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:

Metcalf DJ, Edwards R, Kumarswami N, Knight AE. Test samples for optimizing STORM super-resolution microscopy. J Vis Exp 2013 Sep 6;(79):50579. doi: 10.3791/50579

To make 2.2 ml final volume, which will fill one well of a 4-well ibidi µ-Slide.

• 110 µl Solution A
• 880 µl Solution B
• 220 µl Solution C
• 990 µl PBS

The various solutions above have already been made up and are stored at the appropriate temperatures. For your reference the formulations are listed below.

Solution A (Enzyme Solution)

• 10 µl catalase
• 20 µl 1M TCEP
• 2 ml glycerol
• 125 µl 1M KCl
• 100 µl 1M Tris-Cl pH 7.5
• 50 mg glucose oxidase
• Top up to 5 ml with distilled water

Dispense into 50 µl aliquots and store frozen at -20°C.

Solution B (Glucose Solution)

• 4 g glucose
• 4 ml glycerol
• 36 ml distilled water

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

Sealing a 4-well ibidi chamber for STORM

This video shows how to fill one well of a 4-well ibidi with Glox and then seal the top with a coverslip so there are no air bubbles (and hence no oxygen ingress). It is also possible to seal the chamber by inverting the lid of the ibidi chamber and sliding that over the wells. In that way all the wells can be sealed.

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