What kind of images are you using?

Hi

is better to split channels o deconvolute image, then check the image, correct manually convert in grey scale and measuree the feret mean diametre with imagej.

regards

carlo

Using Image J, it was more difficult to threshold on HE staining than in immunolabelled section using a specific membrane marker.

Here is the example image:

We have optimised a method for semi-automating fibre size and number measurements on immunolabelled images. Refer to our publication Garton et al., 2010, "Validation of an automated computational method for skeletal muscle fibre morphometry analysis" Neuromuscular Disorders 20(8):540-7 · August 2010.

This is useful if you are interested in looking at differentiating fibre size of fast and slow fibres. We are able to count and measure cross-sectional areas of >5000 fibres in ~20 min, using Metamorph software, on muscle cross-sections immunolabelled with antibodies against myosin heavy chain 2B, 2A and type 1, with fibre borders delineated with dystrophin or wheat germ agglutinin.

If you are not concerned with fibre typing or you only have H&E images, then unfortunately you may have to do manual measurements with Image J. You can lessen the pain somewhat by randomly selecting several fields so you don't end up having to measure every fibre on your cross-section.

Try fiji.

I emailed few corresponding authors for measuring csa in H&E in stained slides (as I need it for my research work too), and I heard from one of them that  after setting the scale in um [Analyze-->set scale] [Next click Analyze-->Set measurements, click Area and Display Label.] we can select freehand selection tool and click around the border of a cell and measure using control M (PC) or command M (mac).

Hei Ping, did you manage to measure CSA in your H&E slides by now? Or did you finally turn to different staining options like immunolabeling as Jane suggested? I tried to run our custom-built Makro on your slide, but it turns out to be really tricky, because of weak cell boundaries.

I'd love to hear how you finally addressed this issue. Thx!!

Hey Ping, I do most of our histology (skeletal muscle, regenerative biomaterials) and when I perform CSA measurements, I never use H&E images (just for the figure) but rather immunostained images.  Specifically, collagen III.  This stain gives you perfectly delineated bundles of fibers which you can take to ImageJ and perform thresholding to get your measurements for area.

Hope this helps!