Live-cell imaging, which has proven to be tremendously beneficial in helping scientists understand how cells work, is not without its wrinkles. “Live-cell imaging is key to understanding cellular differentiation and function,” said Magnus Persmark, Ph.D., senior product manager at Life Technologies, “yet cell types like stem cells, primary cells, and neurons have traditionally been quite challenging to label efficiently and without cytopathic effects.”
Another challenge, noted Anna Christensen, Ph.D., imaging product manager at Caliper Life Sciences, is specificity. “In vivo challenges to cell imaging, particularly targeting specific processes within the whole cell, remain,” she said. “When you look at a specific pathway in a specific animal, there are a lot of nonspecific events occurring. The real challenge lies in targeting relevant events and processes, in the cell, in the pathways, and in the whole animal.”
At the “Focus on Microscopy” and “Immunology” meetings held earlier this year, investigators and vendors explored some of the issues facing researchers working with live-cell imaging and offered some solutions to help enhance research efforts.
The evolution of superresolution microscopy has been fairly rapid over the last few years and has lead to several important findings, particularly by using stimulated emission depletion (STED) microscopy, said Tanjef Szellas, product manager, superresolution marketing, Leica Microsystems.
“The implementation of continuous wave (CW) lasers emitting in the visible spectral range has opened up new perspectives for biomedical research,” Dr. Szellas noted.
STED microscopy uses the nonlinear de-excitation of fluorescent dyes to surmount the resolution limit imposed by diffraction with standard confocal laser-scanning microscopes.
The resolution of a confocal laser-scanning microscope is limited to the spot size to which the excitation spot can be focused. However, within the STED microscope, the diffraction limit is surmounted by targeted strong de-excitation of dye molecules, switching them off effectively.
Dr. Szellas discussed the capabilities of Leica’s commercial STED microscope, the TCS STED CW. “The integration of an orange CW laser to increase the resolution has made it possible to investigate fixed and living intact specimens labeled with standard fluorophores like Alexa488 and Oregon Green and also with fluorescent proteins like YFP.”
Dr. Szellas noted that there are quite a few applications where continuous wave stimulated emission depletion microscopy has been used, including cell structure research, vesicle trafficking, and neurobiology, particularly in the areas of synapse assembly and vesicle fusion, and to better understand structures of nuclear pore complexes.
“In general, it can be used anywhere where the following is needed: sub-80 nm optical resolution in an intact specimen—inside a cell, for example—with recording speed of up to 20 frames per second, especially for viruses and vesicles that exhibit average sizes of approximately 50–100 nm. To follow their exo- and endocytosis in real time with “real” sizes requires a set-up like STED.”