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.
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.
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.
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 highspeed 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 livecell 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.
Axio Imager 2
• Light manager can store and reproduce illumination intensities for each magnification
• Motorized stands allow the user to select stored contrasting techniques via contrast manager
• Multi-user capabilities can store vital system parameters for up to ten users
• Permits homogeneous illumination at low magnifications
• Combines a light microscope (LM) and atmospheric scanning electron microscope (ASEM) to reduce sample prep time and allow dynamic observation of real-time processes
• Wide-field LM is co-axially aligned with inverted electron column, making it easy to switch between LM and ASEM without moving specimen dish
Handheld Digital USB
• Plugs into USB port of PC or laptop; images can be viewed at up to 500x magnification
• Active Pixel Technology produces crisp, sharp images and accurate color reproduction
• Features six built-in lights with adjustable brightness
• A model is available with a polarizer for reducing glare on reflective items
Helios NanoLab™ 50 Series Dual-Beam™
• Features a new, high-performance focused ion beam (FIB)
• Features an advanced 16-bit digital pattern generator for very fine and complex patterns to be written directly with the FIB
•Customized detectors let users access precise topographic, chemical or crystallographic information from the sample