Controlled Light Exposure Microscopy

Time-lapse studies of protein interactions can be undertaken without fear of cell degradation, death or bleaching now that the Nikon confocal systems can be configured with Controlled Light Exposure Microscopy (CLEM).

This marks a revolution in the ability to observe dynamic events in living cells, due to the prolonged cell viability, and was first described in the paper recently published in Nature Biotechniques. Observation of cellular targets labelled with a fluorescent peptide, can now be captured with a dramatic reduction in damaging excitation light, reducing photobleaching and enhancing cell survival.
This breakthrough in the study of live cells is solely due to the method by which CLEM operates. In contrast to conventional confocal microscopy, when CLEM is deployed, cellular exposure to laser light is determined on a per pixel basis and excitation light is reduced through two unique strategies. The first of these ensures that, if there is no signal (such as the background), the illumination is switched off. The second detects whether there is sufficient signal to acquire an image, and illumination can be stopped on this basis. Importantly, studies examining the morphology of, for example, pollen grains using both conventional non-CLEM and CLEM microscopy provided comparable images, indicating that use of CLEM does not affect image detail.

Thanks to this exceptional system, studies such as those undertaken with BY-2 tobacco cells expressing GFP-MAP4 proteins, exhibited a resistance to photobleaching that was five times greater when the Nikon C1 confocal system was configured with CLEM in contrast to non-use of CLEM. In addition, time-lapse experiments, conducted on HeLa cells expressing GFP tagged histone-2B proteins in the absence of CLEM, showed membrane blebbing and cell death at a far earlier stage than samples viewed with CLEM configured microscopes.

Experimental imaging results indicate that under the standard operating conditions of point scanning laser based confocal microscopes, the CLEM system can extend the imaging time of living cells by a factor of two.

“It is imperative for the progression of live cell research to reduce the effects of both photobleaching and phototoxicity in order to prolong cell survival, particularly in time-lapse experiments” said Maarten Balzar, Biological Product Support Manager, Nikon Instruments Europe. “In response to this need, we have developed in collaboration with Dr. Erik Manders from the University of Amsterdam, an innovative product in which the Nikon C1 confocal system is configured with Controlled Light Exposure Microscopy. We are very encouraged by these results, and early indications show that CLEM will become an essential tool for live cell imaging studies. This is also an interesting development for us as it builds on our increasing commitment to complete live cell care”

The CLEM control unit is an optional add-on system for the C1. The CLEM unit calculates the integrated detected signal and exposure time for each pixel in real time, performs AOM high speed shutter control and high speed operation processing based on the amount of acquired fluorescence signal, PMT gain and then outputs the resulting fluorescence signal to the C1 controller’s line grabber electronics circuit.