Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells.
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.
1] Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA [2] Department of Neurobiology, Duke University Medical School, Durham, NC 27710, USA.
Mikihiro
M
Shibata
1] Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan [2] Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa 920-1192, Japan [3] CREST/JST, Tokyo 102-0075, Japan.
Takayuki
T
Uchihashi
1] Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan [2] Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa 920-1192, Japan [3] CREST/JST, Tokyo 102-0075, Japan.
Toshio
T
Ando
1] Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA [2] Department of Neurobiology, Duke University Medical School, Durham, NC 27710, USA.
Ryohei
R
Yasuda
D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D015638:Cytochalasin D / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D061251:Primary Cell Culture; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D059008:Time-Lapse Imaging / Q000379:methods*
D007328:Insulin; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D004815:Epidermal Growth Factor