Science in a New Light.(microscopy imaging systems)

Microscopy imaging systems focus on the little things in life

The science of microscopy, especially how microscopic observations are made and data are collected and displayed, has come a long way since the days of Carl Zeiss. The last decade has seen a resurgence in the use of optical microscopy in basic research, due in part to advances in instrumentation. Confocal technology, high resolution solid state cameras such as the slow scan, cooled charge coupled device (CCD) camera, and new probes for small molecules are contributing to this trend.

For many years the still-film camera attached to a microscope was the method of choice for image capture. Although used by many researchers today, the still-film camera is slowly being replaced by video-digital imaging systems. These new imaging systems offer unprecedented opportunities to study the full gamut of cellular structure and physiology. They have expanded the experimental approaches that scientists can use to answer more and more complex biological questions--for example, tracking intracellular localization of enzymes or membrane proteins.

Researchers, no longer bound to the examination of static, 2-D film-based photographic images, now can delve into an image--through, under, over, around--to thoroughly examine all facets of the subject material in real time and over time. Modern microscopic imaging has become and will continue to be an integral part of many 21st-century scientific discoveries, providing new avenues to investigate biological phenomena not possible by other "advanced" methods.

The one key feature of video-digital imaging that distinguishes it from its still-film predecessor is that it acquires images in multiple spatial and temporal dimensions. Images can be captured in the standard x and y axes, in the z-axis (rotating the specimen about a fixed point in space), at different wavelengths, over time, and in narrow- and wide-focus views of the subject matter. This article focuses on basic aspects of image acquisition and analysis and some of the video-digital systems and devices available today. Detailed reviews of microscopy and microscopes are available elsewhere.[1-4]

The modern microscopy imaging system is composed of five basic components: the microscope, a lighting source, a specimen stage control, an image acquisition device, and a postacquisition image analysis system. The microscope can be practically any high-end optical device, such as those offered by Zeiss, Olympus, Nikon, and Meiji, that meets the initial resolution and functionality requirements of the experiments for which the instrument will be used.

For the researcher who has a functional microscope or has one in mind to purchase, the major criterion for choosing an imaging system should be the capability and ease of integration with the camera and other peripherals. Not all microscopy and imaging instruments are physically compatible, and even those that are may lack the device drivers and software necessary for controlling the system from a workstation or other automation device. The lighting source, whether a conventional white light or pulsed field laser, is specified by the experimental application, but also defines the kind of imaging system possible and its limitations and functionality. Control of the specimen stage is extremely important, especially for 3-D imaging.

IMAGE CAPTURE DEVICES

The most common image acquisition devices for microscopy applications are the still-film (photographic) camera, the video recorder, and video-CCD camera hybrids. Photomicroscopy is generally synonymous with the use of a still-film camera, such as those from Nikon, Canon, and Olympus, mounted to the microscope. Pictures are taken as with any hand-held camera, except that the camera is mounted to the viewing field of a microscope. The still-film camera records the image on a standard photographic film or plate, which is subsequently developed, and the image is printed as a photograph. The quality of the photograph is dependent in large part on the quality of the microscope. Other factors include the quality and type of photographic film used and the light source. The film type is generally defined by the amount of light to which the film will be exposed during image capture. Thus it is important that the light source be optimized to yield even illumination over the entire field of view.

Mounting the camera to a microscope is a relatively straightforward process--in fact many quality …