Seeing Science: Confocal Microscopy

Editor’s note: This is the first article in an ASU feature series that will highlight the tools of the trade scientists use in their quest for new discoveries.



Scientists probe the mysteries of the natural world across an astonishing range of dimensions, from the subatomic domain of quarks and leptons to walls or sheets of supercluster galaxies known as filaments—gargantuan structures measuring over a billion light years across.

Cells and other features of the living microworld figure between these extremes. Their domain was first brought into focus when investigators in antiquity began to develop an ever-expanding toolkit to assist their observations.

One of the earliest and most ambitious pioneers of the microscopic realm was a 17th century Dutch tradesman by the name of Antonie van Leeuwenhoek. Often tinkering in his free time at night, he made simple devices that would change the course of science. With little more than strong magnifying lenses mounted on silver or copper frames, van Leeuwenhoek brought into focus a previously unseen world  He made careful observations of muscle fibers, capillary blood flow, free-living and parasitic protists, spermatozoa, nematodes, rotifers and other inhabitants of the microscopic universe.

In one such experiment, van Leeuwenhoek removed samples of his own tooth plaque. Upon examining the gooey substance with his microscope, he was astonished to find a lively zoo of living forms now rendered visible: “The biggest sort. . . had a very strong and swift motion, and shot through the water (or spittle) like a pike does through the water. The second sort. . . oft-times spun round like a top. . . and these were far more in number," he wrote. These were the very first living bacteria ever observed.

From such humble beginnings, the microscope has developed into one of the most valuable tools ever created for the exploration of nature. Today, the field of microscopy continues to evolve, offering researchers a vast array of imaging technologies for probing the very small, with a level of exquisite detail van Leewenhoek and his peers could only have dreamt of. 

Among the powerful techniques available to modern researchers is the laser confocal scanning microscope. One such instrument can be found at Arizona State University’s Biodesign Institute, where it is used extensively for imaging varied biological specimens. The Institute’s Zeiss Duo Scanning Laser Microscope is overseen by Doug Daniel, who also assists with other sophisticated optical instruments used for Institute research.

“Confocal microscopy was invented in 1957 by a guy named Marvin Minsky,” Daniel notes, adding that it took several decades before the technology developed into a practical tool for use in the lab. The instrument allows for high-resolution imaging of biological material.

It is a form of fluorescence microscopy, in which the molecules of the material under study are rendered into an excited state by means of high intensity light. In response to this excitation, the sample will emit light of longer wavelength—this is the fluorescence that will form the image. A small aperture or pinhole a few tens of microns in diameter located in the detection pathway acts to reject of any out-of-focus light, ensuring that the final image will reveal the specimen in vivid detail. This differs from conventional light microscopy in which images are a composite of both in focus and out of focus signal. This is a limiting factor for good imaging, particularly at higher magnifications.

Most biological material is suitable for confocal imaging, providing it is somewhat transparent, though cell and tissue samples are the most common subjects. “You would generally label parts of the cell or tissue with fluorescent dyes,” Daniel notes, explaining that such preparation of material permits distinct imaging of cell structures like the membrane, nucleus or other parts of interest.

One of the critical features of confocal microscopy is its ability to image successive layers through a specimen. These slices can then be reassembled by computer into highly detailed 3-dimensional images. Many post-imaging techniques can also be applied, Daniel notes, which can reveal delicate details that would otherwise be lost.

Imaging of living cells and their dynamics is also possible, providing they can be fixed in place during the imaging process. Daniel teaches these techniques to Biodesign Institute researchers, in addition to basic operation of the instrument. “If you’ve had no background in imaging or microscopy,” he says,  “a lot of these concepts are new to you and you don’t really know what to tell the instrument to do sometimes. It takes some time to understand the problems you’re faced with and how to handle them. ” With patience and some practice however, most researchers can become proficient with the instrument.

The popularity of confocal microscopy in biology and clinical medicine has grown significantly in recent years, thanks to the instrument’s ability to easily produce stunningly detailed images, with minimal sample preparation. By comparing the inner states of normal cells with cells associated with diseases such as cancer, diabetes or Alzheimer’s, scientists have made much progress in identifying components of the cell that may go awry during the transition to a diseased state.

The optical micropscope, which arrived on the scene c. 1600 as little more than an intriguing toy, has been refined into a central weapon in science’s arsenal for probing the structure of the microworld.

 

Neuronal images in the slideshow above were supplied by Dr. Page Baluch at the School of Life Sciences, Keck Bioimaging Laboratory.

 

Written by Richard Harth
Biodesign Institute science writer
richard.harth@asu.edu