Hi everyone,
For some time I have been conducting calcium uptake assays in an immortalized line of astrocytes, I use the 5 μM Fura-2AM probe, I place these cells in glass covers and record for 12 minutes. The cells are in a recording solution [in mM: NaCl (148); KCl (5); CaCl2 (1.8); MgCl2 (1); glucose (5); HEPES (5), pH = 7.4]. It happens that I add the compound that generates the increase in intracellular calcium, by means of a reperfusion system I wash the cells and it is at that moment that instead of having a black background, the background turns blue and it makes it difficult for me to see if cells do or do not respond to a second response. I have not been able to determine what the problem is, I have been adjusting the gain and exposure but still, the problem persists. Can someone guide me to solve this problem?
Thank you, Francisca.
Hi Francisca,
What do you mean with "blue"?
Is it blue through the oculars, or blue in the screen where you are getting the image? Which set of filters/dichroic mirrors are you using for these experiments? Which software you use for imaging?
Can you share a picture of this so we can advice better?
When you are getting images into a computer they are not colored, but pseudo-colored. This means that your image should range between black (value=0) and white (value=255 in 8-bits; value=65535 in 16-bits). Then you can apply a Lookup Table (LUT) to see the image in the desired (pseudo)color.
Do you know which LUT are you working with?
As a fast answer, the first thing that comes to my mind is that you are using some kind of range indicator LUT (this LUT gives blue to value=0, red to value=255, and grays to the intermediate values) in your imaging system. So, during this washing/perfusion step, you are washing out the remaining dye that gives you some background, and decreasing the fluorescence levels of the cell-free (background) regions to zero, and that's the reason why it turns "blue".
Try to save the images, open them in ImageJ and apply a different LUT (like green) to see if this is the case. You can also measure the intensity of background regions to see if they reach a "0" value after perfusing.
Hands-on: Increase the washing time before perfusion so backgrounds levels reach the "0" before you apply the substance.
Cheers,
J
Hello Francisca,
The first thing I think about is just the "saturation" option activated. In all software, you can activate a visualization of the saturated pixels (ofter in red) AND the most unsaturated ones (often in blue or white). It is useful to parameter your recordings but you need to de-activated it during your experiment ;-)
Best regards
Is the perfusion level increasing during washout? It would be convininet to hae a uniform perfusion level along the whole experiment to control light reflections.
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