Extending Visualization into Three Dimensions
Invented in the late 16th century, the microscope is arguably the oldest laboratory instrument. Yet despite its maturity, microscopy continues to evolve toward greater capabilities.
Because microscopy is limited by the physics of light collection and manipulation through lenses, it will never experience the miniaturization and integration we have observed in electronics- based instrumentation. Still, vendors are constantly extending the capabilities of lenses while integrating optics with advanced automation, image acquisition and storage. Microscopy has moved well beyond conventional visual light into fluorescence, infrared and Raman, which provide “spectrum-in- pixel” capabilities that transform 2-D and 3-D micrographs into multidimensional visualization tools.
“Rarely do we find a customer who only needs to purchase a microscope,” observes Kristen Orlowski, product marketing manager for light microscopy at Zeiss (Thornwood, NY). “This is a significant change from years past.” Users, Orlowski explains, are looking for “complete solutions” to their microscopy needs. A typical system will consist of the microscope, a selection of optics, a camera, and software. Choices of system and components are related to price point and, ultimately, system capability. The two factors contributing most are the quality of the optics and the integration of system components with easy-touse software.
Orlowski believes that product support is a critical factor in purchase decisions. Support could be in the form of online FAQs, how-to videos, a support telephone hotline, and knowledgeable and responsive sales consultants and specialists. “Without support, you might just find yourself with a very expensive paperweight.”
One of the fastest-growing applications of microscopy is live-cell microscopy, or live-cell imaging. Most major microscope manufacturers supply systems suitable for analyzing live cells as they carry out reproduction, cell cycle, ingestion, metabolism, apoptosis (cell death), secretion, signal transduction, and other essential functions. Applications span biological disciplines from drug discovery and metabolism to medical diagnosis, immunology, cell biology, neuroscience and pharmacology. With live cells, investigators can visualize not just gross cell features, but can probe deep inside the cell.
Microscopes can be set up to capture video continuously or take snapshots at varying intervals for one cell or a predesignated collection of cells. Live-cell methods most often employ fluorescence techniques using externally added reagents based on green fluorescent protein (GFP), red fluorescent protein, and others, either alone or in combination. The use of multiple fluorescent labels enables analysis of multiple phenomena simultaneously. Visible light may also be used on live cells, but the information it provides is limited to regions where the light penetrates.
Live cell analysis requires important modifications to a microscope’s sample holder, principally the ability to regulate within strict limits culture conditions outside the cells’ natural environment. Controlled conditions include culture media and nutrients, temperature, pH, osmolarity, and dissolved gases, and these conditions need to be maintained for the duration of the experiment—up to several days. “The cells have to ‘think’ they’re still inside the mouse’s body,” comments Stan Schwartz, marketing VP at Nikon Instruments (Melville, NY).
Because live cell microscopy involves trade-offs between image acquisition and cell viability, instrument speed and sensitivity are critical. Instruments need to resolve images in both time and space and do so rapidly and accurately over experiments that last several days.
The heart of a live-cell imaging system is a confocal, inverted microscope. Confocal instruments provide high-resolution 3-D images by eliminating out-of-focus light. Inverted microscopes view the sample from below instead of from above. “Cells are denser than water and tend to sink in an aqueous sample,” says Anthony Santerelli, advanced fluorescence product manager at Leica Microsystems (Bannockburn, IL). Inverting the microscope therefore shortens the distance between the objective lens and the cell(s) under examination.
Other key components include a light source; a fast, high-resolution, low-noise camera; an imaging system; and automation. Camera speed is critical for capturing transient or short-lived events deep within cells. Automation enables investigators to move rapidly from one object to another and back, to keep track of multiple cells or events, and to maintain focus. With the high-speed camera, automation permits time-lapse imaging of events occurring at multiple locations within the field. Tying everything together is software that controls movement of the stage, acquires data, and performs calculations.
Most users interested in live-cell microscopy purchase complete systems rather than a collection of components. This will continue as biology, rather than microscopy, becomes the dominant competency among individuals performing live-cell analysis. “You still have some tinkerers who purchase microscopes and build systems for their unique needs,” Santerelli notes. Many high-end microscopes suitable for cell imaging are, in fact, interoperable with third-party components and software. However, the trend toward complete “solutions” is unmistakable in microscopes, as it is in other instrument markets.