Data analysis super-resolution
The data analysis of the super-resolution data is done in ImageJ, with help of the ThunderSTORM plug. The images below are for a GattaQuant sample, therefore you see 3 spots in a row. Our DNA-paint sample only has two spots in a row.
If you want to do the analysis at home:
Install ImageJ and download the latest version of ThunderSTORM. For installation, copy the downloaded file into ImageJ's plugin subdirectory and run ImageJ.
In ImageJ:
Load the image (can be done by dragging it on the ImageJ taskbar), if possible as virtual stack
to speed up: select the right channel, by selecting it with the rectangle tool, and crop via image>crop
In ImageJ, goto Plugins> ThunderSTORM> run analysis.
You get a settings screen in which you have to enter:
- camera settings, most important: 92.9 nm/pixel. This is measured with a grid. camera pixel size =16 um, f_obj=1.8 mm, f_tubelens= 300 mm (you expect a magnification of f_tube/f_obj)
- the required super‐resolution magnification, default 5. Note that you can change the magnification after getting the results.
Run the analysis, and observe the super‐resolution image being formed (progress is shown in ImageJ taskbar).
Afterwards you see a list of positions, run the drift analysis (with cross correlation default values), and export the list.
Also save the super‐resolution image.
To find out whether you indeed have the super‐resolution image you need, zoom in, and search for tiny spots close together.
As example, below gattaquant DNA paint data is shown (3 spots with 80 nm in between).
Do this by placing a line over two closely positioned spots (our DNA PAINT samples only has two docking locations).
Use ctrl+K to make a line profile, and by hovering over find the peak locations and the distance in between the two peaks.
Once you have a line profile, you can either (less accurately) read of the distance from the graph, or (more accurately) save the data and fit a double gaussian in Python
Don't forget error analysis: find 20 images, extract the wanted distance between spots, and find average and other statistics.
What do you expect as distance for the C1 super-res sample? Do you find this from measurements?