How Can We Measure Brain Transmittivity?

[tfpp]

Does anybody know what is the transmittivity (light) of the brain, and how do you find that out?

I am starting an experiment to image the inside of the mouse brain and most of the methods that I know of rely on external imaging techniques. Any reference for in vivo imaging of the brain.

Thanks.


David

 

[imabug]

depends entirely on what you want to look at.

there are small bore MRI units out there designed for animal imaging. MicroPET systems are available for PET imaging of small animals. Nuclear medicine and x-ray imaging is routinely done in animal studies. In-vivo microscopy techniques can be done, but I imagine that might involve knowing microsurgery techniques depending on the size of the animals you want to peep the insides of.

 

[DocToxyn]

Transmittivity should vary as a function of age. Can you give us some more info on the exact endpoint you are interested in? I'm assuming you're looking for a "live" (as in not dead animal) image? None of the more common brain imaging techniques out there- fMRI, PET, SPECT, CAT- apply to your work? Thanks.

 

[tfpp]

Thank you very much for your replies.

Yes, I am aware of the solutions and techniques that are available for brain scans. However, I have no idea about the details or what are their advantages and disadvantages are. imabug, DocToxyn could you direct me to some sites that explains all these bewildering techniques that you mentioned.

And yes, the ultimate aim is to look at live animals.

As for my original question. I am not looking to study the function of the brain (mouse) per se, but rather to develop a technique for the study. I am developing a solution for optical imaging of the brain. What I hope to do is to insert a camera inside the brain (much like the endoscope). It is extremely invasive, yes, but this is just a continuation of my previous work on studying brain slices in vitro. In that work, I used a CMOS image sensor and placed the brain slice directly on top of the sensor chip. Injected a fluorescent agent, shine a suitable excitation light on it and detected the emitted flourecent light. That's it, not microscope or optics whatsoever. Now, when I tried to use the same chip for in vivo imaging, I run into all sorts of problems like, how to get enough light into the brain, how to properly inject the fluorescent agent etc. Which was why I asked about the light transmittivity in the brain. No matter how hard I tried, I just could get enough light in there.

By the way, you can see the image of the in vitro experiment here.
http://www.aist-nara.ac.jp/~n-david/In-vitro_Imaging_Setup.pdf

Thank you very much.

David

 

[Moonbear]

I'm not sure I understand completely what you're looking for. In the example you described, you're not looking at anything being transmitted by the brain, but at the fluorescent label you've injected. Taking a look at your set-up, I'm still not certain what your intention is. You're showing an approach to imaging of a 400 micron slice. With a 2-photon microscope, you should be able to get good resolution up to a 1 mm slice thickness, but that's REALLY pushing the limits of that instrumentation. I've only tried that with up to 200 micron thick sections to get decent labeling of dendritic spines. So, it really depends on what level of resolution you're interested in. The sort of low resolution "temperature" chart shown as your output from your instrumentation can be achieved with techniques DocToxyn listed. If you're trying to image individual cells, such technology doesn't exist for a whole brain scale. That's why people who wish to do that sort of work often use much smaller organisms, where you can image through the entire brain in a live animal.

 

[DocToxyn]

The closest thing I can remember reading about live, in situ brain imaging was a study looking at microdialysis combined with fura-2 calcium imaging. I think that joined the micridialysis probes with a fiber optic line which transmitted the light and then measured the alteration in excitation of the fluorophore induced by the drugs infused via the microdialysis probe. I think I have this right, it could very well have been carbon fiber voltammetry instead of microdialysis, either way I should be able to find the citation tomorrow.

 

[tfpp]

Hi Moonbear. Yes, what I am trying to find out is to see the limitations of a CMOS image sensor chip in trying to image the brain. I am no expert in the brain, but my biological colleagues wanted to see if the current in vitro brain slice imaging can be further developed into some sort of in vivo imaging technique. They mentioned about neuropsin studies for the brain memory and learning functions, but that is really beyond me. What I am trying to do is develop a system whereby they can achieve their objectives.

The low resolution image is really just a blown up view taken using a 64x64 pixel image sensor array. Each pixels is about 50 microns insize. That's the first prototype. I am currently using a higher resolution image sensor with each pixels at 7 microns and higher pixel count. I am attempting a couple of methods to insert this sensor chip into the brain for some useful imaging. It appears that lighting is a real issue in that dark and damp condition of the brain. So far I have tried using fiber illumination. As the excitation light for the fluorescent agent, I use UV, but I think the brains just absorbs UV like crazy and I am not getting much illumination in there. Which explains my original query.

By the way, I do not have access to expensive equipment like fMRI or PET for comparison work and would really appreciate more information regarding these techniques.

Thanks.

 

[DocToxyn]

I couldn't find the exact article I was thinking of last night, but I gathered some along the same lines. I don't know if these are what you want but check them out if you wish. I didn't go into the abstracts on all, but the last was an interesting review and also touched on MRI, etc. BTW, nice pics, hippocampus I assume?

1: Ashworth R, Bolsover SR.
Spontaneous activity-independent intracellular calcium signals in the
developing spinal cord of the zebrafish embryo.
Brain Res Dev Brain Res. 2002 Dec 15;139(2):131-7.

