Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

Abstract

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy.

Keywords: atomic force microscopy; conventional florescence microscopy; correlation; super-resolution fluorescence microscopy.

Figure 1

Schematic diagram of atomic force microscopy. In AFM, the tip-sample interactions are detected to characterize the topography and biophysical properties of sample. Reproduced from [2] with permission.

Figure 2

Schematic diagram of confocal laser scanning microscopy. In confocal microscopy, the point illumination, the detector pinhole, and the focus in the specimen are all confocal with each other. Reproduced from [28] with permission.

Figure 3

Schematic diagram of total internal reflection fluorescence illumination. When the excitation beam travels across the coverslip-sample interface (n₁ < n₂) with an incident angle θ above the critical angle θ_c (indicated by the dashed line), the excitation beam is totally internally reflected back into the cover slip and an evanescent field is generated in the sample. Only fluorophores that are located in the evanescent field are excited (indicated by the green color). Here, n₁ and n₂ are, respectively, the refractive indices of the sample and the glass coverslip. Reproduced and rearranged from [34] with permission.

Figure 4

The basic principle of stimulated emission depletion microscopy. In STED, the depletion beam is superimposed to the excitation beam to reduce the size of the excitation spot. The higher the depletion beam power, the smaller the size of the excitation spot. Reproduced and rearranged from [36] with permission.

Figure 5

Imaging principle of single-molecule localization microscopy. In SMLM, only a small subset of fluorophores can be randomly switched on using appropriate illumination and localized at high resolution; after the small subset of fluorophores is switched off, a new subset is switched on and localized. This cycles is repeated to record many frames, including the localizations of individual fluorophores (a–c). Therefore, an super-resolution image is reconstructed from all of the successful localizations (d). Reproduced and rearranged from [36] with permission.

Figure 6

Imaging a virus binding to cells using correlative force-distance curve-based AFM and confocal microscopy. The AFM tip was functionalized with a single EnvA-RABV (∆G: eGFP) virus (EnvA: the glycoprotein of the avian sarcoma leukosis vieus subgroup A; RABV: rabies virus). Mixed cultures of wild-type MDCK cells and TVA-mCherry (TVA: the avian tumor virus receptor A) expressing MDCK cells (red) were grown for three days. DIC image (a), fluorescence image (b) and overlay of both images (c) guiding the AFM tip to choose an area of interest (the dashed square), including both cell types. AFM topography (d) and corresponding adhesion map (e) in the dashed square, which were used to evaluate specific and nonspecific virus binding events. Distribution of adhesion forces of specific interactions (f,g) and nonspecific interactions (h). (i) Merged image of the topography and fluorescence images. The adhesion frequency was in line with the relative fluorescence intensity, which meant the specific adhesion events corresponding to specific binding events. Reproduced from [53] with permission.

Figure 7

Imaging mature fibronectin (FN) fibril structure using the correlative TIRFM/AFM technique. In this experiment, the F-actin (red) of rat embryonic fibroblasts (REF52) were stained to visualize cellular structure, the live REF52 cells were incubated on a homogeneous coating of Alexa 488-labelled FN (green) for 4 h, and then fixed before data acquisition. (A) Superimposition of the phase contrast image and the fluorescence image guiding the AFM tip to choose a region including FN fibrils. (B) Three correlative images (fluorescence image, AFM deflection image, and AFM topography) of the same region in the dashed square of (A). (C) The merged image of topography and fluorescence image showing the FN fibril structure and the cellular structure. (D) Correlating the height with the corresponding fluorescence intensity to distinguish the FN fibril structure and the cellular structure using a 30-nm height cut-off (dashed line). (E) Three-dimensional topography intuitively showing the FN fibril structure. Reproduced from [57] with permission.

Figure 8

Nanomanipulation of a 40 nm fluorescent bead using the correlative STED/AFM technique. (A) A comparison of confocal and STED images which shows that STED has a higher resolution. (B) Merged image of STED images acquired before (red) and after (green) AFM dragging of the same area. The overlay of both colors shows stationary beads in yellow. Magnified STED images acquired before (C) and after (D) AFM dragging, and the corresponding merged image (E), which clearly shows that the movement made by AFM at a subdiffraction distance. Reproduced from [64] with permission.

Figure 9

The setup of correlative SMLM/AFM microscopy. (A) Schematic of the optical path which is aligned with the AFM cantilever. (B) Schematic of the AFM integrated with an inverted optical microscope. (C) Photograph of the correlative SMLM/AFM instrument. (D) Magnified photograph showing the AFM cantilever aligned with the optical axis. Reproduced from [15] with permission.