|
Figure 1. Schematic representation of the concept of photoconversion of cells in culture and in embryos. Photoconversion in cells (A): Single cells or group of cells expressing a constitutively active KikGR can be labeled non-invasively. KikGR expressing cells fluoresce green before photoconversion. After exposure to short wavelength light (405 nm) a single or a group of cells of interest can be photoconverted. Photoconverted cells emit red fluorescence and can be tracked over time. Photoconversion in mouse embryos (B): Any KikGR-positive cell or group of cells of interest in a mouse embryo can be photoconverted using short wavelength light (405 nm). Photoconverted red fluorescent cells can be imaged in live static cultured embryos, so that 3D over time (4D) data sets can be acquired. Using live imaging and roller culture, a region of interest (ROI) in multiple embryos (for example all embryos in one litter) can be photoconverted in the same experiment and imaged. This is advantageous for comparing cell behavior in wild type and mutant mice before any visible onset of a phenotype.
|
|
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.
|
|
Figure 3. Photoconversion of widespread expressing CAG::KikGR transgenic ES cells. Panels show 2D (A-F, I) and 3D images (G, H) of the photoconversion of a selected group of cells in an ES cell colony. Bright field image of ES cell colony (A). Image of ES cell colony with green (B) and red (C) channel showing green but no red fluorescence prior to photoconversion. Yellow dashed box show regions of interest (ROI) that is to be photoconverted. After photoconversion, cells in an ROI do not fluoresce green (D) but red (E). Merge of green and red channel after photoconversion, showing complete photoconversion of cells in the region of interest. 3D image of ES cell colony before photoconversion. All cells emit green fluorescence (G). Line plot showing average fluorescent intensity measured along the white arrow in G (G'). 3D image of ES cell colony after photoconversion. Photoconverted cells emit red fluorescence (H). Line plot showing average fluorescent intensity measured along the white arrow in H showing complete photoconversion of cells, i.e. shift of emission spectrum from green to red in photoconverted region (H'). 2D orthogonal representation of photoconverted ES cell colony showing complete photoconversion in the region of interest through entire z-stack (I).
|
|
Figure 4. Efficiency of KikGR photoconversion over time with constant exposure to short wavelength laser light. Continuous scanning over time with 405 nm laser light excitation (30% laser power; 25.0 mW) of an ES cell colony, constitutively expressing KikGR, shows fast and complete photoconversion. Fluorescence in green and red channels at different times of 405 nm exposure (A-H). Yellow dashed ellipse depicts region of interest (ROI) exposed to 405 nm laser light. Plot of time series in A-H shows shift in emission spectra from 513 nm to 596 nm (I).
|
|
Figure 5. Widespread KikGR expression in organs of CAG::KikGRTg/+ adult mice. Panels of bright field and corresponding dark field epifluorescent images of organs taken from a 6 week old CAG::KikGRTg/+ mouse and a non-transgenic littermate. Brain (A, A'), heart (B, B'), liver (C, C'), muscle (D, D'), pancreas (E, E') and testis (F, F'). Panels of bright field and corresponding dark field epifluorescent images (green and red channels) of the brain before and after photoconversion using a UV filter (G-L). Yellow dashed ellipse depicts region of interest (ROI) to be photoconverted (H). Green channel (H, J). Red channel (I, K). Overlay of images of green and red channel (L).
|
|
Figure 6. Widespread KikGR expression in CAG::KikGRTg/+ embryos. Panels of bright field and corresponding dark field epifluorescent images of CAG::KikGRTg/+ embryos and non-transgenic littermates at E7.5 (A, A'), E8.5 (B, B'), E9.5 (C, C'), E10.5 (D, D') and E12.5 (E, E').
|
|
Figure 7. Photoconversion of cells in the lateral plate mesoderm (lpm) of an E8.0 CAG::KikGRTg/+ embryo: live imaging of behaviors of cell cohorts. Panels show 2D (bright field) and 3D (green and red channels) laser scanning confocal images of a posterior view of an E8.0 CAG::KikGRTg/+ embryo (A-H). Bright field image (A). KikGR expression in an E8.0 embryo prior to photoconversion. Yellow dashed rectangle, region of interest (ROI), demarcates population of cells to be excited with laser light of 405 nm wavelength (B). Loss of green fluorescence in boxed ROI after photoconversion (C). Cells in boxed ROI fluoresce red after photoconversion (D). Merge of red and green channels after photoconversion showing labeled lpm cells at start of embryo culture (E). Panels show migration of photoconverted cells in lpm of embryo after 6 hrs. Dimensions of cell population photoconverted have become longer and thinner (F-G). Merge of green and red channel (F). Red channel (G). Overlay of red channel onto bright field image (H). ESom, early somite stage; ps, primitive streak. Scale bar indicates 200 μm.
|
|
Figure 8. Live imaging cell fate during mouse cardiac morphogenesis. Panels show 2D (bright field) and 3D (green and red channels) laser scanning confocal images of photoconversion cells on the right side (ROI) of the cardiac crescent (cc) of an E7.5 CAG::KikGRTg/+ embryo (A-E). After 12 hrs of roller culture the embryo has formed a linear heart tube (lh) and photoconverted cells can be detected on the right side of the linear heart tube (F-J). Widefield image of an E7.5 embryo. Red dashed box shows region of cc depicted in panels A-E (A'). Bright field image of cardiac crescent before culture (A) and embryo after culture (F). Red and green channel (B, E, G and J). Green channel (C and H). Red channel (D and I). White dashed box in panel G shows region depicted in panels H-J.
|
|
Figure 9. Live imaging photoconverted KikGR-positive cells in a mouse embryo: photoconversion, ex utero culture and 3D time-lapse laser scanning confocal imaging do not affect embryonic development. Panels show sequence 3D stills over a time of 640 min of a CAG::KikGRTg/+ embryo developing from an early head fold stage to an early somite stage (A-G). The series depicts the developing cardiac crescent (cc), headfolds (hf) and condensing somites (som). Two regions of photoconverted cells are shown over time. Cells of photoconverted region located in the left side of the embryo migrate anteriorly into the cardiac crescent. Bright field images (A-G). Merge of RFP channel and bright field (A'-G'). RFP channel (A"-G"). GFP and RFP channel (A"'). m, midline; n, node.
|
