Open the images mentioned in the activity.iterations=1, count=1, black, do=Nothing.Appreciate that the signal to noise is very low (CCD noise) and it is hard to decide which background to subtract.Channel 2: Fluorescence image that should be quantified within the object.Channel 1: Transmission image showing the object location.xyc_16bit_embryo_transmission_fluorescence.tif.Inspect images where intensity quantificaion may not be possible.Discuss which values changed and by how much percent.Measure the intensities again, now with the larger label mask.Appreciate that it is not always clear how large exactly the label regions have to be.Open label mask xy_8bit_labels_h2b_dilate_labels.tif.Optional: Repeat measurements with larger labels.Discuss the measurements’ biophysical interpretation.Create new columns for background corrected mean, max, and sum intensity.Add the background measurement as a new column to the table.Manually measure the mean intensity in the background.Exports the results as a table (and open in a spreadsheet software).Using the label mask, measure the mean and max intensities as well as the objects’ pixel area.Open label mask xy_8bit_labels_h2b.tif.H2B-mCherry staining acquired with a widefield microscope.Measure intensities (with background subtraction).“Sum Intensity” (just “Intensity” is not enough)! If you publish or present something, label your measurement properly, e.g.At least, think carefully about whether the mean or sum intensity is the right readout for your biological question.Finding the correct background value can be very difficult and sometimes even impossible and, maybe, the project just cannot be done like this!.Intensity measurements need a background correction.Please consider consulting a bioimage analysis expert.Intensity measurements are generally very tricky and most likely the source of many scientific mistakes.It is thus critical whether you used a confocal or a widefield microscope, because widefield microscope have an unbounded PSF along the z-axis.Essentially, you need to exactly know where your microscope system is measuring the intensities. More specifically, you need to know how the 3D extend of the PSF relates to 3D extend of your biological structures of interest.For the correct biophysical interpretation you need to know the PSF of your microscope.sum often represents the total expression level of a protein. ![]() mean often resembles the concentration of a protein.Sum_corr = mean_corr * num_pixels = ( mean - bg ) * num_pixels = sum - ( bg * num_pixels )
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