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Overcome the Limits of Optical Microscopy.

An optical microscope is a useful and powerful tool, but surprisingly little has changed since its invention. Certainly the resolving power of the optics is better, and a range of additional techniques have been developed. However, two fundamental limitations remain: Users must cope with a limited depth of focus and limited field of view. With the advent of new technologies, both of these limitations can be overcome.

The limitations on the depth of focus have existed ever since the first optical microscope was created. In short, they mean that if one part of the sample is in perfect focus, another part is most likely not in focus. Sometimes focusing at the bottom of a feature leaves the top out of focus; sometimes the opposite is true. Unfortunately, the problem only worsens as one increases magnification to gain better resolution.

Historically, these difficulties have been addressed in a variety of ways that simply avoid optical microscopy. An alternative approach has been to manually create a photographic montage. Here, users focus the microscope at different focal planes and collect a series of micrographs. They then carefully cut out in-focus portions and paste them together in an attempt to form an in-focus montage. This tedious procedure is very difficult to do accurately.

Field of view limitations inherent to an optical microscope have existed since the beginning of microscopy. The typical field of view is usually only a very small portion of the whole sample. This, as with the limitations of the depth of focus, becomes dramatically worse as one increases the magnification in order to achieve better resolution.

Similarly, the only way to address limited field of view was for a long time to do so manually. This involved moving the stage of the microscope and taking several micrographs as the field of view changed. Then these individual micrographs could be connected together to give the appearance of one large montaged image--almost. This process was time consuming and not seamless, often leaving undesirable lines or gaps between images.

Technology, such as sophisticated image-processing algorithms, can take full advantage of the montaging principle by fully automating the process and overcoming the problems historically associated with manual montaging.

Using an analog or digital camera, the system addresses the limited depth of focus by automatically focusing the microscope at different heights on the sample and acquiring a series of images with different portions in focus. It then uses a set of algorithms to identify the in-focus portions of each image in the series and compiles this information to automatically build a perfectly focused composite image. This composite image consists of only the most focused information from the series of images.

The file size is no larger than any one of the original source images, because the out-of-focus information has been discarded. Typically, users also have access to a depth map of the composite image, which shows what parts of individual images were used in the composite. The depth map is coded in shades of gray, where each value relates to a particular source image. Systems such as Frederick, Md.-based Syncroscopy's Auto-Montage give users the ability to view 3-D surface models of a sample, perform calibrated 2- and 3-D measurements, and view samples as a color-relief map.

We can also use imaging systems to address the limited field of view of the microscope. Such systems allow users to build huge (9,000 x 9,000 pixel), high-definition, fully focused images in real time while moving the stage and focus knob of the microscope. A camera provides image data, and the system performs real-time analysis as the X-Y position of the stage and the focus position change. Software extracts the most focused components from the current field of view and correlates them with position. These bits are montaged together to produce a high-resolution, perfectly focused image over a very large field of view.

There are a number of important aspects to consider when choosing a system. The first is the ability to have a live, full-color image preview on the same computer that is used for montaging. This eliminates the need for a second monitor dedicated to the camera on the microscope and yields an incredible increase in efficiency.

A selection of algorithms to choose from allows for the many different types of samples that users may acquire. If the parameters for these algorithms are user definable, that allows for variations in the samples.

Lastly, the option to have the system control the movement of the fine focus on the microscope and automate the capture process via an automated z-stepper provides a hands-off approach and calibrated measurements in the direction of the z-axis.

Applying these new technologies to an optical microscope greatly extends the capabilities of this instrument, indeed overcoming some of the very limitations that have been accepted by users since the beginning of microscopy.

McJonathan is the international product manager at Syncroscopy, Frederick, Md.
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Author:McJonathan, Stephen
Publication:R & D
Article Type:Brief Article
Date:Oct 1, 2000
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