Journal: Scientific Reports

Article Title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

doi: 10.1038/srep14072

Figure Lengend Snippet: TIRF ( a ) and SMLM ( b ) image of mEos2 single-molecule surface. The boxed insets in ( b ) are magnifications of two mEos2 molecules and demonstrate repeated localizations (crosses) of single mEos2 FPs. ( c ) Intensity time traces of mEos2 molecules boxed in ( b ) show distinct blinking after photoactivation before the FPs photobleach irreversibly. Time traces ( c ) and crosses ( b ) having the same color belong to the same molecule. ( d ) Distribution of the number of blinking events N blinks,i upon photoactivation for single mEos2 FPs. The distribution is fit to a geometric distribution with p surface = 0.30 ± 0.01 as fitting parameter (n = 622 mEos2 molecules, adjusted R 2 = 0.995) Note that N blinks,i corresponds to the number of fluorophore reactivations, not total appearances. Scale bars: 1 μm; inset 10 nm.

Article Snippet: For single-molecule mEos2 surfaces, glass slides (PLANO GmbH) were washed in isopropyl alcohol (Sigma) for 30 min, plasma cleaned in N 2 plasma for 10 min (Diener Electronic) and incubated in 100 μg/ml poly-L-lysine solution (Sigma) for 2 h at room temperature.

Techniques:

Journal: Scientific Reports

Article Title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

doi: 10.1038/srep14072

Figure Lengend Snippet: SMLM images of CD86-mEos2 ( a ) and CTLA-4-mEos2 ( b ) at the membrane of Hela cells. ( c , e ) Magnifications of region I or II boxed in ( a ) or ( b ) respectively. The inset shows a magnification of the boxed region. Distributions of N blinks are generated from SMLM data sets. ( d ) For CD86-mEos2, the distribution is well approximated by a geometric distribution and yields p membrane = 0.28 ± 0.01 as blink parameter, which is used as a fixed value for all other membrane proteins (n = 11 cells, adjusted R 2 = 0.986 for CD86-mEos2). ( f ) For CTLA-4-mEos2, the distribution of N blinks fits to a negative binomial distribution with an average number of N = 1.93 ± 0.02 underlying fluorescent probes per complex (n = 11 cells, adjusted R 2 = 0.989 for CTLA-4-mEos2). Scale bars: 2 μm; inset 100 nm.

Article Snippet: For single-molecule mEos2 surfaces, glass slides (PLANO GmbH) were washed in isopropyl alcohol (Sigma) for 30 min, plasma cleaned in N 2 plasma for 10 min (Diener Electronic) and incubated in 100 μg/ml poly-L-lysine solution (Sigma) for 2 h at room temperature.

Techniques: Membrane, Generated

Journal: Scientific Reports

Article Title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

doi: 10.1038/srep14072

Figure Lengend Snippet: VSVG-mEos2 and CD80-mEos2 were expressed in Hela cells and subjected to SMLM imaging. ( a , b ) Resulting distributions of N blinks were fit to negative binomial distributions yielding N = 1.41 ± 0.03 for CD80-mEos2 ( a ) and N = 2.71 ± 0.04 for VSVG-mEos2 ( b ) as fitting parameters (n = 10 cells, adjusted R 2 = 0.983 for CD80-mEos2; n = 11 cells, adjusted R 2 = 0.964 for VSVG-mEos2).

Article Snippet: For single-molecule mEos2 surfaces, glass slides (PLANO GmbH) were washed in isopropyl alcohol (Sigma) for 30 min, plasma cleaned in N 2 plasma for 10 min (Diener Electronic) and incubated in 100 μg/ml poly-L-lysine solution (Sigma) for 2 h at room temperature.

Techniques: Imaging