Figure 2

Comparative photoconversion of PS-CFP2, Kaede and KikGR. Panels show photoconversion of PS-CFP2 (A-F), Kaede (G-H) and KikGR (M-R) in transient transfected COS7 cells. The ROI that was subjected to short wavelength laser light is depicted in the yellow dashed box. Bright field (BF) images of respective cell transfections (A, G and M). Bright field images are 2D representations (A, G and M). Panels B-F, H-L and N-R show 3D images of converted COS7 cells. Upper row shows photoconversion of PS-CFP2. Cells fluoresce cyan before photoconversion and green after photoconversion. CFP channel (B) and GFP channel (C) before photoconversion. CFP (D) and GFP channel (E) after photoconversion. Merge of the CFP and GFP channel (F). Middle row depicts the photoconversion of Kaede, and bottom row shows the photoconversion of KikGR both of which fluoresce green before, and red after photoconversion. GFP channel (H, N) and RFP channel (I, O) before photoconversion. GFP (J, P) and RFP channel (K, Q) after photoconversion. Merge of the GFP and RFP channels (L, R). Note that you see a stronger CFP signal in D due to signal bleed through of the GFP channel. Note that cells were photoconverted with a 20× objective using 35% laser power of the 25 mW 405 nm laser, however, KikGR was imaged with less gain in the channels of its respective colors as compared to PS-CFP2 and Kaede.