Results

Bead experiments

These experiments were performed with the 532 nm setup, and the 100 nm PSF beads prepared in the lab before. 4 different data sets were captured, with different fields of view and exposure times of 10, 30, 60 and 100 ms. These data sets were analyzed using the QuickPALM software as an exercise to better learn the technique.

Aside from the default parameters used for all 4 data sets, we sampled the parameter space with ranges of 3 parameters in the image processing, namely, the minimum signal to noise ratio accepted by the algorithm (values of 4, 8, 12, and 16), the maximum full width at half maximum tolerated (values of 2, 4, 8 and 20 nm), and the expected symmetry of the particles (values of 0, 50 and 90%). Additionally, the SpheroTech beads used for all the previous experiments were compared at different exposure times with the Invitrogen PS-Speck deep red beads using different optics and laser.

If not stated at the bottom right corner of each image, the parameters used for the experiment were SNR of 12, FWHM of 4 nm and Symmetry of 50%.

The figures are located at an external link

Cell experiments

The first two images are of cells treated with microtubule-targeting antibodies conjugated with Alexa 647, while the following two are of mitochondria-targeting antibodies conjugated with Alexa 647 and Cy3. These cells were imaged using a 632.8nm laser without any beam expansion. A stack of thousands of images were taken with an exposure time of 500ms and a gain of 225. The first image of each pair is an example frame from this image stack, while the second image of each pair is the STORM reconstruction image created using QuickPalm. For the QuickPalm software, the following parameters were used: Minimum SNR=9.0, FWHM=8.0, Threshold=0.2, and Symmetry Estimate=50%

Example Frame from Microtubules Image Stack

STORM Reconstruction of Microtubules Image

Example Frame from Mitochondria Image Stack

STORM Reconstruction of Mitochondria Image