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 typical single-molecule calibration to extract the above-mentioned parameters.

Experiment:

1. Clean glass slides using plasma cleaner
2. Incubate cleaned slides with 0.1 % Poly-L-Lysine for 30 min at 37 deg
3. Dry slides
4. Dilute labelled probe (i.e. a conjugated antibody)(~1:10^6)
5. Incubate slides with the diluted sample
6. Wash slides and image under experimental conditions

Data analysis:

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

1.1 Measure track length to get the on time

2. Merge tracks into blinks

3. Perform tracking with gap = maximum number of frames. Combines blinking events per molecule.

4. Measure track length to get the number of blinks

5. Measure the gap between blinks to obtain the dark time

5. Measure the spread of localizations to obtain sigma