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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.