Optical Biology Practical 1
- Andrew Vaughan
Super-Resolution Microscopy
Sample Preparation
Glox Switching Buffer
The Glox buffer formulation is adapted from the following publication:
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 (Reducing Agent Solution)
- 113.6 mg MEA-HCl
- 1 ml distilled water
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.
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
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.