Super-Resolution Microscopy

Sample Preparation

Gatta-STORM 94R

1. Wash one of the central two wells of the Ibidi μSlide chamber three times with 700 μl PBS.
2. Incubate the chamber with 500 μl of BSA-biotin solution (1 mg/ml in PBS) for 5 min.
3. Remove the BSA-biotin solution and wash it three times with 700 μl PBS. Make sure not to scratch the chamber surface. Pick one corner and always pipette at that position.
4. Incubate the chamber with 500 μl of neutravidin solution (1 mg/ml in PBS) for 5 minutes.
5. Remove the neutravidin solution and wash the chamber three times with 700 μl of immobilisation buffer (IB).
6. Dilute 3 μl of GATTA-STORM DNA origami solution in 500 μl of IB and incubate the chamber with this solution for 5 min.
7. Wash the chamber three times with 700 μl IB.
8. Fill the chamber with the required amount of GLOX switching buffer, supplemented with 10 mM MgCl₂.

Glox Switching Buffer

The Glox buffer formulation is adapted from the following publication:

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Dispense into 400 µl aliquots and store at -20°C.

Solution C - Gatta-STORM version (Reducing Agent Solution)

• 113.6 mg MEA-HCl
• 1 ml distilled water with 100 mg/ml MgCl₂

Use fresh or make 100 µl aliquots for storage at -20°C

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In the image below I have sealed the middle two wells by sliding the inverted lid across them.

Focusing on the specimen

Adjust the focus (Z dimension) for both microscopes to the following positions to find the approximate focus for the middle two wells of the ibidi chamber.

• Zeiss Elyra PS.1: 4.622
• ONI Nanoimager: -240.0

Coloc-Tesseler

Levet F, Julien G, Galland R, Butler C, Beghin A, Chazeau A, Hoess P, Ries J, Giannone G, Sibarita J-B. A tessellation-based colocalization analysis approach for single-molecule localization microscopy. Nature Communications, 10, 2379. 2019.

Link to Github downloads.

Colocalisation measurements in Coloc-Tesseler

Spearman Rank Correlation Coefficient

Like the Pearson Correlation Coefficient, this measures the degree to which the values of individual measurements of two channels change together either positively (correlated) or negatively (anti-correlated). A value of +1 indicates 100% correlation and −1 indicates 100% anti-correlation. A value of 0 indicates random correlation. Unlike the Pearson coefficient, the channel measurements do not have to be linearly related to give a correlation close to 1.

Manders Coefficients

The Manders Coefficients show the colocalised values from each channel as a ratio of the total fluorescence for that channel, so there is one coefficient for each channel.

Manders EMM, Verbeek FJ, Aten JA. Measurement of co-localization of objects in dual-colour confocal images. Journal of Microscopy, 169(3), 375, 1993.