Hi,
I am new to fluorescence microscopy. I have been doing some staining with Lysotracker red to visualize lysosomes and hoechst as a reference dye. I have been looking for differences among the control and treatment with respect to lysotracker staining and never thought of keeping the exposure time constant between the two, i.e. I just let the microscope expose it as it thought appropriate. The exposure times are really high for the control compared to treatment. Should this pose a standardization issue? Is it customary to fix exposure time for all of the treatments when doing experiments like mine? I am taking stills and not time-lapse stuff. Any feedback would be really appreciated. Thanks!
First newbie question for all you pros :D
Re: First newbie question for all you pros :D
Hello,
I have only done fluorescence work once, long time ago, back at university, and my recommendations are based on darkfield, where you also have bright speimens on a dark background.
The problem with automatic exposure is that the camera determines an average brightness of the image to set the exposure. As most of the image is black (the background), the camera wants to expose longer, making the bright parts too bright (local overexposure). this causes you to lose image information in these areas, as the sensor goes into saturation at these pixels. Different parts of the same slide will be exposed differently, because of the different number of bright fluorescing spots and also because the center of the field of view is given more importance than the sides, for determining exposure time. The exposure time will therefore change as you move athe slide. What you need is an internal reference, a standard.
Now, if the amount of brightness is important, the degree of fluorescence, then this is definitely a standardization issue. If you just want to know if something is there, then it is not an issue. If you want to combine different images into one larger one (stitching) then a common exposure is also important, but this is a different thing.
Oliver
I have only done fluorescence work once, long time ago, back at university, and my recommendations are based on darkfield, where you also have bright speimens on a dark background.
The problem with automatic exposure is that the camera determines an average brightness of the image to set the exposure. As most of the image is black (the background), the camera wants to expose longer, making the bright parts too bright (local overexposure). this causes you to lose image information in these areas, as the sensor goes into saturation at these pixels. Different parts of the same slide will be exposed differently, because of the different number of bright fluorescing spots and also because the center of the field of view is given more importance than the sides, for determining exposure time. The exposure time will therefore change as you move athe slide. What you need is an internal reference, a standard.
Now, if the amount of brightness is important, the degree of fluorescence, then this is definitely a standardization issue. If you just want to know if something is there, then it is not an issue. If you want to combine different images into one larger one (stitching) then a common exposure is also important, but this is a different thing.
Oliver
Oliver Kim - http://www.microbehunter.com - Microscopes: Olympus CH40 - Olympus CH-A - Breukhoven BMS student microscope - Euromex stereo - uSCOPE MXII