Our images of the same area were made with the confocal and atomic force microscopes simultaneously. Because the scan of the confocal microscope is only 80 mm square while the AFM scan needs to be 20 mm square in order to see DNA molecules, it was important to work very accurately. The scans were often off by a few hundred nanometers, making it difficult to combine the data in order to obtain more information from them. One of the difficulties encountered was the drift of the sample due to the heating of the piezos which control the sample by the equipment. This caused the AFM cantilever tip to be off by several hundred nanometers from the confocal microscope laser so the two microscopes are not scanning the same area at the same time. They need to be aligned better to see nanophotonic effects in the flourescence picture.

Different fluorophores are used with different biological molecules. One of the most commonly used in the lab is l DNA with YOYO-1 as the fluorophore. We studied the effect known as photobleaching, which renders a fluorophore unusable due to it no longer fluorescing. This was done with different laser intensities to get the highest contrast image while preserving the fluorophore for as long as possible with the confocal microscope.

Since the AFM can be used in ambient conditions or in an aqueous environment, we also studied the fluorophores with the confocal microscope using both ambient conditions and in an aqueous environment. Different compounds were tried in the buffer solutions used to maximize the ability to image with both microscopes concurrently. No final conclusions have been drawn yet, although several things have been found that do not work well.
DNA Image taken with the AFM - Notice the single molecules in the upper right corner

DNA on mica with the AFM. The dark spot in the middle is dirt.

The following is a series of images taken with the confocal microscope of DNA molecules. The first two are of the same region, while the third through sixth are of a smaller region within the first two. Notice that the brightness of the images changed over time. This is due to either photobleaching or an interaction between the fluorophore and the buffer solution it is in.

Image 1 - The bright spots are DNA, which likes to fold on itself. There is one stretched strand.

Image 2 - The line in the lower right is a stretched DNA strand

Image 3 - This is a small region on the previous image. We also increased the intensity of the laser.

Image 4 - Notice that the intensity is starting to drop.

Image 5 - As the fluorophores in the DNA begin to dim, more background "noise" becomes visible.

Image 6 - The previous DNA is barely visible.