Journal: bioRxiv

Article Title: PEPCy: Photostable fluoromodules for live cell, super-resolution microscopy of surface proteins

doi: 10.1101/2024.07.03.601615

Figure Lengend Snippet: Applications of PEPCy tags for imaging cell surface receptors. A . Schematic of membrane protein tracking experiment using TIRF microscopy. AlphaFold predicted structures of PEPCy tags. B . Normalized probability distributions of photons detected per trajectory from single particle analyses between PEPCy and HT targeted to their cognate dyes. (N = 1000-1300 single tracks per sample). C. Super-resolution microscopy of Intimin fused to PEPCy3 expressed on E.coli outer membrane. Schematic of the fusion is shown followed by diffraction-limited DIC and fluorscence images of the cells expressing the same. Maximum intensity projections (MIP) compared with a 2D reconstruction of single molecule localization data. Inset compares a cluster observable in the MIP that is resolved into two clusters in the super-resolved image. D . Time-lapse confocal microscopy of HeLa cells expressing PEPCy3-B2AR labeled using Cy3. Numbers on the left of each image denote the time in minutes. Isoproterenol was added at t = 0 and imaged were acquired every 30s for 1 hour. E . Schematic of simultaneous two color tracking of PEPCy3-B2AR and PEPCy5-B2AR-Ala on the same cell. F . Single molecule trajectories of PEPCy3 (left) and PEPCy5 (right) on a HEK cell, color coded by median trajectory speed. All scale bars in the figure unless mentioned otherwise are 5 µ m. A and E were created with BioRender.com .

Article Snippet: Single molecule localization microscopy was performed on a Nikon Eclipse Ti2 microscope using a 100x (1.45 N.A.) oil immersion objective.

Techniques: Imaging, Membrane, Microscopy, Single Particle, Super-Resolution Microscopy, Expressing, Confocal Microscopy, Labeling

Journal: bioRxiv

Article Title: PEPCy: Photostable fluoromodules for live cell, super-resolution microscopy of surface proteins

doi: 10.1101/2024.07.03.601615

Figure Lengend Snippet: Application of PEPCy tags for single molecule and time-lapse microscopy of cell surface proteins. A. Micrographs of HEK cells expressing PEPCy3 or HT on their surfaces labeled with Cy3, and B. cells expressing PEPCy5 or HT on their surfaces labeled using Cy5. The colormap is inverted to clearly show single molecules against the white background. Normalized probability distributions of integrated single molecule signal above the background are shown adjacent to each image. (N = 1200-2000 single molecules per sample for A and B). Scale bar is 5 µ m. C . Probability distributions of the background counts from single PEPCy3 or HT-Cy3 molecules expressed on HEK cell surfaces and imaged via TIRF microscopy (N = 998 for PEP and 1020 for HT) D . Probability distributions of the background counts from single PEPCy5 or HT-Cy5 molecules expressed on HEK cell surfaces and imaged via TIRF microscopy (N = 1133 for PEP and 784 for HT). E . Wash-free extended tracking of single PEPCy5-B2AR-Ala molecules on HEK cell surface. The first image of each acquisition is shown. The black vertical lines represent the instance and duration of the illumination turned off. The graph below shows the number of localizations per frame as a function of time in this experiment. F . Time-lapse confocal microscopy of HeLa cells expressing B2AR-Ala-PEPCy5 labeled using Cy5. Numbers on the left of each image denote the time in minutes. Images were acquired every 30 s for 30 mins. G . Normalized frequency distributions (bars) and cumulative probability (dashed lines) of median speed per trajectory for PEPCy3-B2AR and PEPCy5-B2AR-Ala molecules (trajectory N = 42906 for PEPCy3 and N = 25253 for PEPCy5) H . Normalized frequency distributions (bars) and cumulative probability (dashed lines) of jumps in a 50 ms duration for PEPCy3-B2AR and PEPCy5-B2AR-Ala molecules.

Article Snippet: Single molecule localization microscopy was performed on a Nikon Eclipse Ti2 microscope using a 100x (1.45 N.A.) oil immersion objective.

Techniques: Time-lapse Microscopy, Expressing, Labeling, Microscopy, Confocal Microscopy