Pyxel Edit Full Version 809: Tips and Tricks for Making Pixel Art and Tilesets with this Editor

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In this study, we successfully demonstrated high-speed 4-channels imaging by introducing a mechanical fast-selecting system for either of the two types of NIR laser light beams and an image-splitting detection system into our TPLSM by utilizing a spinning-disk scanner (TPLSM-SD). Moreover, the linear unmixing method developed here canceled out the cross-talks among the 4-channels images in the acquired dataset. Our developed methodology could successfully help visualizations of the 3D dynamics of individual organelles undergoing mitosis with low photodamage.

As a proof of concept, we applied the developed system to conduct xyzt-λ imaging in human HeLa cells expressing Lifeact-mCitrine, EGFP-α-tubulin, and histone H2B-mRFP (labeling filamentous actin (F-actin), microtubules, and chromosomes, respectively)12,13. As shown in Fig. 2A, although the 4-channel fluorescent images were visualized with submicrometer resolutions, inter-channel cross-talks were recorded; the EGFP and mCitrine signals were detected in both Ch1 and Ch2, while the mRFP signals were detected in Ch2, Ch3, and Ch4. To overcome this issue, we applied a linear unmixing method based on firmly established procedure3,14 to the measured raw dataset. Initially, the matrix R was obtained from the image datasets of the HeLa cells labeled with an individual single fluorophore, which was acquired with the same condition as in the subsequent multi-color imaging (Supplementary Table S1A). After normalization to an identity matrix, the matrix was transposed to R+ (Supplementary Table S1B). By multiplying R+ with the 4-channel signal intensity matrices measured in raw images, individual fluorophore images were successfully isolated as shown in Fig. 2B. For instance, in the unmixed images, the mCitrine signals were not detectable in the EGFP-labeled microtubules image, unlike in the Ch1 image without unmixing. For quantitative evaluations, we compared the signal intensities of F-actin, which were arrowhead indicated in Fig. 2A,B, by using the signal intensities of the microtubules in the neighboring cell as the standard. As the result, the leaking signals of the F-actin were decreased by 80% after the unmixing. As for the mCitrine-labeled F-actin images, the mRFP signals were mostly undetectable, unlike in the Ch2 image. We also evaluated the signal intensities of chromosomes indicated with arrowheads in Fig. 2A,B based on the F-actin signal intensities as the standard, realizing that the unmixing decreased the leaking chromosomes signals by 96%. Next, every xyz-λ image at each time point was converted to an individual fluorophore image. Using this approach, the complex real-time morphological changes of different subcellular structures during mitosis, including the formation of the mitotic spindles, segregation of the chromosomes, and reorganization of the actin cytoskeleton, could be traced in a 3D manner (Fig. 2C, Supplementary Video S1).

Next, we observed multi-color labeled tobacco BY-2 cells, of size greater than the mammalian cells. Since plant cells tended to exhibit stronger light refractions and scattering than animal cells in general, intracellular structures were difficult to be visualized by single-photon excitation confocal microscopy, especially at the far side of the cell from the objective lens16. For 3-colors imaging, we prepared a stable transformant of BY-2 cells that expressed H2B-sGFP17, mCitrine-β-tubulin, and mCherry-CenH3; these were transfected into BY-2 cells to label chromosomes, microtubules, and centromeres, respectively. As shown in Fig. 3A, individual organelles were successfully visualized from the surface to the bottom of large cylindrical cells, almost isotropically. As demonstrated for HeLa cells, inter-channel color cross-talks were also recorded in the raw images. Accordingly, we applied the linear unmixing method to the xyz-λ images of 3-color labeled BY-2 cells, and calculated the R and R+ matrices (Supplementary Table S2). By multiplying R+ with 4-channel signal intensity matrices measured in raw images, individual fluorophore images were successfully obtained (Fig. 3B). The calculated 3 images represented clearer morphological structures of individual organelles. Especially, in the image of centromere markers, leaking signals of mCitrine-labeled microtubules were drastically decreased than those in the Ch4 image without unmixing. We evaluated the signal intensities of microtubules, which were arrowheads indicated in Fig. 3A,B, by using the signal intensities of the centromeres as the standard. As the result, the leaking microtubules signals were decreased by 73% after the unmixing. We next applied the unmixing method all xyz-λ images at different time points. Figure 3C and Supplementary Video S2 exhibit the real-time morphological changes in individual organelles undergoing mitosis in a 3D manner. With this approach, detailed 3D morphological dynamics of mitotic spindle-phragmoplast transition, expansion, and disappearance of the phragmoplast, as well as microtubule reorganization around the nuclei at the end of mitosis were successfully traced.

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