Photoactivation and photoconversion with 405nm laser on Zeiss LSM 510 

This is the second LSM 510 laser scanning microscope acquired by the Cell Imaging Facility. Those who are familiar with LSM 510 (details here) will need very little orientation on this scope. In addition to all the capabilities provided by the "old" LSM 510, the new system is also equipped with a 405 nm laser, thus allowing users to perform fluorescent confocal microscopy with blue-emitting fluorophores.

Blue-emitting fluorophores
The use of DAPI, blue fluorescent protein, and Hoechst stain are now possible with this new confocal microscope.

Photoactivation and Photoconversion
This is one of the major reasons why the new LSM 510 is equipped with the 405 nm laser. Taking advantage of the capability of the scanning laser to specifically excite (or photobleach) a user-defined region of interest, users can also specifically photo-activate or photo-convert a subpopulation of molecules in living cells in order to follow the dynamics of the targeted population of molecules. This is a a very important technical leap since the use of green fluorescent protein (GFP) and its variants.

Photoactivation: The photoactivatable GFP is developed by the laboratory of Dr. Jennifer Lippincott-Schwartz (Science 297: 1873-1877, 2003. NU library users can access the PDF through this link). Immediately following intense radiation at 405-413nm, the intensity of PA-GFP increases 100-fold when excited with the regular GFP excitation light of 488nm.



Fibroblast transfected with untagged PA-GFP was photoactivated in the red-square and sequential images were taken immediately after  photoactivation and with 1 min intervals thereafter.    Teng-Leong Chew, unpublished data.


Photoconversion: A similar fluorescent tag is a newly isolated protein from stony coral Trachyphyllia geoffryi (PNAS 99:12651-56, 2002.  NU library users can access the PDF file through this link). Named Kaede, the protein contains a tripeptide which acts as a green chromophore that can be converted into a red chromophore (excited with the 543nm laser) upon irradiation with UV light (our 405nm laser). Kaeda expression vector is commercially available from MBL International. More information about the vector can be obtained as follows:

CoralHue pKaede-MN1
CoralHue Kaede




Spectral signature of Kaede during a stepwise photoconversion of the entire cell using low-power irradiation with 405nm pulses. Pictures courtesy of Carl Zeiss, Inc.


The special properties of both PA-GFP and Kaeda thus allow users to:
1. Follow the rate of diffusion of any tagged protein in vivo
2. Follow specific protein trafficking in live cells
3. Follow a subset of a population of cells without the need of microinjecting fluorescent dyes. Cell labeling can now be conveniently performed with the user-friendly software.

This scope is equipped with 405nm, 458nm, 477nm, 488nm, 514nm, 543nm, and 633nm excitation lasers

Please contact Teng-Leong Chew for initial technical consultation and subsequent training and experimental design. The charge for this microscope is $40/hour.

You can download the general LSM510 confocal manual here.