Hi!
I have a question about how to determine cells with fluorescence in a given image (transfection efficiency calculation ). For example under a given set of conditions (expose time) cells in an image may have different fluorescence intensities. So how do we determine which cells to count as having fluorescence. How do we decide the cutoff? See the two images attached for example base on the pixel values in the give cell area?
Thanks for your time on this.
1: To exclude autofluorescence, look at your untreated controls. Increase exposure time in the appropriate channel (green?) until you see a signal. This is autofluorescence.
Reduce the exposure time until this disappears (no cells visible). Note the exposure time.
Use this same exposure time to image your treated (transfected) cultures. Any cells you see now, should be showing signal above autofluorescence, and therefore transgene associated.
2: Or, presuming you have already taken images of control and treated cultures, and that you used the same exposure settings:
*Within each image*, you should determine the average intensity of the background (area with no cells), and subtract this value from all cell intensity values *within that image*, to get corrected values for each cell. You can use mean intensity, or integrated density, or possibly other measures, but be consistent.
Find the greatest cell/pixel intensity in any of your control images. Use this as a cutoff, and class cells with greater pixel intensities in the treatment groups as transfected. You can then introduce arbitrary thresholds for low/medium/high expression, etc.
Hope that helps,
Stuart
1: To exclude autofluorescence, look at your untreated controls. Increase exposure time in the appropriate channel (green?) until you see a signal. This is autofluorescence.
Reduce the exposure time until this disappears (no cells visible). Note the exposure time.
Use this same exposure time to image your treated (transfected) cultures. Any cells you see now, should be showing signal above autofluorescence, and therefore transgene associated.
2: Or, presuming you have already taken images of control and treated cultures, and that you used the same exposure settings:
*Within each image*, you should determine the average intensity of the background (area with no cells), and subtract this value from all cell intensity values *within that image*, to get corrected values for each cell. You can use mean intensity, or integrated density, or possibly other measures, but be consistent.
Find the greatest cell/pixel intensity in any of your control images. Use this as a cutoff, and class cells with greater pixel intensities in the treatment groups as transfected. You can then introduce arbitrary thresholds for low/medium/high expression, etc.
Hope that helps,
Stuart
Hi Nipuna, we do a lot of fluorescent counting so I hope I can help a bit.
Cells can often have some degree of auto-fluorescence so it's always handy to have the same cells which have not been labelled/transfected that can act as a control when setting the parameters for taking the fluorescent pictures (pinhole size, exposure time, laser power, gain etc) - as mentioned above by Stuart.
After taking the pictures (fluorescent and bright field as you have done) then you have 2 choices a) count the fluorescent cells manually or b) count using a program (such as ImageJ).
Which of these to choose will depend upon: a) cell density b) cell number and c) size of the area to be measured.
As your cells are quite confluent, and you are looking at a single picture I would go for the manual count (i.e. merge images, count non-fluorescent and fluorescent cells).
(You can set parameters in automatic/program counting to help distinguish individual cells such as minimum size, circularity etc but some programs will really struggle in giving an accurate answer when the cells are confluent).
I have attached some merged picture of your images to this file so you can see what I mean. I adjusted the brightness/contrast in the fluorescent image to exclude "non-fluorescent cells": this is the subjective part of the operation - if you have set the fluorescent measurement on the microscope using the "non-transfected" line as a control then it makes things easier. For example compare the first merge with the second merge I have uploaded. In the second merge I was more stringent in setting the brightness/contrast so only strong signals are visible.
A (very) quick look shows ~ 16 fluorescent cells in the second merge and ~ 33 non-fluorescent (giving a transfection rate ~33%). In the first merge I counted ~ 28 glowing cells so transfection rate ~ 55%.
So in brief: setting microscope parameters against a non-fluorescent line is essential for an accurate assessment of signal in the fluorescent line. In the absence of this a subjective setting of the cut off threshold has to be made.
I hope this helps,
Gary
p.s ImageJ is free to download online and if you do not have this program I recommend that you get it as it has a lot of good features.
Hi Gary ,
Thanks for your detail answer.sorry for late reply as I was out of town for last couple of weeks.
- Badges
- Science method