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Volume 7 issue 9 September 2006
Fast and precise protein tracking using repeated reversible photoactivation
Dmitriy M Chudakov, Tatyana V Chepurnykh, Vsevolod V Belousov, Sergey Lukyanov, Konstantin A Lukyanov
Supplementary video 1.
Time series showing photoactivated asFP595-A148G movement within a nucleus of HeLa cell, raw data.
Video 1 (.avi)
Supplementary video 2.
Time series showing photoactivated asFP595-A148G movement within a nucleus of HeLa cell, image averaging from multiple series applied.
Video 2 (.avi)
Supplementary video 3.
Time series showing photoactivated PS-CFP2 movement in the cytosol of HeLa cell.
Video 3 (.avi)
Supplementary video 4.
Time series showing photoactivated asFP595-A148G movement in the cytosol of HeLa cell, raw data.
Video 4 (.avi)
Supplementary video 5.
Time series showing photoactivated asFP595-A148G movement in the cytosol of HeLa cell, image averaging from multiple series applied.
Video 5 (.avi)
Supplementary video 6.
Time series showing photoactivated asFP595-A148G movement within mitochondria of HeLa cell, image averaging from multiple series applied.
Video 6 (.avi)
Supplementary Methods.
Gene construction for expression in mammalian cells. For the expression in eukaryotic cells, the PCR-amplified AgeI-NotI fragment encoding asFP595-A148G or PS-CFP2 was inserted in lieu of the EGFP-coding region in the pEGFP-N1 vector (Clontech). For the construction of mitochondrially targeted asFP595-A148G, PCR-amplified AgeI-NotI fragment encoding asFP595-A148G was swapped with TurboGFP in pTurboGFP-mito mammalian expression vector (Evrogen). For the construction of Bid fusion PCR-amplified AgeI-NotI fragment encoding asFP595-A148G was swapped with DsRed2 in pDsRed2-Bid mammalian expression vector (Clontech).
Live cell imaging. Human carcinoma HeLa cell line was used. Cells were transfected using Lipofectamine reagent (Invitrogen). Tracking experiments were performed 24-48 h after transfection. Leica confocal inverted microscope TCS SP2 equipped with 125 mW Ar and 1 mW HeNe lasers was used for cell imaging. Quantification of image intensities was done with ImageJ software. LaserCheck (Coherent) power meter was used to measure total power of the excitation light after the microscope objective.
Parameters optimized for the fastest protein river tracking : Objective Leica HCX PL APO 63x/1.40-0.60 OIL. Mode xyt, 256x256 format, 1400 Hz scanning, bidirectional scanning pattern, zoom 6x, beam expander 3. Advanced time-lapse program of two steps: 1) Point photoactivation by 100 ms bleach with 543 nm 1 mW HeNe laser line, 100% power (5 kW/cm2 output power) followed by time series of 20 frames (1 frame per 144 ms) using 10% power 543 nm laser for excitation, 570-670 nm emission collected. 2) Quenching of asFP595-A148G with 488 nm laser line, 10 scans in the same mode with 10W/cm2 output power.
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