Precise calculation of photoactivation kinetics

ADVERTISING FEATURE APPLICATION NOTES © 2006 Nature Publishing Group http://www.nature.com/naturemethods Precise calculation of photoactivation kinetics Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate measurements. Researchers hoping to obtain precise kinetic data from fluorescence recovery after photobleaching or photoactivation experiments need an easily controllable system for stimulation of a specific region and subsequent imaging, and the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM) with SIM scanner makes this possible. We describe here how to precisely measure off rates using a cytosolic photoactivatable probe that binds endosomal membranes. The diversity of genetically encoded fluorophores has increased since Measuring rapid kinetic events GFP was first cloned in 1992. There are now a variety of GFP mutants To measure rapid kinetics after photoactivation, it is essential to know spanning much of the visible spectrum. Some of these variants allow the exact interval between stimulation and measurement. The exposure photoactivation, such that they increase their fluorescence intensity after of the stimulating laser can easily be controlled on a millisecond level, exposure to specific wavelengths of light, and using these variants, one and the images acquired using the main scanner also have millisecond can turn on the fluorescent intensity in a region of interest and study the precision. It is therefore straightforward to make an identical, repetitive activated proteins over time (for a review, see ref. 1). The photoactivat- setup in one line of experiments. However, the location of the stimulated able version of GFP (PA-GFP) gains a 100-fold increase in fluorescence region within the image will usually vary for each measurement, result- (with emission at 517 nm) after exposure to light in the ultraviolet-to- ing in different delays between stimulation and start of measurement. If violet range (350–420 nm; ref. 2). the stimulated area appears late in the frame, it will be a longer delay Using standard molecular biology techniques, a protein of interest compared to an area early in the frame (Fig. 1). It is possible to cor- can be linked to a fluorophore. Depending on the fusion partner, this rect for these varying delays by calculating the exact time from stimula- approach can be used to label subcellular organelles, cells of interest and tion to imaging of the region of interest, and then adjusting this time specific tissue regions. This has opened the possibility for in vivo studies for each measurement. With a short interval, the correction will only of, for example, organelle dynamics and function, protein expression have minor effects; but if the interval is longer, a correction will be more and turnover, protein interaction, and cell motility. Chimeric fluorescent pronounced. proteins allow studies of dynamic events that range in duration from less than a second up to several days. The measurement and analysis of rapid Measuring in practice subsecond kinetics, however, necessitates special requirements for both To illustrate this principle we used a coat protein linked to PA-GFP. This the microscope hardware and software. coat protein interacts with a specific lipid in the membrane of early The Olympus FluoView FV1000 cLSM is available with a proprietary SIM scanner, which allows the confocal system to simultaneously stimulate and image. Using this setup one can use an independent laser for light stimulation while recording images with the main scanner. Structures of interest can be selected and stimulated during scanning, facilitating accurate measurements immediately after stimulation. For the highest efficiency in photobleaching or photoactivation, a circular or so-called ‘Tornado’ scan, is possible with the SIM scanner, maximizing the dose of light in the activation or bleaching area. Trygve Bergeland1, Martin Tewinkel2 & Oddmund Bakke1,3 1Department of Molecular Bioscience, University of Oslo, Pb. 1041, Blindern, 0316 Oslo, Norway. 2Olympus Life and Material Science Europa GmbH, Wendenstrasse 14 - 18, 20097 Hamburg, Germany. 3Broegelman Research Laboratory, University of Bergen, N-5021 Bergen, Norway. Correspondence should be addressed to M.T. (). Figure 1 | Varying delays between photoactivation and imaging of activated area. Illustration of how varying locations of a stimulated area can result in different delays between stimulation and scanning of the stimulated area. Blue area indicates start and end of stimulation. The circular enclosed areas indicate two examples where the stimulating light can be exposed. NATURE METHODS | DECEMBER 2006 | iii ADVERTISING FEATURE Figure 2 | Activation of PA-GFP linked to a cytosolic coat protein. Maden-Darby canine kidney cells were stably transfected with a cytosolic coat protein linked to PA-GFP. Region of interest was located, and the SIM scanner allows individual activation of this area independently of the image scanning. Cells were monitored before, during and after photoactivation. Scale bars, 5 µm. endosomes. We marked a region of interest around the area to be acti- the region being stimulated will vary in the image scan between experi- vated, in this case an endosome, and the SIM scanner stimulated the ments, a delay will often appear. If this delay is not taken into consid- area with a short pulse (25 ms) of low-intensity (10%), 405-nm light for eration and corrected for, the kinetic data are wrong—for example, photoactivation. The image scanning, however, was continuous prior in the experiment above, the t1/2 would appear to be too long. Other to, during and after activation, allowing changes in intensity to be moni- techniques may not generate similar delays, and the comparison of dif- tored over time (Fig. 2). The intensities were normalized and plotted ferent measurements requires accurate data. By using the SIM scanner against time3, and the intensity half-life (t1/2) and fraction remaining on on the Olympus FluoView FV1000, one can regulate both stimulation the membrane were calculated4. In the first example, there was a 1.1-s and scanning at the millisecond level. As a result, the output data can be delay between stimulation and imaging of the stimulated area. The t1/2 subjected to the necessary correction, producing precise calculations of before time correction was 4.90 ± 0.25 s, whereas the corrected t1/2 was kinetic parameters in photoactivation. 3.90 ± 0.16 s. In the next example the t1/2 was measured as 3.02 ± 0.19 s 1. before time correction. The delay was 0.33 s, and this resulted in a cor- 2. rected t1/2 of 2.90 ± 0.16 s. The correction is naturally most pronounced 3. Chudakov, D.M. et al. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23, 605–613 (2005). Patterson, G.H. & Lippincott-Schwartz, J. A photoactivatable GFP f (...truncated)