Two-photon fluorescence microendoscopy aids cellular research

Researchers at Stanford University (Stanford, CA) have demonstrated a two-photon fluorescence microendoscope that can capture micron-scale images from deep in the brains of live subjects—in a package small enough to fit in the palm of a hand.¹

There is great interest in imaging individual cells inside living subjects to gain insight into how cellular behavior influences the properties of organisms as a whole. But imaging live cells in vivo has been difficult to accomplish using conventional techniques. Fluorescence excitation offers some advantages; for example, one-photon fluorescence, in which a dye is injected into the sample and then illuminated with a bright light, is easy to use, lower cost, and capable of acquiring full-frame images. But it also has drawbacks—most notably photon scattering in deep tissue, which can limit resolution.

Two-photon fluorescence imaging can overcome these issues. Instead of a single high-energy photon, researchers bombard the molecule with two photons of lower energy to excite the fluorescent-dye molecules. This approach reduces scattering because molecules outside the area of interest are less likely to absorb a pair of photons simultaneously and fluoresce in response.

However, conventional two-photon microscopy is still only able to penetrate brain tissue down to about 500 to 600 µm. To overcome this limitation, the Stanford group initially developed a gradient-refractive-index (GRIN) probe mated to an optical-fiber bundle, which provided improved mechanical flexibility; however, the bundle degraded image quality because of pixelation and distortion of the ultrashort laser pulses commonly used for two-photon excitation.²

Probing below the surface

More recently, the researchers opted to use tiny optical probes that can be inserted deep into live brain tissue. The device combines a compound 1-mm-diameter GRIN endoscope probe, a direct-current micromotor, and a flexible photonic-bandgap fiber for near-distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution (see figure). The excitation light was obtained by coupling femtosecond pulses from a Ti:sapphire laser (with a 790- to 810‑nm spectral band) into the bandgap fiber. Light exiting the fiber reflects off a coated 1‑mm microprism serving as a dichroic mirror and into the GRIN lens probe. A multimode polymer fiber (980‑µm-diameter core) positioned above the microprism captures the fluorescence photons returning through the endoscope probe.

So far, the Stanford group has used this device to obtain detailed images of the blood vessels in the hippocampus sections of the brains of live mice. The mice were injected with a fluorescein dye that labeled the blood plasma so the vessels in the brain could be clearly seen. To make one group of images, the researchers inserted the microendoscope into the hippocampus, about a millimeter below the mouse brain surface, and were able to obtain images another 80 µm below the hippocampal surface.

The researchers conclude that fluorescence microendoscopy enables visualization of biological cells within tissues too deep to access by conventional microscopy. Potential applications include clinical diagnostics and small-animal research.

REFERENCES

1. B. A. Flusberg et al., Optics Letters 30(17) (Sept. 1, 2005).

2. J. C. Jung et al., J Neurophysiol. 92(5) 3121 (November 2004).