Hong Yu shows Manisha Sharma the Confocal Microscope.

In confocal microscopy the collected light is reflected or emitted by a single plane of the specimen image. This generates a high contrast, high resolution image due to effective suppression of background light scattered from outside the focal plane.

A series of such images generated with the focal length shifted in small steps can be combined in a three dimensional stack (a “z-stack”), which is amenable to digital processing. Thus, confocal microscopes have an optical sectioning capability – as slices of the specimen can be examined without mechanical cutting and direct specimen preparation.

With the enhanced optical resolution over conventional wide-field techniques, the confocal microscopy can be used in a wide range of applicationsb including:

• imaging of various Z axis planes (XYZ) to render a 3D image of the cells or tissue in question
• co-localization to determine whether labelled molecules are localized in the same region in multi-stained specimens
• spectral unmixing to separate fluorophores with similar emission peaks at 2nm spectral resolution via emission wavelength detection system (XY)
• time-lapse analysis (XYT) at high speed image acquisition (16 frames/sec) and high-precision achieved by the power of zero thermal drift compensation in Z-direction
• FRAP (fluorescence recovery after photobleaching) to observe rapid movements of fluorescent molecules that come from outside the specific photobleached area
• FRET (fluorescence resonance energy transfer) to visualize the interactions between two molecules tagged with proper fluorophores.

The confocal system used at WMI is equipped with a fully enclosed incubator, temperature controller and heated stage, mini culture chamber, as well as 5% CO2 supply, therefore allowing imaging of live cells and tissues.

There are 405 nm, 473 nm, 559 nm, and 635 nm laser lines and the system allows free emission wavelength range setting rather than fixed filter settings in conventional confocal microscopes, making it possible and easy to work on most frequently-used fluorescent probes.