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Emerging Technologies Impact Confocal Microscopes

Fri, 12/17/2004 - 7:07am

Easier-to-use, affordable confocal microscopes are hitting the market, providing scientists with complete research workstations.

The advantages of confocal microscopes are well known. Allowing scanning in 3-D, these instruments offer an enhanced resolution that yields a better image and a controllable depth of field. Additional benefits, as well as current limitations, of confocal microscopy were recently covered in the Microscopy Pavilion of the online Analytical Laboratory Expo 2004 (archived at www.AnalyticalLabExpo.com). The presentation of "Confocal Microscopy: Practical Considerations in Instrument Selection and Image Acquisition" by Robert Price, Director of the Instrumentation Resource Facility at the Univ. of South Carolina School of Medicine, Columbia, discussed the basics of image acquisition and the different types of confocals available in today's market.

Supplementing this discussion with their views of the confocal microscopy market are Bill Fester (B.F), Director of Biomedical Marketing at Olympus America Inc., Scientific Equipment Group., Melville, N.Y., Stan Schwartz (S.S), VP of Product Marketing at Nikon Instruments, Melville, N.Y., and Sebastian Tille (S.T), Product and Applications Support Manager at Carl Zeiss MicroImaging, Inc., Thornwood, N.Y.

Q. How much of an issue is ease-of-operation in the purchase of a confocal system and are new systems becoming simpler to operate?
B.F: While the science is becoming more and more complex, we always strive to make systems easier to operate. Researchers today are demanding new capabilities and versatility, and so it is always a challenge to make our systems both robust and simple to use.

S.S: Ease of operation is very important. What makes people feel that an instrument is easy to operate is the user interface, which should be simple and intuitive. For example, with Nikon software, you don't have every available window open all the time. We put controls behind tabs. This keeps the interface uncluttered and prevents it from being threatening, especially to new users.

S.T: At Carl Zeiss, we see ease-of-use as one of the essential aspects for design and implementation of confocal imaging systems.

Q.How much of an issue is cost and will future systems see any significant reduction in price (for systems with comparable performance)?
B.F: Confocal instruments today are trending upward in terms of capabilities. A great diversity of scientists at all levels of expertise use confocal technology, and manufacturers need to be able to adapt to these various levels of demand. The pricing for systems of comparable performance could indeed be reduced.

S.S: Certainly cost is an issue. Are things going to get cheaper? Like the computer industry, the cost will not increase, but the value will rise. Instruments using current technologies are about as cheap as they can be.

S.T: We did not see a lot of change in the price of confocal microscopes, but certainly an increase in diversification and additions of new imaging and analysis methods, such as multi-spectral and multi-photon imaging, fluorescence correlation spectroscopy, and lifetime imaging.

Q. How pervasive are confocal microscopes in the life sciences R&D arena? Cell biology lab? What percent of confocal systems sold go into life sciences?
B.F: As Olympus America markets microscopes, imaging equipment, and software only in the life sciences arena, all of our confocal instrumentation is sold into life sciences laboratories and centers of research.

S.S: Confocal microscopes today function like research workstations, and more than 95% of these systems are sold into the life sciences area. Confocal for live cell microscopy is essential and is becoming ubiquitous as an everyday tool for doing live cell investigation. To view a cell that's stained in multiple florescence proteins and have closely spaced emissions spectra, you're going to need spectral imaging to unmix these emissions, which is why we've introduced Nikon's C1si spectral confocal.

S.T: Confocal microscopy has almost become a standard research tool for biomedical sciences. More and more scientific questions are answered using the help of imaging methods that are complemented by a variety of other analysis routines. We especially pay attention to fluorescence microscopy; a microscopy contrast method that is superior due to its specificity, hence the new 'Carl Zeiss: FluoresScience' focus and philosophy.

Q. Are improvements in CCD detector technology expected to make significant changes in future confocal systems?
B.F: Yes. CCDs are always improving in sensitivity, and these improvements in CCD detector technology will be an important contributor to the future of confocal systems.

S.S: What's expected are sensitivity improvements in CCD cameras, which will allow confocal microscopes to approach the resolution achieved with point scanning confocal microscopes, enabling them to be used for good structural imaging. I also think that you'll see multiple detector technologies used with a single microscope.

S.T: The answer is 'yes' and the future is now. We just released a new fast confocal system, the LSM 5 LIVE, which makes use of an advanced line detector setup. This system is an addition to our family of laser-based confocal microscope systems, with its flagship, the LSM 510 META, which features single and multi-channel photo multiplier tube technology. Multi modality in confocal imaging will combine even more different detector types in the future.

Q. Are dramatic changes (or additions) to current spinning disk and point scan technologies expected in the future?
B.F: Yes. Olympus is continuing to advance its R&D and engineering efforts focused on both spinning disk and point scan type confocal systems.

S.S: About five spinning disk companies now essentially use the same components and strategy. Spinning disk is getting old because current spinning disk designs offer only a single pinhole size. Nikon's Swept Field Confocal (SFC), however, is a field scanned confocal that is similar to a spinning disk confocal microscope, except that it has four different pinhole choices and two slit sizes available.

S.T: We believe that Carl Zeiss' LSM 5 LIVE is one of those dramatic additions to current scan technologies, encouraged by the feedback of scientists. Our engineers were able to achieve what seemed to be impossible: boosting speed AND sensitivity in a confocal setup—something that will improve live cell imaging.

Q. Were any new technologies/ applications in confocal microscopy presented at the Cell Biology Show?
B.F: Olympus showed its Fluoview FV1000 confocal system at Cell Biology, which provides improved sensitivity and high scan speeds for imaging live organisms with minimal specimen damage. Its SIM Scanner incorporates two independent, fully synchronized laser scanners in a single compact design for simultaneous laser stimulation and confocal observation. As a result, rapid cellular reactions that occur during or immediately following laser stimulation may be captured without a time lag.

S.S: One technology that Nikon finds interesting is a software technique Nikon calls two-dimensional real time deconvolution (2DRT). With this software, you can improve image sharpening in real time and at the camera's frame rate. Another thing we see emerging is a stable focus assist, which can maintain the user's selected train of focus over days so that long-term experiments can be run with the specimen held in perfect focus. This will be increasingly important because it extends the useful time of live cell imaging.

S.T: Scientists nowadays look not only for structure but morphology, nevertheless 2-D and 3-D imaging of fixed cells and tissue are still in use. However, the trend clearly goes toward more in vitro and in vivo imaging and quantitative analyses of image data acquired in time series measurements. The viability of cells is very important for such research, which is supported by advances of technology and confocal imaging systems, such as the LSM 5 LIVE.

—Danielle Sidawi and Tim Studt

Resources
Carl Zeiss, 800-233-2343, www.zeiss.com/micro
Nikon Instruments, 631-547-8500, www.nikonusa.com
Olympus America, 631-844-5000, www.olympusamerica.com
Univ. of South Carolina School of Medicine, 803-777-7000, www.med.sc.edu

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