In many research arenas, image analysis tools have become as important as microscopes.

Software has become a major component of most research light microscope systems today. With the development of digital cameras and other capture methodologies that rely on the power of computing to capture, analyze, and organize data, it is rare now to find a research-level stand that is sold without being considered as part of a comprehensive package that includes peripherals, computer controls, advanced data storage, and software.

Cornerstones of use

The Image Xpress Micro automated imager driven by MetaXpress is the latest in a line of imaging systems offered by Molecular Devices.

Both microscope manufacturers themselves and third-party organizations including corporations and academics are involved in software development. While applications may range from laser scanning, deconvolution, and widefield-CCD-based applications to high-throughput/high-content screening systems, software has three major purposes:

• First, it provides organizational support to and configuration of the experiment being performed. Such uses as control of hardware components, setting intervals for time lapse experiments and other similar functions are included in this type of software, along with data acquisition itself.

• The second type of software involves image processing and data analysis-taking raw data and using it to generate further images, reports, and visual analysis.

• The third aspect of software is documentation of the raw image and analysis data, along with storage, archiving, and retrieval.

Image analysis software, the second of these two steps, adds meaning and depth to the raw data scientists seek to analyze. "Whether it's something as simple as a 2-D measurement, or as a complex as measuring the spatial relationship of proteins via a ratiometric measurement of two intensity values, digital microscope imaging systems that are being built today require the support of an image analysis program," says Edward Lachica, Imaging Specialist for Olympus America Inc., Melville, N.Y.

"Software makes it easier for customers to get the most out of their instruments, allowing them to see and do things we couldn't do before," adds Stan Schwartz, VP of Product and Marketing, Nikon Instruments Inc., Melville, N.Y. "We can capture images at numerous depths of focus and display them all as a single image in a 3-D projection. In industrial research, we can get the top and bottom of the pipe to both be in focus in the same image. We can provide good data, in six dimensions, with precise measurements of intensity and XYZ dimension. Such information is invaluable."

With the imaging software often the most important aspect of users' interface with a microscope system, many microscope manufacturers now have software R&D groups that focus on making it easy to get the most out of their powerful hardware. In addition, some companies have close relationships with dedicated third-party software developers and have specific software devoted to their research hardware. There are also independent companies that specialize in this field.

Image evolution

"Technology has come a long way," says Geoff Jenkinson, a product manager with Leica Microsystems, Bannockburn, Ill. "Twenty years ago, the image analysis computer was large enough to nearly fill an office. For its price it was possible to buy a small house. Now a standard mid-range PC is all that is needed." One of the early systems available was Image-1 by Universal Imaging Corp., (now owned by Molecular Devices Corp., Sunnyvale, Calif.). Image-1 was replaced by what became one of the most famous names in biological research level software, Metamorph. Numerous functions now available in several of the companies' latest software offerings were unheard of in the early versions-including montaging or stitching, a practice of "sewing" numerous images of tiny areas together to form a single, high-resolution image of a larger area; and working with ever-fainter fluorescence, seeking the imaging of cellular events and structures in real-time.

Nikon’s NIS-Elements imaging software has multi-image window capability with simple drag and drop, along with multi-channel wavelength merge/unmerge functions.

"The applications in biomedical sciences are ranging from sub-micron to macro dimensions, covering research on cells, tissue, and embryos-fixed, cultured, or in situ-for single experiments as well as sample arrays," says Sebastian Tille, Product & Application Support Manager, Carl Zeiss MicroImaging, Inc., Thornwood, N.Y. Among the most respected of the company-developed software packages is the Zeiss suite of solutions for biomedical and material research. Axiovision release 4.5, for widefield/CCD-based imaging microscopy, includes multifluorescence imaging, colocalization, FRET, deconvolution, 3-D and 4-D rendering, and interactive automated measurements. There is also proprietary software for TIRF, particle analysis, cellomics, digital slide scanning for pathology, and confocal and multi- photon work.

Nikon has recently introduced its NIS-Elements, a comprehensive software suite that includes image acquisition, analysis, and data management. Key features include image stitching, the creation of an all-in-focus image from a series of Z-axis image stacks and a multi-dimensional image viewer. A real-time 2-D deconvolution module is available that supports live on-the-fly or captured deconvolution of either an entire image or specific regions of interest.

Olympus MicroSuiteFIVE software offers multidimensional time-lapse analysis, 3-D rendering, slide- or multi-well processing, and simple deconvolution as part of its Advanced Suite.

Olympus offers a pair of comprehensive solutions. MicroSuiteFIVE is a software package that is designed to address the needs of the majority of microscope users that capture images and perform simple 2-D operations such as measuring the distance between two objects, measuring the perimeter and area of an object or counting objects in 2-D space. Olympus has also formed a strategic alliance with Intelligent Imaging Innovations, Inc. (better known as 3i), to sell the software company's respected SlideBook product. SlideBook software is designed to address the needs of microscope users who conduct live cell experiments requiring exquisite control of digital cameras and peripherals.

The Leica Application Suite integrates Leica automated microscopes, digital cameras, and software into one common micro-imaging environment to provide a consistent platform. The image processing functions make it suitable for a diverse range of imaging tasks such as visualization, enhancement, measurements, and documentation.

The MetaMorph imaging package, now offered through Molecular Devices, has been a staple tool for R&D imaging for more than 20 years. Since MetaMorph has embedded journaling functionality and user-defined toolbars, it is useful for research and prototyping purposes. Many researchers know how to use MetaMorph, which is important, considering that the software packages are tools that take a while to learn.

Analysis options
Many dedicated software companies also have exciting offerings in the image analysis field. These include Advanced Imaging Concepts, Inc., Princeton, N.J., Alicona, Grambach, Austria, Applied Imaging Corp., San Jose, Calif., in the genomics field, Applied Precision, Issaquah, Wash., in the semiconductor and life science imaging arenas, and Clemex Technologies, Longueuel, Canada, in quality control and research labs.

Down the road
"Today, with mature operating systems, powerful processors, ample amounts of RAM, and fast frame rate cooled CCDs, research imaging is a whole new ball game," says Michael Sjaastad, Director of Imaging Marketing for Molecular Devices. But tomorrow, the largest issue in imaging will certainly be data handling, with gigabytes of new image data requiring not only analysis, archiving, and storage, but easy retrieval as well.

-Ilene Semiatin
Ilene Semiatin is a freelance writer
based in White Plains, N.Y.