By Stan Schwartz
July 20, 2005 | Over the last 30 years, we’ve made enormous strides in digital imaging by improving image analysis techniques, alleviating imaging bottlenecks in the research pipeline, and enabling digital image transmission over global networks for remote analysis. The convergences among digital imaging, information technology, and the public and private sector will continue to accelerate — providing us with new and imaginative imaging solutions for years to come.
For example, recent progress in microscope design is opening new doors to multidimensional microscopy. We all know that the standard confocal microscope provides data in the X, Y, and Z dimensions, but new techniques are expanding this capability. Think of the fourth dimension as X, Y, and Z plus time; the fifth dimension as the fourth plus wavelength data; and the sixth dimension as the fifth plus data from multiple locations acquired through the use of the microscope’s motor stage.
So, by adding additional fields of view for obtaining data on multiple cells, and the lamda dimension for acquiring various wavelengths, we can now capture six-dimensional live-cell images over an extended time period while minimizing the problems associated with photobleaching.
Specially designed confocal systems such as Nikon’s Swept Field Confocal system permit multidimensional confocal imaging in a fraction of the time required by traditional scanning systems — all while preserving longer cell viability.
| ||CATCH A WAVELENGTH: New |
spectral imaging solutions such
as NikonÕs C1si system rapidly
record multiple wavelength
images without the need for
switching optical filters.
New screening tools that promise high-resolution imaging within a cell are now commercially available. These high-content screening systems present information on multiple spatial and sequential cellular events. Their design comes from expertise in the fields of fluorescent microscopy and cell biology to create a new set of research tools aimed at solving the bottlenecks commonly found in the drug discovery laboratory. They offer significant advantages because they measure biological variability of individual cells within a well as opposed to a single intensity per well.
A typical high-content screening system consists of a microscope, fluorescent reagents, probes, and software and can become a fully automated system using multiwell and robotic sampling techniques. An automated high-content screening system is an ideal tool for pharmacological research because it shortens research cycle times while increasing the probability of clinical success. Combing fluorescence labeling with automated high-content screening has the potential to change dramatically the way drug discovery research will be performed.
Across the Spectrum
Major progress continues to be made with spectral imaging, which combines conventional live-cell three-dimensional imaging with spectroscopy. Imaging data are acquired in a “true color” stack of images, each recording a single spectral “color” channel. Earlier techniques required researchers to acquire spectral images using a charge-coupled device detector through a series of dielectric filters. Each frame was stored, and the composite image was analyzed offline. To increase the number of spectral data points, expensive automated filters were sometimes used. Each of these filter methods acquires the same image at the first wavelength and then the next wavelength, the next wavelength, and so on. Depending on the signal strength and the number of acquisitions needed to secure the optimum wavelength coverage, this method could be very time consuming, using anywhere from 10 MB to 100 MB of file space before the initial computation is made. The major drawback to this technique is the inability to take a spectral “photograph.” You must wait until the very last scan before you get a good first impression of the image quality. This is a major drawback of using filters. Because each part of the spectrum must be acquired sequentially, deterioration of the image caused by photobleaching often occurs.
Today, new spectral imaging solutions such as Nikon’s C1si system rapidly record multiple wavelength images without the need for switching optical filters. The technique is especially useful in time-lapse observations of living cells and has present and future applications in fluorescence resonance energy transfer, surgical pathology, multicolor fluorescence immunohistochemistry, and DNA expression arrays, among others.
But, without question, the most dramatic change we’ve seen over the last 30 years is in our ability to digitize image specimens. Digitized images captured through an optical microscope result in a dramatic increase in our ability to enhance features, extract information, or modify images. Digitized specimens can be shared with remote observers for automating and streamlining workflow, for image acquisition, and for storage and re-transmission. We call this capability “virtual microscopy,” and Nikon’s virtual microscopy system is the COOLSCOPE VS.
Virtual microscopy allows professionals to create Internet-accessible virtual slides and virtual slide databases, viewable at any time from anywhere, using a private network or Internet access. Research professionals can share an entire slide or set of slides over the Internet, a vast improvement over individual photographs or images of single fields of view. In research applications, a great limitation is access to a large sample population. Through the use of virtual microscopy and database technology, clinical professionals can access a large number of specimens from diverse sources. Sharing specimens using virtual slides can also further increase a sample size when specimens are archived on a database that stores slide specimens on a network or local drive for easy specimen retrieval.
Vast numbers of biological specimens are prepared on slides, yet much of this material degrades over time as samples fade, glue dries and cracks, and glass breaks. There is tremendous benefit in putting the original material online in permanent digital databases. Other clinicians now have access to difficult-to-create specimens without wasteful duplication of effort.
With a virtual slide database, users around the world can share the very best specimens, making the highest-quality and rarest preparations available.
Innovations in digital imaging will have a profound influence on biological research in the years to come, making possible new discoveries with global benefits. Light-based imaging has always been used extensively in biological research, and pioneering advances in optical microscopy are opening new avenues for visualizing, recording, and analyzing living specimens. The future has never looked brighter.
Stan Schwartz is vice president, microscopy division, of Nikon Instruments Inc. E-mail: email@example.com.