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High-speed atomic force microscope combined with single-molecule fluorescence microscope
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10.1063/1.4813280
/content/aip/journal/rsi/84/7/10.1063/1.4813280
http://aip.metastore.ingenta.com/content/aip/journal/rsi/84/7/10.1063/1.4813280
View: Figures

Figures

Image of FIG. 1.
FIG. 1.

Dichroic mirror tilter for laser-beam tracking of lateral cantilever motion. (a) Schematic showing the working principle of laser-beam tracking by mirror-tilting method. (b) and (c) Side views of the two-dimensional mirror tilter when viewed from two orthogonal directions.

Image of FIG. 2.
FIG. 2.

Dynamic response of X- and Y-tilters. (a) Frequency spectra of tilting motion of the X-tilter (black line, gain; blue line, phase). (b) Tilting motion of X-tilter (red line) driven by 1 kHz isosceles triangle wave signal (black line). (c) Tilting motion of X-tilter (red line) driven by 1 kHz isosceles triangle signal filtered through inverse compensation (black line). The orange line shows the tilting motion when the filtered driving signal is further modified for the elimination of the curved response (blue line). (d) Frequency spectra of tilting motion of the Y-tilter (black line, gain; blue line, phase). (e) Tilting motion of Y-tilter (red line) driven by 10 Hz sawtooth wave signal (black line) whose precipitous downward part is slightly blunted.

Image of FIG. 3.
FIG. 3.

Effect of laser-beam tracking of lateral cantilever motion on the optical lever detection of cantilever oscillation amplitude. (a) Amplitude image captured at 1 fps for 650 × 650 nm (256 × 256 pixels) without laser-beam tracking. The bottom shows a height profile of the image along the diagonal red line. (b) Amplitude image captured at 1 fps for 650 × 650 nm (256 × 256 pixels) with laser-beam tracking. The bottom shows a height profile of the image along the diagonal red line. (c) Amplitude image captured at 10 fps for 250 × 250 nm (128 × 128 pixels) without laser-beam tracking. The bottom shows a height profile of the image along the diagonal red line. (d) Amplitude image captured at 10 fps for 250 × 250 nm (128 × 128 pixels) with laser-beam tracking. The bottom shows a height profile of the image along the diagonal red line.

Image of FIG. 4.
FIG. 4.

Schematics of fast tip-scanner. (a) Bottom view. (b) Side view.

Image of FIG. 5.
FIG. 5.

Schematics showing the structure of tip-scan HS-AFM unit. (a) Optical lever detector with dichroic mirror tilter for laser-beam tracking. (b) Whole structure of the tip-scan HS-AFM unit. The numbers attached to respective parts correspond to the numbers shown in (a). (c) Optical microscope stage with wide-area XY-scanner.

Image of FIG. 6.
FIG. 6.

Successive AFM images showing processive movement of M5-HMM along actin filament. The images were captured at 5 fps for an area of 150 × 75 nm (100 × 40 pixels).

Image of FIG. 7.
FIG. 7.

Position-correlated TIRFM, AFM, and HS-AFM images of rhodamine-labeled actin filaments placed on a cover slip via avidin. (a) TIRFM image. The inset shows a wide-area AFM image captured at 10 min/frame for an area shown by the white rectangle in the TIRFM image. (b) HS-AFM image captured at 8.3 fps with 100 × 45 pixels. The position of cantilever tip was set by reference to the TIRFM image. (c) HS-AFM image captured at 2 fps with 100 × 100 pixels. The position of cantilever tip was set by reference to the TIRFM image. (d) The overlaid TIRFM/AFM images. The imaged areas of (b) and (c) are those shown by the black rectangles.

Image of FIG. 8.
FIG. 8.

Simultaneous HS-AFM/TIRFM imaging of proteins in action. (a) Cy3-labeled chitinase A on a chitin microfibril. The HS-AFM images (left) captured at 3 fps with 150 × 35 pixels show a single chitinase A molecule moving toward the top-right (arrows). The TIRFM images (right) with 40 × 15 pixels also show the same moving chitinase A molecule (arrows). (b) Cy3-labeled M5-HMM on an actin filament. The HS-AFM images (left) captured at 3 fps with 100 × 36 pixels show a single M5-HMM molecule moving toward the bottom-left. The TIRFM images (right) with 17 × 9 pixels shows corresponding correlated fluorescent spots (white arrows) slightly moving from right to left.

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/content/aip/journal/rsi/84/7/10.1063/1.4813280
2013-07-17
2014-04-16
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: High-speed atomic force microscope combined with single-molecule fluorescence microscope
http://aip.metastore.ingenta.com/content/aip/journal/rsi/84/7/10.1063/1.4813280
10.1063/1.4813280
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