Andras,

>  >
>>>Hi all,
>>>
>>>
>>>We are looking at performing a quantitative blood flow analysis by
>counting fluorescent microspheres (15 microns size, made of polystyrene and
>coated with flourescent dye) in the nerve of a rat after injection into the
>abdominal aorta.

These are very large spheres and carry a lot of dye. It is quite
possible that you can count them in a fluorometer, if you don't mind
collecting to create "average" values. You can actually calibrate
your fluorometer with a known dye concentration with similar spectra
as your beads to see if this is practical. Alternatively, perhaps you
can use a flow cytometer, which would have to be adjusted for the
large signal you'd expect to get from the beads, but you could
actually count the beads individually.

As for the microscopy, what if you tried a 5x lens with 0.2 NA?
Alternatively, if you can afford a motorized x-y stage and
z-controller, as well, you could work at 100 mag. and collect
adjacent areas and several thick sections (perhaps 4 sections 15 um
thick) then use a macro in NIH-Image or its Windows analogs to count
the particles. It is really quite easy. 3 cm is admittedly quite
long, but most 10x objectives (100mag) will cover a millimeter so you
are talking about 30-60 images times the number of z-sections.

>
>  >>our nerve section is 3cm long.  We tried using a fluorescent microscope to
>count the spheres but that was quite difficult due to the 3D distribution of
>the spheres, and the limited field of view especially at the requires 100x
>mag resulting in overlap in counting of the spheres.
>  >>

See above

>  >>Other options include:
>>>
>>>digesting  the tissue to extract the dye from the spheres and then using a
>fluorometer but we believe the low concentration of the spheres in our
>tissue amy be the limiting factor.

Extract dye or extract spheres?

>  >>
>>>filtering the solution containing the digested tissue, and analysing the
>filter under a microscope.

Don't see the advantage to doing this over just reading the images
manually with some up and down focus to pick out overlapping
particles.

A few more details might provide more suggestions such as are you
using a CCD for image capture, video, totally by eye? What "color"
beads are you using?

Regards,



--

Mario M. Moronne, Ph.D.
NanoMed Technologies
ph (510) 528-2400
FAX (510) 528-8076
Berkeley, CA
94706