However, always make sure you save the file of the original “raw” image, and save the manipulated images with different file names. Cropping an image to focus the eye only on the area important in the field of view, or adjusting the brightness and contrast to clean up the background and make the details more visible, are very commonly done and can help the viewer understand your image better. There are many situations where adjustments are perfectly within the bounds of good scientific conduct. Most image acquisition software will allow you to adjust the brightness, contrast, and color of your image after you have taken it, or to crop the image to retain just the areas you want to emphasize. True co-localization requires more specialized microscopes as well as plenty of technical skill, controls, and math. So practically, if you have labeled protein X with a fluorophore that emits in the green channel and protein Y with a fluorophore that emits in the red channel, and in the image you see that the green and red pixels are on top of each other, you can definitively say only that both proteins exist within a specific 3-dimensional volume, and how big or small this volume is will depend on the resolution limits of your microscope. To complicate things even more, this limit extends to the entire 3-dimensional volume (x, y, and z planes). Most microscopes cannot distinguish between two objects that are less than 200 nm apart. When imaging with epifluorescence illumination, it’s actually quite difficult to determine co-localization, mainly due to limits in resolution. Finally, subtract this value from the intensity values for the regions of interest.Ĭell biologists often do experiments to discover causal relationships between various molecules, and showing that two molecules are very close to each other in the cell-called co-localization-is one piece of evidence that they are interacting with each other. Use the fluorescence intensity in this neighboring region as the value for the background intensity. For cells, this would be an area that has no cells present. To subtract background, first determine the fluorescence intensity of the background by drawing a region that is close to the defined region of interest (see previous section) but doesn’t have a fluorophore specifically bound to a target. Skipping this step is really at the discretion of the experimenter, but before you do so, make sure you have checked (and can prove you have checked) that the background signals were the same (or very similar) between the control and treated samples. When this is the case, it can be okay to skip the background subtraction step. In reality, in some experiments, the background in both the control and treated samples is very similar. Background needs to be subtracted from true signal within the region of interest otherwise, background will contribute to the apparent “signal” and give you an incorrect value. The specific fluorescence that you do want is often called “signal”. Background fluorescence is the fluorescence that is not specific to your experiment.
0 Comments
Leave a Reply. |