Caltech researchers developed a type of microscopy that simultaneously achieves high resolution, penetration depth and imaging speed, filling biologists’ need for fast 3-D imaging methods of organisms that don’t perturb the sample.
Three-dimensional live imaging of zebrafish showing both the embryo’s tissue (in white) and a fluorescent label driven by a gene-specific promoter (in orange).
Caltech researchers developed a type of microscopy that simultaneously achieves high resolution, penetration depth and imaging speed, filling biologists’ need for fast 3-D imaging methods of organisms that don’t perturb the sample. By combining two-photon excitation with light-sheet microscopy, Scott Fraser’s group demonstrated that the method provides deeper penetration than single-photon light-sheet microscopy. It also works much faster than point-scanning two-photon microscopy and doesn’t compromise normal biology (Nature Methods 8, 757; doi:10.1038/NMETH.1652).
The method combines two previous microscopy designs. Two-photon laser point-scanning microscopy uses two photons to excite fluorescence and achieves excellent penetration but with slow-image acquisition times. The new method is similar to light-sheet microscopy, which illuminates the sample with a plane of visible light, generating single-photon-excited fluorescence from a thin optical section and capturing the fluorescence with a wide-field camera above the light sheet. Light-sheet microscopy is fast, but it lacks the penetration depth of two-photon laser scanning method. Both systems minimize light damage to the sample.
“The conceptual leap for us was to realize that two-photon excitation could also be carried out in sheet-illumination mode,” says Truong. Two-photon scanned light-sheet microscopy uses femtosecond near-infrared laser pulses that enter the sample from two sides and are scanned to create the light-sheet. When parts of the sample absorb two photons, they fluoresce at a longer (visible) wavelength that is captured by a camera above the sample. With the aid of fluorescing tags, this allows resolution below the level of individual cells and can be used to reveal the action of certain genes. The group used the method to capture a 3-D video of the development of a fruit fly embryo.
Scanning to create the light-sheet offers advantages over using a cylindrical lens, in that it provides more fluorescence with less input light (and therefore less photo-induced damage). Using two-photon excitation reduces the incidence of fluorescence from unwanted areas—which helps maintain the resolution in the axis of the camera.
While imaging fruit fly embryos, they found that they could image as deep as 60 µm into the sample in the axis of the camera. Because the photodamage is low using the scanning two-photon design, excitation power could be increased to provide more fluorescence, allowing for higher acquisition speed: With 200 mW excitation power and volumetric pixel (voxel) sizes of 0.635 x 0.635 x 1 µm, the researchers image a volume measuring 400 voxels wide x 900 voxels long x 200 voxels deep at 10 frames per second.
Speeds even higher than video rate are possible. They demonstrated 70 frames/second imaging of a beating heart in an embryo. The limiting factor was the camera readout speed.
Two drawbacks are the expense of ultrafast lasers and the fact that multicolor imaging is more difficult to execute using two-photon excitation.
Next, the group plans to improve the system, possibly by incorporating multi-angle illumination and adaptive optics or by engineering the excitation beams to manipulate the focus.
Yvonne Carts-Powell is a freelance science writer who specializes in optics and photonics.