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Step Eight: Remove the sample from the vacuum system to view it using the atomic force microscope(AFM) to observe the topography of the vanadium droplets.
microscope setup
{IMG00014-20100622-1127_copy.jpg} Atomic force microscope {carbon_spectrum.JPG}
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{IMG00012-20100622-1125.jpg} Atomic force microscope setup
{IMG00014-20100622-1127_copy.jpg} Atomic force microscope
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AFM and confocal microscopes together.
Atomic Force Microscope:
Atomic force microscopy, or AFM, is a type of scanning probe microscopy. With AFM a very small, sharp probe is scanned across a surface, and small deflections of the probe are measured to detect changes in the topography of the surface.
With soft biological samples such as macromolecules and
cells, AFM can routinely resolve structures that are tens or
hundreds of nanometers in size. Because the tip touches
the surface, the technique can reveal not only information
about shape and size, but also about tip-sample interactions involving texture, adhesion, and elasticity of the sample.
Advantages: (1) provides better resolution than light
microscopy, (2)samples need not be fixed or stained, nor
imaged under vacuum as for electron microscopy,
(3) samples can be viewed in air or in liquid medium
(perfect for biological samples.)
Disadvantage: the sample must be supported by a solid surface.
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Confocal Fluorescence Microscope:
If you shine light on some molecules, you may see light of a different color emitted from those molecules. This is known as fluorescence. The molecules absorb high-energy light (blue, for example). This increases the energy of the molecules. Some of the energy from the blue photon is lost and then emits a photon with less energy. Florescent dyes act this way, emitting photons when hit with excitation light that can be read by a detector.
Advantages: (1) dye molecules can be attached to
specific parts of your sample, so that only those
parts are the ones seen in the microscope.
(2) more than one type of dye can be used and by
changing the excitation light, you can cause one
type of dye to fluoresce, and then another, to
distinguish two different parts of your sample.
Disadvantage: Fluorescent molecules can get
bleached by light, giving them a limited lifetime.
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=WHAT TYPES OF EXPERIMENTS ARE HAPPENING IN THE LAB? ----
=Adhesive strength of cells
The AFM can be used to find the adhesive forces between cells and specific proteins. The cell is fixed to the end of the AFM tip and then placed on a surface that has been prepared with proteins for the cell to bind with. The AFM tip can then be retracted, pulling the cell from the surface until it is detached completely. The resulting data reveals information about the adhesive force between the cell and the binding proteins.
Imaging of DNA:
This image was taken using AFM. The original image is created by using shades to represent heights from the surface. The image is then enhanced by using software that filters out background noise and creates a colored representation.
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Mechanical properties of Cancer Cells:
Various Cells were imaged using the confocal Fluorescence Microscope to yield the image below. Two different florescent dyes were used to mark the DNA (yellow) and the RNA (blue). Then the AFM tip was used to apply force to the membrane of the cells at different locations. The curve is a force vs. depth graph. The slope of the graph represents Force per unit of distance pushed or the “squishiness” of the cell. In general, as cells progress from normal to precancerous to cancerous, they become increasingly squishy (the slope is less steep.)
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Single molecule imaging using fluorescence and AFM
By using the confocal microscope, it is difficult to resolve molecules that are very close. The confocal microscope detects the intensity of the florescence, but molecules on top of each other would yield similar intensity to a single molecule. By using the AFM tip, it is possible to “quench” the intensity of one molecule at a time and therefore concentrate on the single molecules. This technique may be useful when marking proteins to be identified within a chromosome, since these proteins can be very close to each other.
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The image above shows fluorescence of a molecule that is only 1-2nm wide, but gives fluorescence about 200nm wide. The graph shows the intensity at a single point over time until bleaching
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