High-speed atomic force microscopy imaging of live mammalian cells

Abstract

Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.

Keywords: AFM; Bio-imaging; Probe microscopy; live-cell imaging; nanotechnology.

Figure 1

HS-AFM setup for observations of live mammalian cells. (A) Photograph of a wide-area scanner (B) Scanning electron microscopy (SEM) imaging of the end of the cantilever with and without an electron-beam-deposit (EBD) tip. The tip length is about 3 μm for live-cell HS-AFM. (C) Epi-fluorescence images of a COS-7 cell transfected with mEGFP. The white broken lines highlighted the base of a cantilever. The white square indicates a HS-AFM scanning area. HS-AFM images corresponds to Figure 2.

Figure 2

HS-AFM images of a living COS-7 cell. A HS-AFM topographical image acquired from the area indicated in white box in Figure 1C before the addition of cytochalasin D (Top), after application of 20 ng/mL cytochalasin D (middle) and following washout for 30 min (bottom). HS-AFM images taken at the indicated times (green) and the image taken at 0 s (magenta) are overlaid. White arrows indicate newly appeared structures at the leading edge. HS-AFM imaging rates, 10 second per frame. HS-AFM pixel resolutions, 200×200 pixels².

Figure 3

HS-AFM images of living COS-7 and HeLa cells in response to extracellular stimuli. (A) Fluorescence image of a COS-7 cell transfected with mEGFP. The white square corresponds to the HS-AFM scanning area. A HS-AFM topographical image acquired after application of 20 mg/mL insulin. HS-AFM images taken at the indicated times (green) and the image taken at 0 s (magenta) are overlaid. Before the addition of insulin, cells were starved with serum-free medium at least for 1 hour. White arrows indicate newly appeared structures. Dashed circles indicate the pits formation. (B) Fluorescence image of a HeLa cell transfected with mEGFP. The white square corresponds to the HS-AFM scanning area. A HS-AFM topographical image acquired after application of 20 ng/mL EGF. HS-AFM images taken at the indicated times (green) and the image taken at 0 s (magenta) are overlaid. Before the addition of EGF, cells were starved with serum-free medium at least for 1 hour. White arrows indicate accelerated surface flow on cell surface. HS-AFM imaging rates, 10 second per frame. HS-AFM pixel resolutions, 200×200 pixels².

Figure 4

Pits formation on the plasma membrane of a living COS-7 cell. (A) Fluorescence images of a COS-7 cells transfected with mEGFP. The area indicated with the white square was subjected to HS-AFM imaging. (B) Time courses of the depth of pits with and without closure caps. Bars indicate the formation of closure caps. Arrows indicate pit formation. (C) The number of observed pits per min per μm² area before, after and washout of dynasore application. (D) The histogram of the lifetime of pits for COS-7 cells transfected with mEGFP (control, upper) and mEGFP-Rab5(Q79L) (bottom). The number of total analyzed pits are 101 and 106 for COS-7 cells transfected with mEGFP and mEGFP-Rab5(Q79L), respectively (3 cells each). (E) A sequence of HS-AFM topographical images of a COS-7 cell transfected with constitutively active Rab5 mutant [mEGFP-Rab5(Q79L)]. The white arrows indicate pits formations. HS-AFM imaging rates, 6 second per frame. HS-AFM pixel resolutions, 200×200 pixels².

Figure 5

HS-AFM images of endocytosis on the plasma membrane. (A, B) A sequence of magnified HS-AFM images of a living COS-7 cell transfected with mEGFP, taken at 6 seconds per frame, during the pit formation and the closure of the pit with a cap. Arrows indicate the formation of the pit. Dotted circles indicate the formation of the closure cap.

Figure 6

SEM imaging of fixed cultured hippocampal neurons. (A) Overall images of cultured hippocampal neurons at 2 days in vitro. (B, C) SEM imaging of cultured hippocampal neurons at 21 days in vitro.

Figure 7

HS-AFM images of a living cultured hippocampal neuron at 9 days in vitro. (A) Fluorescence image of a hippocampal neuron transfected with mEGFP. The white square indicates the HS-AFM scanning area. (B) A sequence of HS-AFM images of a dendrite. The white arrows indicate the growth of dendrite. HS-AFM imaging rates, 5 second per frame. HS-AFM pixel resolutions, 200×200 pixels².

Figure 8

HS-AFM images of a living cultured hippocampal neuron at 13 days in vitro. (A) Fluorescence image of a hippocampal neuron transfected with mEGFP. (B) A sequence of HS-AFM images at 5 s per frame. White arrows indicate the thin, sheet-like ruffling structure. White dotted box at 360 s indicates the magnified region shown in C. (C) A sequence of magnified HS-AFM images during the pits opening and clouser. White arrows indicate the pit formation and dotted red circles indicate the closure cap. (D) Time course of the depth and height of the pit with and without the closure cap. Bars indicate the formation of the closure cap. Arrows indicate pit formations. HS-AFM imaging rates, 5 second per frame. HS-AFM pixel resolutions, 200×200 pixels².

Figure 9

HS-AFM images of a living cultured hippocampal neuron at 15 days in vitro. (A) Fluorescence image of a hippocampal neuron transfected with mEGFP. Corresponding HS-AFM images for A is shown in B. (B) A sequential HS-AFM topographical images are taken at 5 s per frame. White arrows indicate the dynamics of spine-like structure. HS-AFM imaging rates, 5 second per frame. HS-AFM resolutions, 200×200 pixels².