2: Helmchen F, Fee MS, Tank DW, Denk W.
A miniature head-mounted two-photon microscope. high-resolution brain imaging
in freely moving animals.
Neuron. 2001 Sep 27;31(6):903-12.

3: Neunlist M, Zou SZ, Tung L.
Design and use of an "optrode" for optical recordings of cardiac action
potentials.
Pflugers Arch. 1992 Apr;420(5-6):611-7.

4: Bowmaster TA, Davis CC, Krauthamer V.
Excitation and detection of action potential-induced fluorescence changes
through a single monomode optical fiber.
Biochim Biophys Acta. 1991 Jan 10;1091(1):9-14.

5: Kudo Y, Akita K, Nakamura T, Ogura A, Makino T, Tamagawa A, Ozaki K,
Miyakawa A.
A single optical fiber fluorometric device for measurement of intracellular
Ca2+ concentration: its application to hippocampal neurons in vitro and in vivo.
Neuroscience. 1992 Oct;50(3):619-25.

6: Hirano M, Yama****a Y, Miyakawa A.
In vivo visualization of hippocampal cells and dynamics of Ca2+ concentration
during anoxia: feasibility of a fiber-optic plate microscope system for in vivo
experiments.
Brain Res. 1996 Sep 2;732(1-2):61-8.

7: Thayer SA, Sturek M, Miller RJ.
Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and
electrophysiology.
Pflugers Arch. 1988 Jul;412(1-2):216-23.

8: Ligeti L, Mayevsky A, Ruttner Z, Kovach AG, McLaughlin AC.
Can the Indo-1 fluorescence approach measure brain intracellular calcium in
vivo? A multiparametric study of cerebrocortical anoxia and ischemia.
Cell Calcium. 1997 Feb;21(2):115-24.

9: Shortreed M, Kopelman R, Kuhn M, Hoyland B.
Fluorescent fiber-optic calcium sensor for physiological measurements.
Anal Chem. 1996 Apr 15;68(8):1414-8.

10: Thorsrud BA, Harris C.
Real time micro-fiberoptic monitoring of endogenous fluorescence in the rat
conceptus during hypoxia.
Teratology. 1993 Oct;48(4):343-53.

11: Papworth GD, Delaney PM, Bussau LJ, Vo LT, King RG.
In vivo fibre optic confocal imaging of microvasculature and nerves in the rat
vas deferens and colon.
J Anat. 1998 May;192 ( Pt 4):489-95.

12: Thomas TP, Myaing MT, Ye JY, Candido K, Kotlyar A, Beals J, Cao P, Keszler
B, Patri AK, Norris TB, Baker JR Jr.
Detection and analysis of tumor fluorescence using a two-photon optical fiber
probe.
Biophys J. 2004 Jun;86(6):3959-65.

13: Libersat F, Mizrahi A.
In situ visualization and photoablation of individual neurons using a low cost
fiber optic based system.
J Neurosci Methods. 1996 Aug;67(2):157-62.

15: Brecht M, Fee MS, Garaschuk O, Helmchen F, Margrie TW, Svoboda K, Osten P.
Novel approaches to monitor and manipulate single neurons in vivo.
J Neurosci. 2004 Oct 20;24(42):9223-7. No abstract available.

 

[Moonbear]

Yes, you can get quite a bit of autofluorescence with UV in brain tissue. Old immunocytochemistry protocols with weak fluorophores in the UV range used to include quenching steps to eliminate this background. These wouldn't be practical in vivo though. Brain tissue is one of the easier tissues to penetrate though. When our university applied for funds to get our 2-photon microscope, the PI on it was really pleading with the neuroscientists to get involved as primary users since preliminary data in brain tissue really could show off its capabilities (as I mentioned, it's supposed to be able to operate down to optical depths of 1 mm, whereas in other tissues, I think the upper limit was more like 200 microns or so). From your set-up, it sounds like you're just using regular fluorescent light (a mercury lamp?). Confocal and 2-photon microscopes use lasers and pinholes to achieve the appropriate wavelengths for optimal excitation of the fluorophores. The problem with working with thicker tissues is that you get a trade-off between the energy you need to penetrate the tissue and the energy that is going to bleach out your fluorescent label, especially in vivo where you can't use any agents to prevent photobleaching. But it doesn't sound like you're interested in very high resolution. I'm really not certain of the purpose though, as it's hard to imagine why you would be injecting a fluorescent dye and tracking it in vivo. Are you measuring something like tortuosity with regard to CSF flow in the brain?

I'm not sure how inserting a fiber optic probe is going to be useful for in vivo imaging studies though, because just the insertion of the probe is going to do a lot of damage in the process, and it would still be pretty directional in where the beam is focused.

I realize you may be hesitant to explain your application on an open forum, on the other hand, it's hard to be much more helpful than this without knowing the intended application.

 

[tfpp]

Hi DocToxyn, Moonbear. Thanks for all your input. I finally found the information that I needed. It seems that there has been a whole lot of studies in the topic of light propagation in biological tissues especially in the brain where optical topmography is used. There appears to be plenty of ugly maths in it too in the theory side of it (you guys wouldn't be familiar with transport theory of light would you?)

DocToxyn thanks for the list. I'll try to pull out whatever that I can read up on.

I know who to ask again if I got any problems in this brain imaging issue.

Thanks guys.