Journal: Light, science & applications

Article Title: Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing.

doi: 10.1038/s41377-021-00536-3

Figure Lengend Snippet: Fig. 4 FRET imaging in live cells via resolving the excitation spectrum. a Excitation spectrum measured by our spectral microscope (black solid line) and its unmixing (dash lines) for non-interacting mRuby2 and Clover co-expressed in the cytoplasm of a live COS-7 cell. b Measured excitation spectrum and its unmixing for a directly linked Clover-mRuby2 construct expressed in a live COS-7 cell. c, d Measured excitation spectrum and its unmixing for the Clover-mRuby2 FRET crowding sensor in the cytoplasm of a live COS-7 cell, before (c) and ~25 s after (d) 150% hypertonic treatment by adding into the cell medium an equal volume of medium that was supplemented with 300 mM sorbitol. e Color-coded FRET maps for the crowding sensor, for two live COS-7 cells before (left), ~10 s after (center), and ~25 s after (right) the 150% hypertonic treatment. f Color-coded FRET maps for the crowding sensor, for two live COS-7 cells before (left), 500 s after (center), and 1650 s after (right) 50% hypotonic treatment by adding into the medium an equal volume of water. g, h FRET value time traces for the boxed regions of cytoplasm (solid lines) and nuclei (dash lines) of the four cells (e, f). Experiments were performed with 8-wavelength excitation cycles at 10 fps (0.8 s acquisition time for each spectral image). Scale bars: 10 µm (e, f). See also Supplementary Videos 8–9

Article Snippet: The co-expression plasmid of free Clover and mRuby2 was constructed by inserting an internal ribosome entry site (cloned from Addgene #51406; a gift from Kathleen L Collins) between the Clover and mRuby2 of ClovermRuby2-FRET-10 at the AgeI site.

Techniques: Imaging, Microscopy, Construct

Journal: Scientific reports

Article Title: ShadowR: a novel chromoprotein with reduced non-specific binding and improved expression in living cells.

doi: 10.1038/s41598-019-48604-4

Figure Lengend Snippet: Figure 4. FRET efficiency and maturity of ShadowR in tandem fluorescent proteins. (a) A schematic drawing of the constructs used to evaluate the FRET efficiency and fraction of the mRuby2 or mScarlet undergoing FRET. (b) Representative fluorescence lifetime images of the tandem proteins in HeLa cells; the images were taken at 1000-nm two-photon excitation. Because the expression level of mRuby2-Ultramarine and mScarlet- Ultramarine was low, we used different laser powers for each condition (5 mW for mRuby2-Ultramarine, 4 mW for mRuby2-ShadowR, 3 mW for mScarlet-Ultramarine, 2 mW for mScarlet-ShadowR). Scale bar, 50 µm. (c,e) Quantification of FRET efficiency of the tandem proteins. The fluorescence lifetime over the whole image was used for the analysis (See Materials and Methods). The number of images used for the analysis are indicated in the figure. Each image contains 4–12 cells, and the data are presented as mean ± SEM. Asterisks denote statistical significance (t test, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant). (d,f) A comparison of the fraction of mRuby2 or mScarlet fluorescent protein undergoing FRET (chromophore maturation efficiency of Ultramarine or ShadowR) analyzed in individual cells; data were plotted in the descending order. The FRET fraction is directly related to the maturation efficiency of an acceptor, i.e., Ultramarine (d) or ShadowR (f). Means ± SD are also plotted on the right (t test, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant). The number of samples (n) and mean ± SD are also indicated. (g) Comparison of E. coli expressing Ultramarine, ShadowR, or dUltramarine2. Purple colonies indicate that the E. coli cells express the respective chromoproteins. After transformation, the cells were incubated for 16 hr at 32 °C and imaged.

Article Snippet: The mRuby2 gene construct was a gift from Michael Lin (Addgene plasmid #40255).

Techniques: Construct, Fluorescence, Expressing, Comparison, Transformation Assay, Incubation

Journal: Scientific reports

Article Title: ShadowR: a novel chromoprotein with reduced non-specific binding and improved expression in living cells.

doi: 10.1038/s41598-019-48604-4

Figure Lengend Snippet: Figure 5. Epifluorescence analysis of Ultramarine and ShadowR fusion proteins. (a) Tandem constructs of mEGFP (green), mRuby2 (red), and mScarlet (red) with Ultramarine or ShadowR were expressed in HeLa cells and imaged under an epifluorescence microscope. All cells in the image field were identified by Hoechst 33342 staining (blue). Scale bar, 100 µm. (b–d) The fluorescence intensities of mEGFP (b), mRuby2 (c), and mScarlet (d) in the image field were measured and divided by the cell number determined by Hoechst staining and counting. Ten images per condition were used for the analysis. Each image contains 500–1000 cells, and the data are presented as mean ± SEM. Asterisks denote statistical significance (t test, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant).

Article Snippet: The mRuby2 gene construct was a gift from Michael Lin (Addgene plasmid #40255).

Techniques: Construct, Microscopy, Staining, Fluorescence