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BioScience AFM


Figure 7 : AFM image of the cytoskeleton structure of a KPG7 fi broblast. (a) Height and corresponding (b) phase channel from a scan of living cells cultured at 37°C in a Petri dish heater recorded at a line rate of 6 Hz. The recording of the cytoskeleton fi laments with such a high contrast at a cantilever velocity of 600 µm/s is possible with fast-scanning AFM. Z -scales in (a) and (b) are 300 nm and 10 degrees, respectively.


where each monomeric bundle of seven alpha helices packs a pigment molecule called retinal [ 14 ]. Upon absorption of a photon, the chromophore undergoes an all-trans to 13-cis conformational change, which triggers a proton flow directionality and a cascade of changes in the BR structure. In wild-type BR, this process is extremely quick (10 ms), but the existence of point-mutations in specific BR types substan- tially inhibits their response to light. In particular, the D96N mutant BR carries an Asp96 point mutation to Asn, which slows down one of the longest intermediates of the phototo- cycle (M) in the mutant compared to the wild type protein by about 50 times [ 15 ]. The two recurring frames in Figures 6 a and 6 b depict the structural/conformational differences in the BR trimers between their photolyzed ( Figure 6b ) and non-photolyzed state ( Figure 6a ). As seen from the outlined sector in Figures 6 a and 6 b, the structural change is associated with an outward displacement of the monomers with respect to the trimeric center (approximately 0.7 nm) and is rotated counterclockwise.


Morphological changes in living cells . Conventional AFM imaging of live cells in dynamic (and static) mode is challenging because of the rather long image acquisition times and the relatively slow feedback being unable to cope


with the soſt and topographi- cally inhomogeneous samples. Fast-scanning AFM can now be applied for study of weak and rapidly changing signals such as dynamic cellular processes at near-physiological conditions. Figures 7 a and 7 b highlight a 1024-pixel 40 µm scan of a liv- ing KPG7 fi broblast recorded in DMEM/F-12 cell culture medium imaged at 37°C. T e fi lamentous structure of the underlying transverse and radial cytoskeletal arcs is immediately resolved. T e cell was imaged for a few hours at diff erent speeds and scan sizes to verify that the tip-sample interaction is not leading to a specifi c cell retrac- tion or introduction of further


morphological artifacts. T e direct observation of cell membrane dynamics is another example discussed. Living cells are constantly interacting with their surroundings by exchanging molecules and signals. Membrane ruffl ing or vesiculation of external molecules and vesicles (endocytosis), release of metabolitic degradation products, and release of signaling molecules to other cells (exocytosis) are mostly associated with membrane turnover. T e timescale of most of these processes can depend on the type of membrane fusion or secretion and normally ranges from seconds to minutes [ 16 , 17 ]. T e example below shows a plausible exocytosis event on the timescale of 30–40 seconds, which is associated with a morphological change happening directly on the cell surface ( Figures 8a–e ). T e budding vesicle can be clearly resolved in AFM phase images because of the much higher sensitivity of the phase channel to the rapidly changing and weak signals from the morphological diff erences in the cell surface. T e same features are resolved in the height channel as well, corresponding to a gradual increase of the height of the budding area from 10 to about 25 nm.


Dynamic AFM modes . The most commonly used dynamic AFM mode is amplitude modulation AFM


Figure 8 : Fast scanning of surface dynamics of KPG7 fi broblasts with high spatiotemporal resolution (a–e). Phase images acquired with a temporal resolution of ~8.5 s per frame. Yellow circles emphasize the budding event discussed in the main text. Scans in (a) through (e) contained 256 × 256 pixels per frame and were acquired at a line rate of 30 Hz. Image width = 5 µm.


2015 November • www.microscopy-today.com 23


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