Microscopic Brain Imaging In The Palm Of Your Hand

Researchers want to image individual cells inside living subjectsbecause it will give them insight into how cellular behavior gives riseto the properties of organisms as a whole. For instance, the nervecells of the hippocampus region of the brain give rise to importantmental processes such as learning and memory.

Imaging living cells below the surface has been difficult to accomplishusing conventional techniques. Electron microscopy can't be used onliving tissue, and optical (light) microscopy can't penetrate verydeeply into tissue because light scatters as it travels through tissuenear the surface. Thus traditionally microscopic images of the livingbrain have only been made near the surface. Yet researchers would liketo know more about certain deep-tissue areas of the brain, which arecritical to understanding Alzheimer's and Parkinson's disease, forexample.

Scientists often use some form of fluorescence microscopy to imagetissue. In conventional "one-photon" fluorescence imaging, thescientist injects a dye into tissue and then shines a bright light. Thetissue fluoresces, or radiates, light of a different color in response.However, a problem with one-photon fluorescence is that the deep tissuecauses the photons to ricochet, or scatter, as they return to thedetector. The result is a background haze in the images, almost likeviewing the sample through a cloud.

It's possible to get rid of background haze and reduce thescattering using two-photon fluorescence imaging. Instead of onehigher-energy photon, researchers bombard the molecule with two photonsof lower energy. Their combined energies total the energy required toexcite the fluorescent-dye molecules used to mark the tissue. Thetechnique gets rid of the background haze and reduces scattering,because molecules outside the area of interest are much less likely toabsorb a pair of photons simultaneously and fluoresce in response.

While two-photon microscopy offers an alternative to traditionalone-photon fluorescence microscopy, it still only penetrates braintissue down to about 500-600 microns -- barely scratching the surface.To get at the deep structures, the Stanford researchers turned tomicroendoscopy, tiny, minimally invasive optical probes that could beinserted deep into living brain tissue. To make one group of images(figures 1c-1e), the researchers inserted the microendoscope into thehippocampus, about a millimeter below the mouse brain surface, to imagethis part of the brain. The two-photon imaging provided an additional80 microns of depth, below the hippocampal surface.

When combined with two-photon fluorescence, the result is a system thatbrings the power of a cutting-edge imaging technique to the deeptissues of the brain. By creating a handheld device based on some ofthe latest advances in micromotors, lensing and fiber optics (seeaccompanying article for more information), the researchers were ableto establish a new technique that enables them to obtain microscopicimages deeper in the living brain than was possible beforemicroendoscopy.

"We're bringing two-photon imaging to endoscopy and we're putting itall into a miniaturized package," says Mark Schnitzer, the team leaderon the Optics Letters paper.

The Stanford researchers have used their two-photon microendoscopytechnique to glean the detailed images of the blood vessels in thehippocampus sections of the brains of live mice. The mice were injectedwith a fluorescein dye -- an FDA-approved contrast agent that is mostcommonly used for retinal exams in humans. The fluorescein labeled theblood plasma so the vessels in the brain could be clearly seen.

There are many different options for further exploration, now that thetechnique has been successfully demonstrated, ranging from biomedicalresearch to clinical imaging applications. The Stanford researcherswill be looking into several of those options.

"This is a portable handheld device with the power of two-photonimaging -- the full functionality of a microscope that fits in the palmof your hand," says Schnitzer, indicating that this is what makes thetechnology eminently marketable.

Article: "In Vivo Brain Imaging Using a Portable 3.9-gram Two-PhotonFluorescence Microendoscope," by Benjamin A. Flusberg, Juergen C. Jung,Eric D. Cocker, Erik P. Anderson, and Mark J. Schnitzer, OpticsLetters, September 1, 2005.