What depth are you thinking of imaging at? Surface? 1mm deep? Intact skull?

Hi Susan, it depends on the preparation method. What's the model you're using? Do you want to image in vivo/in vitro/ex vivo a.s.o., is it 3D you want to do?

If it is in vivo with a opened skull (model organism mouse or equivalent), you'd have a good chance using 70% glycerol which first enhances your penetration depth. Next you can apply a simple fluorescence microscope using e.g. 532nm for absorption imaging and e.g. 635nm for scattering Image using the erythrocytes as a native contrast agent. Overlay of the images should give a nice contrast.

If you you can provide more Information, I might be able to give a more specific answer under consideration of different imaging techniques.

Dear Susan,

maybe you have seen the recent posting-thread on ResearchGate (only for Ab's):

https://www.researchgate.net/post/Is_it_somehow_possible_to_differentiate_blood_vessels_from_lymphatics_in_CD_34_stained_samples,

which contains a lot of information on antibodies visualizing endothelial cells (besides localisation of lymphatic vessels) and thus microvessels.

I second the opinion of my fore-posters that it might be helpful to know about your task a bit more in detail (e.g. in vivo/in vitro/ex vivo, evtlly.video imaging / performance; whole body/brain perfusion and further processing of tissue pieces by Histological (IHC) standard procedures, cryo-/ paraffin sections L-Microscopy [eg.semithin resin sections followed by TEM].

And, with honest respect, there comes to my mind the technique of micro-corrosion casting (morphological viewing, calculating, etc) by means of special resins injected into e. g. an ascending stem vessel, polymerisation of the fluid resin and afterwards mazeration of whole tissue fraction, followed by viewing the vessels' casts by SEM (cf. e.g. Google search for: ).....e.g.

(1989) http://www.nature.com/eye/journal/v3/n5/pdf/eye198992a.pdf

(1992, german) http://www.ncbi.nlm.nih.gov/pubmed/1283929

(2002) http://www.researchgate.net/publication/10778183_Application_of_a_mixture_of_glycol_polyethylenes_for_the_preparation_of_microcorrosion_casts--an_observation

(2013) http://www.oapublishinglondon.com/images/article/pdf/1381679714.pdf

and last but not least: cf: https://www.researchgate.net/profile/Alois_Lametschwandtner/ and scroll through Prof. Lametschwandtners .

Best wishes and good luck,

Wolfgang

Article Application of a mixture of glycol polyethylenes for the pre...

Thanks all! Wolfgang, thanks for pointing me to that thread... i'll take a look. I'm curious about methods other than resin casting, mainly because i'm not sure how morphometrics would be calculated from them. can they be digitized somehow?

i am interested in creating reconstructions of microvessels at varying tissue depths with and without infarct and TBI. thanks in advance!

Dear Susan, you are welcome!

Principally 3D-Measurement and perhaps reconstruction from SEM microcorrosion cast preps should be possible. For some reason I will not go into depth here, but would like to point you

i) to at least a reference included in ResearchGate, cf.:

The Vascularization of the Anuran Brain (https://www.researchgate.net/publication/230173078_The_Vascularization_of_the_Anuran_Brain?ev=prf_pub),

Ursula Albrecht, Alois Lametschwandtner, Hans Adam

ABSTRACT: The angioarchitecture of the cerebellum of the toad, Bufo bufo (L.) (Amphibia, Anura) was studied by scanning electron microscopy of vascular corrosion casts. An arterial supply similar to that in the frog by small branches of the superior mesencephalic artery and by terminal branches of central arteries was found. The drainage was demonstrated via mesencephalon (rostro-dorsal cerebellar regions), rhombencephalon and choroid plexus of the fourth ventricle (auricular areas) as well as via numerous branches of the lateral mesencephalic veins. These vessels leave from different levels of the cerebellum.By comparing corrosion casts with light microscopical sections a correlation between histological structures and vascular patterns was found. The dominating transverse course of the vessels of the Purkinje cell layer was pointed out. Some advantages and limitations of the method used were discussed. To solve the most deplorable disadvantage—the impossibility to receive information about the direction of blood flow—a speculative consideration was presented and opened to discussion.

Acta Zoologica 04/2010; 59(3‐4):239 - 245.

and furthermore to ii)

M3 a New Software to Quantify Blood Vessels in Three Dimensions Examined in the SEM / ESEM which also Allows to Render 3D Models of Vascular Trees,

B Minnich; A Lametschwandtner; E Bernroider; H Mayer

Microscopy and Microanalysis, Volume 15, Supplement S2, July 2009, pp 932 - 933, ONLY extended Proceedings ABSTRACT (http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=5908504).

Naturally, classical approach by measuring and 3D-documenting serial sections (paraffin or semithin resin sections, e.g. additionally with any (bio- or physical) marker for vessels) will be perhaps the option of choce for several reasons….

I’ll get into touch with you via internal ResearchGate Messaging system….

Best wishes and regards,

Wolfgang

Article The Vascularization of the Anuran Brain

Ah, yes, I'm familiar with the possibility to reconstruct from thinly sectioned material , but i'd like to optically section a bigger/thicker specimen, and reconstruct from that to create 3D models. I'm not super familiar with SEM though, you can't see the interior of the structure with SEM, correct?