New Label-Free Imaging Method Differentiates Quiescent and Activated T Cells

Researchers at Morgridge Institute for Research have developed a label-free imaging technique that can differentiate active T cells from inactive. The method could help assess T cell involvement in immunotherapies for cancer treatment or autoimmune diseases.

Most methods for characterizing T cells are antibody-based, such as flow cytometry or immunohistochemistry. These require staining with antibodies or contrast agents, a process that is destructive to the cells. This new method detects autofluorescence from molecules within the cell that naturally emit light when imaged by a microscope paired with an infrared laser. This label-free process is non-damaging and doesn't alter the behavior of the cell, according to Melissa Skala, principal investigator and senior author on the paper published in Nature Biomedical Engineering today. The technique could be adapted to image cells in a plate or dish, tissue samples, or even in vivo imaging of a complete organism.

To validate their approach, the researchers acquired blood samples from healthy donors, isolated the T cells, and measured autofluorescence of NAD(P)H and FAD, two molecules that are involved in cellular metabolism. Images of the quiescent cells versus the activated cells revealed differences in metabolic function, most notably through a change in NAD(P)H autofluorescence in the activated T cell populations. They also observed that active T cells were slightly larger in size than quiescent cells.

The activation protocol and imaging capabilities will be useful for manufacturing the CAR-T cells used in immunotherapies, says Skala. These re-engineered T cells are often co-cultured with other cells, like cancer cells, to test their reactivity. However, using additional harsh reagents or antibody-labels to further characterize the T cell is a bottleneck for CAR-T cell manufacturers. The autofluorescent approach provides an attractive way to perform those experiments by imaging the same cells across multiple timepoints in a way that's non-damaging.

While this new technique offers many advantages over traditional methods, there are still limitations. For one, autofluorescence imaging isn't very sensitive. "We aren't relying on really specific labels, we're relying on the metabolism of the cells," Skala says. "That's only going to get you so far in differentiating the cell types." Additionally, the technique requires experienced people to perform the microscopic imaging and analyze the data.

The team is working on developing a prototype to take the imaging capability of their large-scale microscope and translate it into a "box-sized" system. "You won't have to be a specialized optical engineer to use it," Skala says. "That's the direction we're trying to go. We're trying to make it more accessible."

Image: A novel label-free imaging technique differentiates T cells from blood when in a quiescent state (illustrated in blue) and when activated by antibody-stimulation (illustrated in red). Images are captured using a microscope paired with an infrared laser to capture autofluorescence of proteins involved in cellular metabolism. Image courtesy of Alexandra Walsh.