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Fluorescence detection and high resolution AFM imaging of nano structures
AFM imaging of nano structures
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Imaging structures in the nanometer range as beads or quantum dots is an exemplar for the use of AFM technique. To get a first impression of the composition of the sample and to localize the particles or rather to find the optimal sample position, sensitive fluorescence microscopy emerges as a useful tool.
In the first example 40 nm red fluorescent spheres were deposited on a mica substrate. The spheres were focused and an optical image taken using the ProgRes® MFcool (exposure time 1 sec). After the optical system was calibrated to the SPM software using the DirectOverlayTM feature an interesting scan region was specified. AFM scanning of this region revealed clusters of beads, appearing as high fluorescent spots within the optical image, but also single beads showing only weak fluorescence.
In the next example sensitive fluorescence detection was used to localise quantum dots deposited on mica in a low concentration to finally resolve their topology and composition by AFM imaging. In low concentrations quantum dots are almost impossible to be visualised by eye. Then it helps to use sensitive fluorescence detection as shown here using the monochrome camera ProgRes® MFcool.
Since relatively long exposure times (around 7 sec) are necessary to take high quality images the use of the binning option is very helpful. The light information of several pixels is summarised and the exposure time automatically decreased to keep the chosen brightness of the raw image constant. If for instance 3fold binning is chosen the resolution decreases by summarising squares of 3x3 pixels. But the exposure time also decreases to the same extent, allowing to focus the right plane. When the optimal focus and sample region is found the maximum resolution (no binning, low gain, high exposure time) can be used to get high quality images. To take high resolution and quality images it is recommended not to use binning for such small structures like quantum dots, where the size of the structure is similar to the pixel size. When using the binning option anyway, it is essential to use the same binning for the calibration images, since the overlay feature is based on the correlation of the pixel position of the antilever tip to the pixels within the snapshot of the sample.
The blinking nature of quantum dots makes it difficult to localize all dots in one region at the same time. However, using optics it is possible to distinguish between areas of high and low quantum dot concentration and the optical image helps to orientate and choose an adequate scan region.
In the first example 40 nm red fluorescent spheres were deposited on a mica substrate. The spheres were focused and an optical image taken using the ProgRes® MFcool (exposure time 1 sec). After the optical system was calibrated to the SPM software using the DirectOverlayTM feature an interesting scan region was specified. AFM scanning of this region revealed clusters of beads, appearing as high fluorescent spots within the optical image, but also single beads showing only weak fluorescence.
In the next example sensitive fluorescence detection was used to localise quantum dots deposited on mica in a low concentration to finally resolve their topology and composition by AFM imaging. In low concentrations quantum dots are almost impossible to be visualised by eye. Then it helps to use sensitive fluorescence detection as shown here using the monochrome camera ProgRes® MFcool.
Since relatively long exposure times (around 7 sec) are necessary to take high quality images the use of the binning option is very helpful. The light information of several pixels is summarised and the exposure time automatically decreased to keep the chosen brightness of the raw image constant. If for instance 3fold binning is chosen the resolution decreases by summarising squares of 3x3 pixels. But the exposure time also decreases to the same extent, allowing to focus the right plane. When the optimal focus and sample region is found the maximum resolution (no binning, low gain, high exposure time) can be used to get high quality images. To take high resolution and quality images it is recommended not to use binning for such small structures like quantum dots, where the size of the structure is similar to the pixel size. When using the binning option anyway, it is essential to use the same binning for the calibration images, since the overlay feature is based on the correlation of the pixel position of the antilever tip to the pixels within the snapshot of the sample.
The blinking nature of quantum dots makes it difficult to localize all dots in one region at the same time. However, using optics it is possible to distinguish between areas of high and low quantum dot concentration and the optical image helps to orientate and choose an adequate scan region.