New frontiers for microscopic imaging

Since their invention about four hundred years ago, microscopes have afforded scientists glimpses into cellular processes unknowable by the naked eye. Now, recent advances in microscopy arm center scientists with tools for probing deeper into understanding physiology than ever before.

The center's Scientific Imaging Facility — a resource shared by center investigators — provides scientists access to some of the most sophisticated imaging technologies currently available.

These microscopes have proven to be uniquely powerful tools for exploring minute biological events with unprecedented clarity. Watching cellular events as an organism develops from a single cell provides a dramatic example of how these technologies are being used to explore new frontiers in biology.

Dr. Cecilia Moens, investigator in the Basic Sciences Division, uses Scientific Imaging to visualize the migration of nerve cells in developing zebrafish embryos. "The development of the nervous system is complex," she said. "A lot of that complexity arises because cells migrate from where they are born to some other position where they carry out their functions.

Confocal flagship

"If we want to understand the genetics that control migration of neurons, we need to be able to manipulate those genes in the context of the whole animal and to observe the effects in the living embryo. Because even in the early embryo the developing nervous system is a complex, three-dimensional structure, we need a microscope that can look deep inside the tissue, focus on a single neuron at one specific focal plane and watch its behavior over a period of several hours. This is something only a confocal microscope can give us power to do."

Scientific Imaging's flagship technology — a newly acquired Zeiss confocal and two-photon microscope — may help scientists understand how the genes of developing zebrafish embryos create the roadmap outlining each neuron's trip.

"We want to be able to see not just that the cell is moving, but also to watch changes in morphology (shape) as well," Moens said.

Two essential innovations in microscopy provide the clarity and live-cell imaging capabilities needed for these studies, said Dr. Julio Vazquez, manager of the Scientific Imaging Facility.

"For the past 100 years, optics have really not changed that much. What has changed are automation and sensitivity," Vazquez said. "More importantly, the past 20 years saw the development of confocal and deconvolution technology by independent laboratories.

"When you initially collect the image, it is blurred due to the presence of out-of-focus information. John Sedat and his collaborators at the University of California San Francisco invented a way to computationally get rid of the background haze, making the image clean and sharp," Vazquez said.

Removing out-of-focus haze

At the same time, several laboratories developed the confocal microscope, an instrument that removes out-of-focus haze through a special design, and also allows greater sample penetration. Whereas the eye alone has no way of eliminating noise from above and below the plane of focus, the confocal and deconvolution technologies provide clean, crisp images of individual planes from thick samples. This process allows the microscopic study of whole biological samples.

Another major innovation in the past 15 years has been the development of fluorescent probes, enabling researchers to label and track individual proteins and genes inside cells.

While many flourescent probes are useful for examining immobilized tissues, the green fluorescent protein (GFP) has proven invaluable for examing living cells and tissues.

"The true advantage is in looking rapidly at changing events in living tissues," said Dr. Jim Priess, whose Basic Sciences Division laboratory studies development in the worm Caenorhabditis elegans. "What you can do with GFP is image things as they actually happen. A lot of events in the cell are so dynamic that they are impossible to follow in fixed tissue. Also, some cellular structures are fragile and difficult to reconstitute.

Together with the highly sensitive detector technology currently available, fluorescence technology captures images much faster than standard film approaches, allowing Priess and other investigators means of imaging cellular events as they occur.

Sharp, real-time movies

"The amount of light is often so faint that a single exposure might take several minutes with film," Vazquez said. In contrast, the Zeiss microscope can snap images at up to 10 frames per second, making it possible to create crisp, three-dimensional images and real-time movies of such minute developmental events.

A researcher can focus down through a region of interest, collecting a stack of images through the sample. Using imaging software programs such as Volocity, the investigator then renders the stacks into a 3D picture. An investigator can also render the fourth dimension — time — from living samples and play the rendering back as a movie.

"Some events happen too slowly to see changes with conventional microscopy," Vazquez said. "For example, in the development of the fruit fly Drosophila, the embryo takes about 24 hours to develop and hatch from a fertilized egg. If you just look as the events happen, you will miss the big picture. Because it is very sensitive, the Zeiss confocal allows you to collect sequential images over long time periods that you can play forward at a faster rate to watch changes."

The Zeiss microscope stage can accept special incubation chambers that are specially designed to keep the conditions as close to normal as possible while an investigator images the specimen.

"Most preparations can last from a few hours to a couple of days," Vazquez said. "You can take hundreds of pictures, then speed up the frames from beginning to end to see the whole process."

Longer-term imaging

One technological development facilitating longer-term imaging has been two-photon technology.

"The laser gives short pulses of infrared light, which is less toxic to the sample compared to conventional illumination with visible light," Vazquez said. "Infrared light also penetrates deeper inside the sample, because it is less absorbed. This technology has allowed researchers to image samples up to several hundred microns thick, compared to the maximum of 50-100 microns for conventional confocal microscopy. It has also allowed investigators to image developing vertebrate embryos without killing them, something that had not been possible before."

The decision to create the imaging facility as a shared resource provides investigators access to technology otherwise difficult and expensive to attain.

"The Zeiss two-photon microscope alone, for example, costs nearly $700,000, and also requires a lot of maintenance. Its operation is complex and requires training. With the shared facility, an investigator can image samples for many years, for a fraction of what they would pay to acquire a microscope, and without having to worry about maintenance," Vasquez said.