Volume 111, Issue 5, Pages 1014-1025 (September 2016) Photoconvertible Behavior of LSSmOrange Applicable for Single Emission Band Optical Highlighting  Herlinde De Keersmaecker, Eduard Fron, Susana Rocha, Takako Kogure, Atsushi Miyawaki, Johan Hofkens, Hideaki Mizuno  Biophysical Journal  Volume 111, Issue 5, Pages 1014-1025 (September 2016) DOI: 10.1016/j.bpj.2016.07.033 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Fluorescence spectra of LSSmOrange in vitro. Excitation (dotted lines, fluorescence detected at 570 nm) and emission (solid lines, excited at 440 nm and 550 nm, respectively) spectra of LSSmOrange before (green) and after (magenta) photoconversion are shown. LSSmOrange is shown before (u) and after (c) photoconversion under ambient light (left), under illumination at both 445 nm and 559 nm (right top), and under illumination at either 445 nm (middle) or 559 nm (bottom). Images were taken with a digital camera (Stylus TG-3 Tough; Olympus). For fluorescence images (right column), a bandpass filter (HQ590/40 M-2p; Chroma Technology, Bellows Falls, VT) was placed in front of the lens. To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 In cellulo photoconversion of LSSmOrange. (A) Gradual photoconversion of LSSmOrange in living cells. Cells were scanned with a 445-nm laser (56.0 kW/cm2) focused with an objective lens. The scanning speed was 82.5 nm/μs, the zooming factor was 1.5 (pixel size was calculated to 165 nm), and the scanning region was 512 × 512 pixels. Images were acquired after every 10 scans under alternate illumination at 445 nm (green) and 559 nm (magenta). The images shown were acquired before and after 30 and 90 scans. The graph shows time traces of the average fluorescence intensities excited at 445 nm and 559 nm. The scale bar indicates 10 μm. (B) Correlation between the conversion rate and the laser power density under illumination at 488 nm (green rectangle), 445 nm (blue triangle), and 405 nm (red circle). All measured points (open) and their average (solid) are plotted. To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Propagation of photoconverted LSSmOrange in living cells. Time-lapse images for unconverted (green) and converted (magenta) LSSmOrange were acquired every 2 s (frame rate of 1.9 s with a 0.1 s interval) under alternate illumination with 445-nm and 561-nm lasers, respectively. Photoconversion was performed by 100× scanning with a 405-nm laser (2.0 MW/cm2) at a scanning rate of 65.9 nm/μs in the region indicated in cyan in (A). (A–D) Snapshot images at the indicated time points. (E) Time traces in the region of photoconversion (cyan), a cytosolic region outside the photoconversion area (yellow), and a nuclear region (magenta). The scale bar indicates 20 μm. To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Equilibrium radial absorbance profile of unconverted LSSmOrange. The profile of the absorbance at 440 nm (lower panel, blue) was successfully fitted to the single ideal species model (red line). The upper panel shows the residuals. To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Diffusion of LSSmOrange in living cells. (A) Fluorescence image of HeLa cells expressing LSSmOrange. The magenta line indicates the region of line scanning. The scale bar indicates 5 μm. (B) Kymographic display of the propagation of photoconverted LSSmOrange after pulsatile conversion (10 μs, a combination of 405-nm and 458-nm laser lines with power densities of 2.0 MW/cm2 and 49.5 kW/cm2, respectively). The violet rectangle indicates the region of photoconversion. The experiment was performed at 37°C. (C) Distribution of photoconverted LSSmOrange immediately after photoconversion (blue circles). Fitting to a Gaussian function is shown in red. (D) Gaussian fitting at various time points. (E) MSD plot of the propagation of photoconverted LSSmOrange (data, blue circles; fit, red line). To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Mitochondrial fusion visualized by photoconversion of LSSmOrange. LSSmOrange