Thank you David.

we have 2 problems.

The first one concerning aSMA. In my mind (but may be i am wrong) aSMA characterize cells producing ECM and not really fibrosis and it is higly related to blood vessels basal membrane.  The second one with red sirius is related to the size of the samples. We want to charaterize fibrosis in thick section between 500 to 1000µm so it is quite difficult to deal with polarised light that's why we are looking for a fluorescent probe asI want to develop a macro with imageJ to an automated quantification method.

David, the issue I see is likely how you are imaging your samples.  Regardless of brightness of the probe imaging through 500-1000 microns of collagen-containing tissue is going to be difficult for any single-photon confocal microscope, where the PMT sensitivity is rather low and the penetration depth is likely only 200-300 microns if you're lucky .  Two-photon is the way to image the sample, using a long-working distance water objective if possible.  If a two-photon confocal is available, you could look into using second harmonic generation, where a two-photon laser tuned to 900 nm for excitation and observed at exactly half the wavelength (450nm).  Collagen and many other dense proteins can observed this way.  Patricia Keely's lab and University of Wisconsin has used this technique successfully to quantify the amount of aligned collagen surrounding breast cancer tumors (see attached). They also used other techniques to verify their two-photon results which may be helpful. I would also think about sectioning the tissue to increase your chances at imaging.

Thank you Andrew, I heard about bi photon methods to quantify fibrosis but the availability of bi photon is not routinely easy. I think you are right concerning the thickness of the samples. However to increase the lazer penetration we are now adapting clarity methods to the sample transparization and it seems to be very usefull and we use a confocal with GaSP technology increasing sensitivity compared to PMT Zeiss 780). Do you think it will be feasible by using these kind of approaches (i.e. increase transparency and detection sensitivity). May be we might reduce the thickness but we want to have whole size adipocyte (maximum cell size is around 160µm) at least on 2 layers to quantify peri-adipocyte fibrosis.

Dear David,

The fluorescent label at CNA35 can be replaced by a MRI tagged label such as gadopentetate dimeglumine (Gd-DTPA; a gadolinium-based contrast agent).

Take a look to the following article

S. de Jong et al., Monitoring cardiac fibrosis: a technical challenge. Neth Heart J (2012) 20:44–48