Introduction
Live cell fluorescence imaging is an increasingly popular technique for obtaining dynamic information about cellular processes. However, imaging live cells creates many challenges, not the least of which is keeping the cells alive. The excitation light required for imaging can cause photobleaching and phototoxicity, which seriously degrades image quality and can ultimately lead to cell death. Innovative strategies to reduce photobleaching and phototoxicity are needed in order to realize the full benefits of live cell fluorescence imaging. One approach is Controlled Light Exposure Microscopy (CLEM), exclusively available from Nikon Instruments1, 2,. Methods
CLEM is a laser scanning confocal technique which modulates the light incident on the sample on a pixel by pixel basis. Nikon's C1 CLEM confocal system uses electronic feedback between the detector's analog to digital electronics and the laser's acousto-optical modulator (AOM). This additional feedback allows CLEM to monitor the intensity of the emission during first portion (∼20%) of the pixel period and stop the illumination at that pixel under two conditions: when no signal is detected, or when the signal detected is high enough to saturate. In comparison to conventional laser scanning confocal microscopy, which illuminates all pixels equally, CLEM affords several advantages: it limits the light to the sample lessening photobleaching and phototoxicity. Additionally, by attenuating the laser illumination at pixels that will saturate, dynamic range is greatly increased. Some specific examples are discussed below. Results
Figure 1 shows cells that were imaged at 2 different stage positions, one with CLEM and one without. While both fields initially look identical, after nearly 90 minutes of imaging, the non-CLEM cells have rounded up and died, while the CLEM cells remain largely unaffected. Cells could be images approximately six times longer with CLEM than without it. Figure 2 demonstrates the improvement in dynamic range that can be achieved with CLEM. Images taken at the top of a brain slice without CLEM have to be acquired with oversaturated pixels in order for any signal to be detectable deeper in the sample. Using CLEM, however, the laser illumination is attenuated at the top of the sample where the signal would saturate, while sections deeper in the sample are sufficiently illuminated.
Figure 1. (Click to enlarge)
Figure 2. (Click to enlarge)
Summary and Conclusions
Using CLEM, decreases in photobleaching and phototoxicity, as well as increases in dynamic range are achieved, potentially enabling experiments not possible with standard confocal protocols.
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