targeted to the mitochondrial matrix was expressed in HeLa cells. Time-lapse images were acquired at 37°C for every 5 s (frame time of 1.9 s with a 3.1 s interval) under line alternate mode illumination with 458-nm (green) and 561-nm (magenta) lasers. For local photoconversion, the 405-nm laser scanned the regions (5 times, 2.0 MW/cm2, scanning rate of 115 nm/μs) indicated in cyan in (A). (A and B) Images before and 5 s after photoconversion. (C–F, C’–F’) Expansions of the regions drawn with frames in (A), at the time points indicated. The arrowheads indicate the region in which mitochondrial fusion took place. (G) Time trace of the fluorescence intensities for converted (magenta) and unconverted (green) LSSmOrange in the regions of interest indicated by yellow circles on the images. Images correspond to the yellow frame in F’. The scale bars represent 5 μm (A) or 2 μm (C). To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Multicolor imaging of LSSmOrange with eYFP and mRFP. (A–H) Triple-color fluorescence imaging of cells. LSSmOrange targeted to mitochondria, eYFP-tubulin, and mRFP targeted to ER were coexpressed in HeLa cells. (A–H) Images before (A–D) and after (E–H) photoconversion were acquired under 488-nm and 561-nm illumination, respectively. The cyan frame indicates the region of photoconversion by 100× 458-nm laser scanning at 36.6 kW/cm2 with a scanning speed of 24.5 nm/μs. Images in real color (A, A’, E, and E’) and pseudocolor images after linear unmixing (B–D and F–H) are shown. (I) Emission spectra of the respective fluorescent proteins. The spectra of eYFP (red) and unconverted LSSmOrange (green) were acquired under 488-nm illumination with a dichroic mirror (MBS488), whereas those of converted LSSmOrange (magenta) and mRFP (cyan) were acquired under 561-nm illumination with a dichroic mirror (MBS488/561; note that the left side of the spectra obtained with MBS488/561 is distorted due to its reflection band). The color codes used are the same as for the unmixed images. (J–L) Linear unmixing of unconverted and converted LSSmOrange. The cyan frame indicates the region of photoconversion by 458-nm laser scanning. (J) Pseudocolor image acquired using sequential illumination with 488-nm or 561-nm lasers. (K) Image in real color acquired under simultaneous illumination with 488-nm and 561-nm lasers. (L) Separation of unconverted (green) and converted (magenta) channels by linear unmixing, shown in pseudocolor. Scale bars indicate 10 μm (A and J) or 2 μm (A’). To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 8 Photoconversion of LSSmOrange via a two-photon process. (A and B) Profiles of LSSmOrange photoconversion via a one- or two-photon process. LSSmOrange was embedded in a polyacrylamide gel and photoconverted by a single line scanning with a 458-nm continuous laser (laser power density of 36.6 kW/cm2) (A) or 850-nm femtosecond pulse laser (average laser power density of 59.0 kW/cm2 and pulse power density of ≤6.15 × 109 W/cm2) (B). Photoconversion was performed 10 μm above the glass surface. The left panels show x projections of photoconversion profiles, and the right panels show axial profiles of the center along the z axis (blue circles) with fitting to a Gaussian function (red lines). (C–E) Two-photon conversion of LSSmOrange in living cells. LSSmOrange targeted to mitochondria in HeLa cells was subjected to two-photon conversion with a femtosecond pulse laser at 850 nm (average power density of 59.0 kW/cm2 and pulse power density of ≤6.15 × 109 W/cm2). Regions indicated with cyan boxes were converted by 100× scanning with a speed of 83.5 nm/μs. Panels (D) and (E) show an expansion of each converted area. Unconverted and photoconverted LSSmOrange is shown in green and magenta, respectively. The scale bars indicate 2 μm (A, B, D, and E) or 10 μm (C). To see this figure in color, go online. Biophysical Journal 2016 111, 1014-1025DOI: (10.1016/j.bpj.2016.07.033) Copyright © 2016 Biophysical Society Terms and Conditions