Atomic Force Microscopy of Live Neurons

Summary

Researchers have succeeded in imaging structural dynamics of living neurons with an unprecedented spatial resolution.

The observations of structural dynamics in live neurons present the possibility of visualizing the morphology of synapses at nanometer resolution in real time providing many new insights into mechanisms of how neurons store information in their morphology, how it changes synaptic strength and ultimately how it creates new memory.

Scientific Reports 3/4/15

(a) Scanning electron microscopy (SEM) images of the end of the cantilever without an electron-beam-deposit (EBD) tip (left) and with ~3 μm EBD tip (right). (b) A schematic illustration of LT-HS-AFM system. An arc lamp for fluorescence excitation and a CCD camera to capture bright-field and fluorescence images were installed under the LT-HS-AFM system. (c) Fluorescence images of a COS-7 cell transfected with mEGFP. The white broken lines show the shadow of the cantilever.  Credit: Mikihiro Shibata, Scientific Reports

(a) Scanning electron microscopy (SEM) images of the end of the cantilever without an electron-beam-deposit (EBD) tip (left) and with ~3 μm EBD tip (right). (b) A schematic illustration of LT-HS-AFM system. An arc lamp for fluorescence excitation and a CCD camera to capture bright-field and fluorescence images were installed under the LT-HS-AFM system. (c) Fluorescence images of a COS-7 cell transfected with mEGFP. The white broken lines show the shadow of the cantilever.
Credit: Mikihiro Shibata, Scientific Reports

 

Press Release

Max Planck Florida Institute for Neuroscience News 3/12/15

Scientists have developed atomic force microscopy for imaging nanoscale dynamics of neurons

Researchers at the Max Planck Florida Institute for Neuroscience and Kanazawa University (Japan) have succeeded in imaging structural dynamics of living neurons with an unprecedented spatial resolution

Researchers at the Max Planck Florida Institute for Neuroscience– Atomic force microscopy (AFM) is a leading tool for imaging, measuring, and manipulating materials with atomic resolution – on the order of fractions of a nanometer.
– AFM images surface topography of a structure by “touching” and “feeling” its surface by scanning an extremely fine needle (the diameter of the tip is about 5 nanometers, about 1/100 of light wavelength or 1/10,000 of a hair) on the surface.
– This technique has been applied to image solid materials with nanometer resolution, but it has been difficult to apply AFM for a soft and large sample like eukaryotic cells and neurons without damaging the sample. Additionally, image acquisition with conventional AFM is too slow to capture fast cellular morphology changes.
– Researchers have extensively modified the AFM system for imaging eukaryotic cells and neurons with high spatial and temporal resolution.
– This new system allows for analysis of cell morphology changes with a spatial resolution ~20-100 fold better than that of a standard light microscope.

Movie 1

Imaging structural dynamics of living cells and neurons

While progress has been made over the past decades in the pursuit to optimize atomic force microscopy (AFM) for imaging living cells, there were still a number of limitations and technological issues that needed to be addressed before fundamental questions in cell biology could be address in living cells.

In their March publication in Scientific Reports, Drs. Ryohei Yasuda andMikihiro Shibata (pictured above) at Max Planck Florida Institute for Neuroscience in collaboration with Kanazawa University describe how they have built the new AFM system optimized for live-cell imaging. The system differs in many ways from a conventional AFM: it uses an extremely long and sharp needle attached to a highly flexible plate. The system is also optimized for fast scanning to capture dynamic cellular events. These modifications have enabled researchers to image living cells, such as mammalian cell lines or mature hippocampal neurons, without any sign of cellular damage.

“We’ve now demonstrated that our new AFM can directly visualize nanometer-scale morphological changes in living cells”, explained Dr. Yasuda, neuroscientist and scientific director at the Max Planck Florida Institute for Neuroscience.

Movie 2

In particular, this study demonstrates the capability to track structural dynamics and remodeling of the cell surface, such as morphogenesis of filopodia, membrane ruffles, pit formation or endocytosis, in response to environmental stimulants. An example of this capability can be visualized in movie 1, where a fibroblast is imaged before and after treatment with insulin hormone, which intensely enhances the ruffling at the leading edge of the cell. Another example is seen in movie 2, where the morphological changes of a finger-like neuronal protrusion in the mature hippocampal neuron are observed.

According to Dr. Yasuda, the successful observations of structural dynamics in live neurons present the possibility of visualizing the morphology of synapses at nanometer resolution in real time in the near future. Since morphology changes of synapses underlie synaptic plasticity and our learning and memory, this will provide us with many new insights into mechanisms of how neurons store information in their morphology, how it changes synaptic strength and ultimately how it creates new memory.

Article in Scientific Reports (cc)

By Mikihiro Shibata, Takayuki Uchihashi, Toshio Ando, Ryohei Yasuda

Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells

Abstract

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons.

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