| Microscopy
Nikon Instruments Inc. Confocal Microscopy
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www.nikoninstruments.com
Nikon’s C1-CLEM System Decreases Photodamage and
Increases Dynamic Range
Introduction Summary and Conclusions
Live cell fl uorescence imaging is an increasingly popular technique Using CLEM, decreases in photobleaching and phototoxicity, as well
for obtaining dynamic information about cellular processes. However, as increases in dynamic range are achieved, potentially enabling
imaging live cells creates many challenges, not the least of which is experiments not possible with standard confocal protocols.
keeping the cells alive. The excitation light required for imaging can
cause photobleaching and phototoxicity, which seriously degrades
Acknowledgements
image quality and can ultimately lead to cell death. Innovative
We would like to thank Dr. Erik Manders and his team at the University
strategies to reduce photobleaching and phototoxicity are needed
of Amsterdam in the Netherlands, creators of the CLEM techniques. Dr.
in order to realize the full benefi ts of live cell fl uorescence imaging.
Manders has provided data for this protocol as well as invaluable insight.
One approach is Controlled Light Exposure Microscopy (CLEM),
exclusively available from Nikon Instruments
1,2
.
References
1. R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J.
Methods
F. Van Noorden, E. M. M. Manders (2007). Controlled light-exposure
CLEM is a laser scanning confocal technique which modulates microscopy reduces photobleaching and phototoxicity in fluorescence
the light incident on the sample on a pixel by pixel basis. Nikon’s
live-cell imaging. Nature Biotechnology, Vol. 25, pp 249-253.
C1 CLEM confocal system uses electronic feedback between the
2. J. L. Peters (2007). Controlled light exposure microscopy for confocal
detector’s analog to digital electronics and the laser’s acousto-
imaging. Biophotonics International, Vol. 14, pp 36-39.
optical modulator (AOM). This additional feedback allows CLEM to
monitor the intensity of the emission during fi rst 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
Figure 1: Cells imaged with and
sample lessening photobleaching and phototoxicity. Additionally,
without CLEM, shown initially (top),
by attenuating the laser illumination at pixels that will saturate, and after 80 minutes (bottom). Cells
dynamic range is greatly increased. Some specifi c examples are
were imaged every 35 seconds.
discussed below.
Results
Figure 1 shows cells that were imaged at 2 different stage positions,
one with CLEM and one without. While both fi elds 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
Figure 2: Confocal sections of a
with CLEM than without it. Figure 2 demonstrates the improvement
brain tissue sample, taken at the
surface (top) and deeper into the
in dynamic range that can be achieved with CLEM. Images taken
tissue sample (bottom). Sections are
at the top of a brain slice without CLEM have to be acquired with approximately 100 m apart.
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 suffi ciently illuminated.
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 57
PGNov08-Nikon.indd 57 10/26/07 11:58:02 AM
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