Structured Illumination Microscopy
Another commonly used tool for live-cell imaging is wide-field optical microscopy, but there are some limitations for this method as well, noted Lin Shao, research specialist at the Howard Hughes Medical Institute’s Janelia Farm Research Campus. “The main problem is that its resolution is limited by the visible light’s wavelength and the objective’s aperture.”
Also referred to as wide-field structured illumination (SI), this approach, as a means to working around the diffraction limit, depends on both specific microscopy protocols and extensive software post-exposure.
Dr. Shao and his team, led by Mats Gustafsson, have made a number of discoveries using SI. “We have shown that, when using SI in a linear regime, that the resolution limit of the wide-field microscopy can be improved by a factor of two both laterally and axially.
“Data acquisition, however, is not quick enough to achieve live imaging. The reason for this is that reconstructing a 2-D image requires multiple exposures under different illumination patterns.” He added that 15 images are required per every 2-D section in 3-D imaging. Mechanical pattern switching is also slow.
Dr. Shao also spoke about an SI microscope developed to use a ferroelectric liquid crystal-based spatial light modulator as the pattern generator. “The image-acquisition speed is greatly enhanced by the greater than 1 KHz pattern-switching speed of the spatial light modulator and the total internal reflection mode that allows 2-D imaging and thus only nine exposures per time point. As a result, the microscope is capable of 100 nm resolution at frame rates up to 11 Hz for several hundred time points.”
Dr. Shao demonstrated the speed and resolution of the microscope by imaging tubulin and kinesin dynamics in live Drosophila melanogaster S2 cells.
“Speed is an issue, since to keep up with fast moving molecules inside cells requires fast cameras with high resolution. The dynamic nature is also a challenge—how do you maintain the level of fluorescence without bleaching the phosphores. The additional problem of high resolution applies to both issues. And another obstacle is how one can keep the cells happy while being imaged, which involves mounting schemes for temperature control (mammalian cells are happy around 37ºC; S2 cells are happy at RT) and being careful about phototoxicity, i.e., not too high-intensity excitation light should be allowed.”