Cell membrane sample preparation method of combined AFM and dSTORM analysis

Abstract

A major role of cell membranes is to provide an ideal environment for the constituent proteins to perform their biological functions. A deep understanding of the membrane proteins assembly process under physiological conditions is quite important to elucidate both the structure and the function of the cell membranes. Along these lines, in this work, a complete workflow of the cell membrane sample preparation and the correlated AFM and dSTORM imaging analysis methods are presented. A specially designed, angle-controlled sample preparation device was used to prepare the cell membrane samples. The correlated distributions of the specific membrane proteins with the topography of the cytoplasmic side of the cell membranes can be obtained by performing correlative AFM and dSTORM measurements. These methods are ideal for systematically studying the structure of the cell membranes. The proposed method of the sample characterization was not only limited to the measurement of the cell membrane but also can be applied for both biological tissue section analysis and detection.

Keywords: Atomic force microscopy (AFM); Cell membrane; Combined technique; Direct stochastic optical reconstruction microscopy (dSTORM); Super-resolution microscopy (SRM).

Figure 1

The device for cell membrane sample preparation

Figure 2

Schematic diagram of the preparation process of the APTES-coverslip. The reagents DIPEA and APTES were placed in small containers on the bottom of the desiccator. Silylation solution: 3-aminopropyltriethoxysilane (APTES); N,N-diisopropylethylamine (DIPEA)

Figure 3

Schematic illustration of the workflow for preparing the cytoplasmic side of cell membranes by using the sheared-open method

Figure 4

Schematic diagram of the combined AFM and dSTORM technique

Figure 5

Depiction of the custom MATLAB program with a GUI to achieve registration between the AFM and the dSTORM images

Figure 6

Depiction of the custom MATLAB program with a GUI to achieve registration between the low- and high-resolution AFM topography

Figure 7

Identification of Glut1 and Band 3 in the topography of the cytoplasmic side of the erythrocyte membrane. A Topography of the cytoplasmic side of the erythrocytes membrane. B, C Distribution of Glut1 and Band 3 in the same erythrocyte membrane. D Merged image of Panels A−C where the locations of Glut1 and Band 3 are identified in the topography of the cytoplasmic side of the erythrocyte membrane. E High-resolution topography of the corresponding area (white box in Panel A). F–H Localization of Glut1 and Band 3 in the high-resolution topography, respectively and the merged image. Scale bars: 3 μm (Panels A–D) , 300 nm (Panels E–H)

Figure 8

Localization of AnkG and NKA in the high-resolution cell membrane morphology images. A–C The correlative high-resolution AFM image and dual-color dSTORM images of AnkG and NKA. D–E The merged image of AnkG and NKA within the membrane topography. F The merged image of Panels A–C. G–I Depiction of clusters of the specific proteins on the correlative maps. The green (blue) dots represent the true locations of each AnkG (NKA) molecule and green (blue) polygon boxes circle the clusters of AnkG (NKA) molecules. J The correlation analysis among AFM height, AnkG and NKA positions in the white barred region of Panel I