can do lifetime imaging at the same time, creating a 3-D lifetime map of the sample. We’re working toward getting images of whole mice.” Dr. Kumar said a potential application would be to record response to a drug or a particular activity.