The Future of Microscopy is Light

In the modern era of cellular research, electron microscopes have been a primary source for the most meaningful investigations.  Despite their proud past, light microscopes had been largely relegated grade school labs, held back by physical limitations on their resolution.  New techniques, however, are challenging these assumptions and bringing about a renaissance for the tool that first peered into the universe of the unseen.

Most students could explain that the light microscope was the tool first used to see cells and establish the cell theory.  However, as study turned toward the inner structures of the cell, electron microscopes and x-ray crystallography supplanted the light microscope as the favored research tool.  A microscope’s ability to magnify is limited by resolving power, the smallest separation the microscope can clearly view.  Because of interference between light waves, the resolving power of light microscopes sits close to 200 nm.  While many cellular organelles fall just within this range, studying anything finer than the larger organelles with traditional light microscopes is effectively impossible.

In 1931, a pair of German scientists realized they could achieve greater resolving power by shooting electrons at a sample instead of light waves.  The wavelength of an electron beam is much shorter than visible light, decreasing diffraction and enabling clear magnifications of up to 2 million times.  The improved resolution doesn’t come without a price, however.  Electron microscope samples must be prepared in a way that kills the specimen, meaning scientists can’t view living cells or watch cell functions as they occur.  Consequentially, much of cell research of that era centered on documenting the structure and minute details of subcellular components.

In a paradigm shift of cellular research, many scientists are now turning their focuses back to questions about how cell functions occur.  Researchers keen on studying cellular events as they happen have fueled a revolution of microscopy, bringing light microscopes, with their ability to study living samples, back into vogue.  Light microscopes have benefited from the improved design of the confocal microscope, the use of fluorescent molecules and the integration of computing power into the image formation process.  Collectively, these three improvements have restored the light microscope’s role as essential equipment.

Confocal microscopes represent a significantly different light microscope design than most people are familiar with.  This technique involves cutting thin pieces of a sample and viewing each individually, preventing interference from above and below the plane of view.  To further improve the image, confocal microscopes also focus narrow lasers on the sample, rather than blanketing the entire area with light waves.

Use of fluorophores, molecules able to emit light, has revolutionized light microscopy.  Organic molecules such as green fluorescent protein can be genetically engineered into organisms, enabling specific cell parts or cell types to glow on their own, aiding in locating and focusing.  Scientists such as Sunney Xie of Harvard have perfected the technique of adding fluorescent labels to a sample and using the emitted light to locate even individual molecules within a cell.

Improved computer technology has added the final piece to the light microscope puzzle, powering most modern techniques.  By filtering her microscope data through computer programs, Xiamowei Zhuang, another Harvard researcher, is able to focus in on individual fluorophores smaller than the microscope’s limit of resolution.  Using this initial data for calibration, Zhuang’s STORM technique is able to clear away the interference from the rest of the image.   

New technology and lab strategies have helped light microscopes reemerge as a powerful tool in examining the cell.  Despite the superior power of electron microscopes, they can’t help scientists watch neurons interact or genes activate, as light microscopes can.  While the scopes in the Zhuang lab bare no resemblance to the simple versions in classrooms across the country, they carry on the proud tradition of cellular research that reaches back to when Robert Hooke first placed a cork sample under his simple light microscope.

For more information:

Interactive confocal microscopy tutorials from Nikon :

http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html

Fluorophore information from Olympus :

http://www.olympusfluoview.com/theory/fluorophoresintro.html