Mapping GPIIb/IIIa on murine blood platelets with expansion microscopy

Mapping GPIIb/IIIa on murine blood platelets with expansion microscopy

We visualize the membrane architecture of platelets by mapping platelet integrin GPIIb/IIIa by super-resolution fluorescence. Our approach combines colocalization and expansion microscopy and successfully interrogates highly abundant receptors and their interactions where conventional methods fail in such dense 3D scenarios.

Here we provide a selection of typical images of receptor distributions on unexpanded and expanded murine platelets and the code for simulation of platelet receptor distributions including a user manual.

Sample images: 

Unexpanded, 4x expanded and 10x expanded resting murine platelets on glycine coated coverslips were imaged on a SP5 confocal microscope (detailed methods can be found in the manuscript).

There are three different labeling combinations:

• Case I: directly immunolabeled with monoclonal antibodies antibodies carrying Alexa Fluor 488 (MWReg30, green channel) or Alexa Fluor 594 (JON6, magenta channel) can bind simultaneously on the same GPIIb/IIIa receptor -> high colocalization
• Case II: directly immunolabeled with a monoclonal antibody targeting GPIIb/IIIa (MWReg30) carrying either Alexa Fluor 594 or Alexa Fluor 488) -> low colocalizaiton
• Case III: directly immunolabeld with monoclonal antibodies carrying Alexa Fluor 488 targeting GPIIb/IIIa (JON6) and carrying Alexa Fluor 594 targeting GPIX (p0p6) -> low colocalization

The provided sample images are .ims (Imaris) files contain the raw data, chromatic aberration corrected data & deconvolved data as consecutive time points, as well as the cropped image stack of a single platelet with the masked data used for colocalization analysis.

MatLab Scipts: 3D-Image simulation of Platelet Receptor Distributions

The provided MatLab scripts allow to create 2-color, 3D image stacks based on simulated platelet receptor distributions and the image resolution provided by confocal microscopy and sample expansion. Refer to the user manual for a detailed description of how to set up and run the simulation.