Confocal Microscopes: Getting More Data Out Of Your Images

A second way in which Larson believes that Nikon is meeting the challenge to produce data-rich images is through spectral confocal microscopy, which he sees as a significant recent confocal innovation. Nikon’s spectral imaging confocal microscope, the C1si, can acquire spectral data in 32 channels in a single pass, with three channel widths available. “It is possible to follow changes in the fluorescence emission peak over time as physiological conditions change,” explains Larson. “The 5 nm channel width is used to unmix or separate the signals from multiple fluorescent probes with overlapping fluorescence emission spectra. Two probes with emission peaks as close as 5 nm of each other can be easily separated. Three probes with emission peaks spread over a 25 nm range can also be separated. Because of this, it is no longer necessary to think in terms of blue, green, and red emitting fluorescent probes. Probes can now be selected on the basis of ease of transfection as is the case with fluorescent proteins, ease of loading as with calcium indicator dyes, resistance to photobleaching, or any other parameter important to the researcher, without having to consider the probes emission spectrum.” Larson says that with a 10 nm channel width, data can be acquired from up to six fluorescent probes in a single scan, with even more possible in two or three scans.

Orange thinks that a promising innovation is photoswitching (or photoconverting) proteins—proteins that change the color they fluoresce when illuminated with a laser. “They are a relatively recent development so I think that their full utility has yet to be seen, but to a certain extent, the equipment to fully make use of these proteins hasn’t been available.” Orange believes that such technology will make it easier to track organelles and proteins within cells: “Within our UltraVIEW range of confocal imagers, we have just launched the PhotoKinesis accessory, which allows you to do experiments like FRAP, FLIP and photoswitching on a spinning disk system.”

Andor’s Revolution confocal systems use an optically enhanced Yokogawa CSU, Andor’s award-winning iXon+ EMCCD, and Andor’s laser combiner technology (ALC-400) for a combination of up to six laser lines (four solid state and two from an external Argon ion are possible). Other features are a fast piezo stage and objective movers, and the Andor iQ workstation. “Andor is the only spinning disk systems supplier to manufacture the key system components including software, which is a radical approach in this market,” says Browne. “[It] allows us to provide the highest specifications of stability, sensitivity, speed and synchronization. Revolution systems are not only highly specified, but also cost-effective, and have shown high levels of reliability in the field.”

For those who image tissues in research or clinical applications, Glenn Smith, director of sales and marketing at Biomedical Photometrics Inc. (BPI), notes “a widespread need for microscopic imaging of large tissue specimens, fuelled by the rapid increase in research for understanding disease, diagnosis, monitoring and treatment. For example, fluorescent biomarkers will quantify pathology, both for diagnosis and treatment monitoring, and panoramic fluorescence microscopy can ultimately move into the clinic as these biomarkers are incorporated into the diagnostic and treatment regimen.”

Most people who look at specimens on slides first use the microscope to search for areas of interest using low power, then switch to high power, which has a very limited spatial view. Smith maintains that much can be missed with this method. One alternative is to use tiling microscopes, which create a high resolution digital image of the specimen by stitching together (using software) many small digital tiles. Tiling can be problematic, however, because the microscope’s focus can change from one tile to the next, resulting in artifacts.

Instead of tiling, BPI offers their confocal MACROscopeR technology. “An important difference between the MACROscopeR and a tiling microscope is the use of a telecentric, f theta laser scan lens instead of a microscope objective. This results in a viewing area that is 100 times that of a microscope with the same resolution. Multiple contrast modes such as transmission, brightfield, fluorescence, reflectance and differential phase contrast are available in one instrument.” BPI’s TISSUEscope applies the MACROscopeR technology to tissue imaging, and won a Frost & Sullivan Technology Innovation of the Year Award in 2005.