AFM probes cells as never before
Image:High speed AFM captures live neuron dynamics in real-time [Yasuda et al, Scientific Reports]
US and Japan-based researchers have built a fast-scanning AFM to image living cells without damage to specimens.
Called 'long-tip, high-speed AFM', the instrument has captured dynamic cellular processes within cells and neurons at nanometre resolution.
Long-tip high-speed AFM movie of the leading edge of a living COS-7 cell under a control condition (left) and after the application of the drug cytochalasin D (right). [Yasuda et al Nature Scientific Reports]
AFM has been widely used to image solid materials, but using the technique to study soft, large samples, such as eukaryotic cells and neurons, has proven difficult.
Image acquisition with conventional AFM can take minutes - too slow to capture fast cellular processes - while tip motion can damage the sample.
Meanwhile, tapping-mode high-speed AFM (HS-AFM) has been introduced to image proteins at much faster scanning speeds and with minimum biological damage, but researchers have struggled to image larger cells.
With this in mind, researchers from the Max Planck Florida Institute for Neuroscience, Bio-AFM Frontier Research Center, Kanazawa University, and colleagues, have developed HS-AFM with a wide-area scanner and an extremely long-tip cantilever.
Using phenol deposition in a SEM, they first grew a 3 µm long, 5 nm diameter carbon tip onto a soft cantilever.
As Dr Ryohei Yasuda from Max Planck Florida explains: "This new tip design enables imaging of cells with high spatial resolution and with minimum collision to the cell when scanned under tapping-mode."
Yasuda and colleagues also adapted a wide-area, high speed piezo-scanner to scan a 5 x 5 µm² area within five seconds and combined the HS-AFM with fluorescence microscopy to locate the long-tip to the target area.
Using the instrument the researchers first imaged a mammalian cell, placing the cantilever tip at the leading edge of the cell, and scanning a 10 x 10 µm² area to examine surface topology dynamics at 10 s a frame.
LT-HS-AFM images of a living COS-7 cell. [Yasuda et al, Scientific Reports]
In this way, the researchers could image the cell for more than an hour without any obvious cellular damage, revealing membrane ruffling and microspike, or filopodia, extension and retraction over a few seconds.
"Our long AFM tip robustly stepped over cells on a substrate without collisions between the cantilever and cells, during HS-AFM scanning," highlights Yasuda.
The researchers went onto image membrane dynamics close to the nucleus of these cells, as well as morphological changes of live neurons.
(a) Fluorescence image of a GFP-labelled hippocampal neuron. Corresponding LT-HS-AFM images for a is shown in b. (b) A sequence of LT-HS-AFM topographical images at 5 s per frame. White arrows indicate the dynamics of spine-like structure. [Yasuda et al, Scientific Reports]
"Combining [the AFM] with fluorescence resonance energy transfer (FRET) imaging or optical nanoscopy techniques could add further information about molecular interaction and intracellular signal transduction," say Yasuda.
Given his team's successes, Yasuda also reckons the HS-AFM could be adapted to visualise the morphology of synapses in real-time at nanometre resolution.
Research is published in Scientific Reports