2. Photophysics

Quantification of SMLM data often requires calibration measurements to characterize the photoswitching properties of a certain dye in your experimental imaging system (buffer, microscope, etc.).

An example would be the determination of the localization precision (sigma). Here, single labeled proteins (i.e. a conjugated antibody) are spotted on a cleaned glass surface and then imaged under experimental conditions.

The scripts in the folder Photophysics help to analyze such datasets and extract characteristic blinking propeperties such as:

• the localization precision sigma
• the number of blinking events typically observed per dye molecule
• the length of a blinking event (i.e. on time). This can be useful when setting the optimal integration time of your camera.
• the number of frames between blinking events (i.e. gap/dark time)

Protocol to perform a single-molecule calibration to extract the above-mentioned parameters.

1. Perform tracking gap = 0. The first tracking operation will be used to combine blinking events.

2. Merge tracks into blinks.

3. Perform tracking with gap = maximum number of frames.