fiji (imagej) and matlab scripts  (MathWorks Inc)

Journal: STAR Protocols

Article Title: Enhanced fluorescent imaging of proteins in live yeast cells using fluorescently labeled scFv

doi: 10.1016/j.xpro.2023.102299

Figure Lengend Snippet: Optimized detection of RPA foci with 3HA-labeled Rfa1 and 2E2-NLS-FP variants (A and B) Representative fluorescent microscopy images of cells expressing Rfa1-3HA and (A) 2E2-NLS-Envy or (B) 2E2-NLS-HALOtag with the addition of the far-red dye SiR-HALO, from the weak URA3 promoter, before (T = 0 min) and 25 min after irradiation with 390 nm light at 1-min intervals. The appearance of multiple Rfa1 foci is visible (white arrows). A white, dashed box surrounds the region shown in ). (C and D) Detection of cells with Rfa1 foci in Rfa1-3HA cells labeled with 2E2-NLS-Envy (C) or 2E2-NLS-HALOtag + SiR-HALO (D) expressed under the weak URA3 promoter after 10, 15, 20, and 25 min of irradiation. For calculating the percentage of cells with Rfa1 foci, the number of cells with foci was divided with the total number of cells on the imaging field for the presented timepoints. A combination of automated and manual foci identification was performed. Results shown are averages of triplicates, with at least 170 cells in each replicate. Statistical analysis was performed with One-Way ANOVA. Error bars are standard error of the mean (SEM). Different illumination conditions were applied to 2E2-NLS-Envy and 2E2-NLS-HALOtag + SiR-HALO Rfa1-3HA labeled cells during DNA damage induction. (E) Examples of correct (middle) and false (right) foci identification inside the white dashed region of (left) using our custom-made MATLAB-ImageJ combined script “TokyoGhoulRe”. Yellow-white crosses mark the identified foci (local maxima). Right panel shows false-positive foci identification as a result of small noise parameter value selection during identification (see text and Problem 5 for more details).

Article Snippet: Different illumination conditions were applied to 2E2-NLS-Envy and 2E2-NLS-HALOtag + SiR-HALO Rfa1-3HA labeled cells during DNA damage induction. (E) Examples of correct (middle) and false (right) foci identification inside the white dashed region of ( ) (left) using our custom-made MATLAB-ImageJ combined script “TokyoGhoulRe”.

Techniques: Labeling, Microscopy, Expressing, Irradiation, Imaging, Selection

Journal: STAR Protocols

Article Title: Enhanced fluorescent imaging of proteins in live yeast cells using fluorescently labeled scFv

doi: 10.1016/j.xpro.2023.102299

Figure Lengend Snippet:

Article Snippet: Different illumination conditions were applied to 2E2-NLS-Envy and 2E2-NLS-HALOtag + SiR-HALO Rfa1-3HA labeled cells during DNA damage induction. (E) Examples of correct (middle) and false (right) foci identification inside the white dashed region of ( ) (left) using our custom-made MATLAB-ImageJ combined script “TokyoGhoulRe”.

Techniques: Recombinant, Staining, Microscopy, Agarose Gel Electrophoresis, Labeling, Amplification, Plasmid Preparation, Expressing, Software, Membrane, Stripping Membranes, Transferring, Imaging, Spectrophotometry