Figure 2
Expression and photo-conversion of mEosFP::FABD-mTn following its transient expression in an onion epidermal cell. Photo-conversion was carried out using a D-excitation filter (UV/Violet; Ex: BP355–425/Dich: 455/LP 470) on a Leica DM600B epi-fluorescent microscope. Pre- and post-conversion images were acquired using 488 and 543 nm laser lines. Images were acquired within 10 seconds after photo-conversion. A.B.C. Pre-conversion fluorescence status 'A' in the green fluorescence acquisition channel; 'B' in the red channel; C depicts the fluorescence intensity along a straight line ROI (panel B, D) for both channels. Note the very low level of fluorescence picked up by the Leica fluorescence quantification tool for the red channel as compared to the green channel. D.E.F. Post conversion fluorescence status for green ' D' and red 'E' channels. Color profile shows a complete inversion in the relationship between green and red fluorescence emissions along the selected ROI. Images D and E were captured within 10 seconds of photo-conversion. The fluorescence intensity scale is based on a 0–255 RGB colour code. Bar = 50 μm. G. An Adobe Photoshop-based approach to draw out information on colour quality and the relative red/green values in an internationally accepted colour code. Absolute green is 00ff00 or 0/255 while absolute red is ff0000 or 255/0. The 'eye-dropper' tool in Photoshop provides direct readouts of R/G values. Arrows point to examples of some readouts from cropped regions of A and E. H. An imageJ-based approach for creating colour histograms from an image. The red and green components in the rectangular ROI in panels 1–3 are depicted on a scale of 0–255 for each colour. Panel 1 and the histogram below it depict greenness within the image (cropped portion of panel D); Panel 2 (cropped portion of panel E) depicts redness in the same region after photo-conversion whereas panel 3 is a merge of panel 1 and 2 and accurately reflects the merged mean values obtained for them.