Scanning microscope captures large images quickly
Detailed color images of biological specimens are easily acquired with a conventional microscope paired with a CCD camera.
Detailed color images of biological specimens are easily acquired with a conventional microscope paired with a CCD camera. Obtaining high--magnification images of specimens that are larger than the objective's field of view, however, is more difficult. In this case, multiple images must be taken and their boundaries "stitched" together with software. Just acquiring the images can take hours.
Engineers at Biomedical Photometrics (Waterloo, Ont., Canada) have developed an alternative technique that relies on raster-scanning of laser light through a telecentric lens, coupled with a precisely moving specimen-bearing stage, to capture confocal images as large as 22 × 70 mm in a matter of minutes (see figure). The system-called the Tissuescope-has just been commercially introduced.
Conventional image tiling involves the capture of hundreds or thousands of small areas of a microscope slide, explains Glenn Smith, an engineer at Biomedical Photometrics. In the large digital-image mosaic (the “big picture”), the small images generally must overlap one another. A typical 15‑mm2 section requires the capture and stitching of more than 2000 individual small images. One big problem occurs when automated focusing is used, often resulting in overlapping adjacent images focused at different depths.
The image captured by the Tissuescope has dimensions determined in one axis by the field size of the telecentric lens, and in the other by the length of a microscope slide. One microscope version has a telecentric lens with a numerical aperture of 0.35, a best-pixel resolution of 1 µm, a working distance of 6 mm, and a 20- to 22-mm field, depending on the type of contrast mechanism used. For brightfield, the image size with this lens is 20 × 70 mm, while for fluorescence it is 22 × 70 mm. Another version of the instrument contains a telecentric lens with a numerical aperture of 0.5, a best-pixel resolution of 0.25 µm, a working distance of 3 mm, and a field size of 10 mm. The microscopes can also be operated at reduced resolution, with pixel sizes ranging from 2 to 100 µm.
- A large-image-area laser-confocal microscope can capture many types of images. Monochrome transmitted-light (top) and autofluorescence (bottom) images were obtained of an unstained ovarian tumor section, both at an optical resolution of 2 µm. The 10 × 30-mm specimen was scanned with 532-nm laser light. The transmitted-light image was enhanced with equalization contrast, while the fluorescent image was enhanced with logarithmic contrast and a false-color palette. The specimen was provided by David Hedley of the Princess Margaret Hospital (Toronto, Ont., Canada). The microscope can also produce full-color images.
The standard light source is a combination of a 20-mW red (635 nm) laser diode, a green (532 nm) frequency-doubled Nd:YAG laser, and a 15-mW blue (488 nm) solid-state laser. Other possible laser wavelengths include violet (405 nm), yellow (561 nm), red (680 nm), and IR (780 nm); four lasers can be used at the same time in one instrument.
Many types of images
Transmission images (most similar to conventional brightfield microscope images) can be captured in full color. Fluorescent images (in which a biological specimen is prepared with a fluorescent dye) are obtained in a reflection mode; simultaneous three-channel fluorophore detection is possible. Transmitted-light and fluorescence images can be acquired at the same time and the images merged with software, highlighting the exact location of labeled cells within the tissue specimen. Because some types of tissue naturally fluoresce without the need for dyes, autofluorescent imaging is sometimes possible. Nonfluorescent reflected-light imaging can be carried out with the tissue in an unstained state or labeled with a reflective probe substance. Finally, a differential-phase-contrast approach can be used to enhance edges.
The ability to image large-area specimens is important particularly in examining whole-organ tissue slices, notes Smith. “A common desire in drug development and cancer-therapeutics research is to see the localization of a drug throughout a sample or the effect of the drug throughout the organ,” he says. “Also, in the area of stem-cell research, there is a desire to see where the stem cells go and how they congregate through the specimen.”
The ability to image simultaneously with two laser wavelengths is important for fluorescence imaging, says Ted Dixon, the company’s chief executive officer. “One of our customers uses fluorescence to detect and monitor the relationship between the density of blood vessels in a tumor and regions of hypoxia,” he explains. “By using fluorescent stains with two different colors, one stain is preferentially attached to the blood-vessel walls and the other to regions of hypoxia. When the specimen is scanned with two different lasers, the two stains fluoresce with different colors and the resulting image clearly shows the relationship between the number of blood vessels and the hypoxia resulting where the number of blood vessels has been reduced.”