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Optics lie at the heart of microscopy, and advances in optics design continue to push the limits of resolution. For instance, the use of motorized microscopes and laser light sources now help in defining the plane of focus.

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From Optics Design to Imaging Software, Accessories Accentuate the Capabilities of Microscopy

Optics lie at the heart of microscopy, and advances in optics design continue to push the limits of resolution. For instance, the use of motorized microscopes and laser light sources now help in defining the plane of focus, even as microscopes are exposed to routine mechanical and thermal stress. Joseph LoBiondo, product planning manager at Nikon Instruments, Inc., talks about Nikon’s Perfect Focus system, which can readjust to the same position on the same specimen and enable the focus to be maintained at a point of interest for long-term imaging. “In the past, in order to do long-term time-lapse imaging, someone had to be sitting at the microscope adjusting the focus,” says LoBiondo. “Now all you have to do is set the motorized stage to scan and take images at a certain position and then just leave the instrument alone.”

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Automated, hands-free imaging has led to improved speed and ease of use, and now the field is leaning toward miniaturization to enable applications at even higher throughput. “The whole workflow in microscopy—the LEDs, the cameras, the microscopes— has become miniaturizable and user friendly,” says Mike Ignatius, Ph.D., product manager of Applied Visualization Technologies at Invitrogen (now a part of Life Technologies). The field of optics is also shifting toward incorporating more instruments that have capabilities for micromanipulation and microinjection. Lo- Biondo says, “The equipment that we provide is moving toward incorporating smaller devices that are less susceptible to vibration, are more stable and are able to maintain precise mechanical movements.” Major advancements in microinjection systems are related to increasing throughput and automation by providing devices that can be programmed. Eppendorf ’s InjectMan micromanipulator system offers a programmable interface and can be integrated with microinjection products in order to both manipulate and inject samples.

Computational tools and software packages are playing an important role in all aspects of microscopy. Three of the major tasks that image analysis software packages now perform include deconvolution, three-dimensional visualization and image quantification. Deconvolution software improves both contrast and resolution, while reducing image blur from out-of-focus light. Software tools for rendering or visualization of three-dimensional structures— to interpret cellular organelles or arrangement of biological moieties— are being used routinely in life sciences research.

The increased use of fluorescent and time-resolved labeling technologies has also made image quantification programs essential in analyzing microscopic images. The ability to quantitate motion or time-resolved images is an important consideration for end users who are looking to purchase image analysis software for biological applications. MetaMorph is used to analyze and visualize timeresolved and motion data, making the software useful for live-cell imaging applications. Software packages such as MATLAB and Adobe Photoshop are also used to analyze images as more dedicated options continue to populate the image analysis landscape. ImageJ, developed by Wayne Rasband of the National Institute of Mental Health, is an open source application used to acquire, process and analyze images. Other software vendors such as Volocity, Amira and Huygens SVI also offer a variety of packages that can be customized, depending on end users’ needs.

Some other considerations when purchasing imaging software include compatibility with specific hardware systems, ease of use and availability of add-ons. For example, the MetaMorph package supports microscope hardware accessories—such as an automated slide handler—and allows users to create Visual Basic programs to automate and analyze images. A majority of image analysis software programs now available are compatible with multiple operating systems (e.g., MacOS, Windows, Linux). Next-generation software products will probably incorporate more learning algorithms that are capable of pattern recognition, which will have direct benefit for live-cell imaging applications.

Tanuja Koppal, PhD, is a freelance science writer and consultant based in Randolph, N.J.

Leica Microsystems
MM AF imaging systems combine Leica’s high-tech microscopy and imaging with the breadth of applications and analysis of MetaMorph® software. The product line offers tightly integrated systems based on the excellent ease-of-use and optical performance of the Leica microscope range, giving researchers an efficient platform for many types of imaging experiments. An integrated journaling capability is included that allows flexible customization of the platform for virtually any imaging application. “For many years, the MetaMorph range of products has played a leading role in all aspects of imaging and data analysis,” said MDS Analytical Technologies president Andy Boorn. “MDS Analytical Technologies welcomes the opportunity to team up with Leica to add the functionality, flexibility, and market reputation of MetaMorph software to the excellence in design automation and user friendliness of Leica microscopes and cameras.”

Michelson Diagnostics
High Speed imaging software for the EX1301 OCT (Optical Coherence Tomography) microscope allows images to be captured at up to 40 frames per second, enabling imaging of dynamic processes such as the heartbeat of a fruit-fly. “We believe that this capability will prove very useful for biologists, for example those who are researching the genetic origins of heart disease”, said Jon Holmes, MDL’s CEO, “The high frame rate, coupled with market-leading optical resolution from our patented Multi-Beam OCT optics, in an off-the-shelf package, will open up new opportunities for the developmental biology scientist” The new high-speed processing software is four times faster than the previous version, and was originally developed for the company’s new hand-held OCT scanner. Actual frame rates depend on the image width: from 7.5 fps (image width 1,250 pixels), to 40 fps (image width 125 pixels).

Nikon Instruments
VAAS detection is a confocal imaging method for post acquisition recovery of data normally lost during the course of the experiment. This is accomplished by collecting emission photons that normally are rejected by the emission side pinhole, traditionally used at the 1 airy disk size. This unique technology allows for virtual adjusting of the confocality and sensitivity of data postacquisition, as well as collecting more photons during the initial image acquisition to enable image reconstruction with greatly improved signal-to-noise. VAAS, which stands for Virtual Adjustable Aperture System, uses two pinholes to collect emitted photons: a standard-sized one to enable confocality and one with a diameter three-times larger to capture more of the emitted fluorescence without increasing the out of focus background detection. VAAS is an effective method to reduce the out of focus haze signal without reducing the signal from the focal plane.

The Phenom™ Remote Assistant is a service enhancement that allows for remote tracking, diagnostics and repair of Phenom personal scanning electron microscope (SEM) systems. The device is powered by RAPID (Remote Access Program for Interactive Diagnostics), which is FEI’s secure infrastructure for remote diagnostics. RAPID provides a highlysecure, encrypted, VPN (virtual private network) connection between the customer and FEI service engineer. FEI service engineers can remotely track key performance metrics over time, run service test diagnostics on the system, check and modify tool settings, and even view microscope images to access quality and provide expert operations advice. The Phenom imaging tool combines light optical and electron optical technologies in one integrated, easy-to-use microscope system. It offers magnification of up to 24,000x and 30 nm resolution (about 100,000 times smaller than the diameter of a human hair) in 30 seconds.

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