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Exploring light and dark with two-photon microscopy

Apr 2007
Technique offers insight into the behavior of cells in the immune response

Gary Boas

Immunologists have long been interested in germinal centers, specialized structures that develop as part of the immune response. In the classical view of these structures, antigen-specific cells divide and mutate their receptors in one region — known as the “dark zone” — and then move into another — known as the “light zone” — to test the affinity of antibodies to a specific antigen. The rule is thought to be “survival of the fittest” because the B cells with highest affinity antibodies continue to multiply, whereas B cells that make the same or lower affinity antibodies drop out.

Traditionally, researchers have used histology to study germinal centers, but these investigations tend to generate hypotheses based on a static view of what is happening. In truth, little is known about the dynamics of the structures.

In the March 1 issue of Nature, researchers with Rockefeller University and New York University School of Medicine, both in New York, and with NovImmune SA in Geneva reported a study in which they examined the behavior of antigen-specific cells in germinal centers, essentially in real time, with two-photon laser scanning microscopy.

“Our labs have had a long-standing interest in how immune cells function in vivo,” said Michael L. Dustin, a researcher with New York University School of Medicine and one of the authors of the study. “In the last few years, the technology for imaging deep in tissue has advanced to a point where we can actually look at cell-cell interactions at high resolution.”


Using two-photon laser scanning microscopy, researchers have tracked the movements and interactions of cells in specialized structures involved in the immune response. By exploring the dynamics of the structures, they generated new hypotheses that contrasted with conventional wisdom. Images reprinted with permission of Nature.

Two-photon microscopy has been used in neuroscience for the past 10 years or so and in immunology for the past five. Use of the technique in immunology is beginning to mature, Dustin said. He has observed that there are more centers that want people with experience than there are people with the training. The New York University School of Medicine group began to set up instrumentation in 2001, published its first papers with the Rockefeller group — with some surprising findings about dendritic cells — in 2004 and, in 2007, is reporting the “paradigm shifting” work about germinal centers.

“We have in every case seen more movement within and between light and dark zones than we anticipated,” Dustin said. “Histology has put a sort of hyper-organization onto the germinal centers that’s really not true, in particular regarding exclusion of bystander B cells. These bystander B cells that did not have a particular affinity for the antigen were still wandering into light zones and bumping into follicular dendritic cells,” which carry the antigenic complexes.

Earlier hypotheses, based on histology, posited a closed system in which antigen-specific cells divide and mutate their receptors in the “dark zone” (DZ) and then move into the “light zone” (LZ) to test the affinity of antibodies to a specific antigen. In the present study, the researchers found much more movement within and between dark and light zones than was previously believed.

In the Nature study, the researchers used two-photon laser scanning microscopy in a mouse model to examine the behavior of antigen-specific B cells in germinal centers. They injected GFP-expressing 4-hydroxy-3-nitrophenylacetyl (NP)-specific B cells and CFP-expressing control wild-type B cells into the mice and compared their interactions. The germinal centers themselves were located by injecting mice with either Alexa-633- or Alexa-546-labeled antibodies to endogenous follicular dendritic cells.

The scientists imaged microsurgically exposed lymph nodes in the mice with a Bio-Rad Radiance multiphoton microscope outfitted with a Tsunami pulsed laser made by Spectra-Physics of Mountain View, Calif., tuned to 870 nm. They acquired a single plane at a time and then reconstructed the images. They obtained a roughly complete 3-D data set in about 30 seconds; then they recompiled the rendered 3-D images into a time-lapse series.

The study also used classical immunofluorescence histology to visualize fixed germinal centers using a Zeiss system with 488-, 543- and 633-nm excitation lines. Z-stack images were acquired with either a 40×, 1.2-NA or a 20×, 0.75-NA objective. These experiments enabled formulation of new hypotheses and design of experiments based on the dynamic images, which then could be evaluated using more traditional methods.

The two-photon laser scanning microscopy images showed that, in the absence of a specific antigen, both the NP-specific and the control B cells passed continually through the light zone. However, the former cells became physically restricted when the antigen was introduced, exhibiting significantly decreased speed with respect to the control cells and a twofold increase in cell volume. These observations confirmed that germinal centers are open structures continually visited by B cells, a conclusion that stands in contrast to the conventional wisdom, which holds that they are closed systems.

These still images show tracks of and interactions between antigen-specific B cells (green) and control B cells (blue), reflecting the significant movement observed within the structures. The white arrowheads point to the position of the tracked cell.

The researchers plan to continue working to apply two-photon microscopy to questions in the area of immunology. First they plan to expand the scope of the present study.

“This was really a vaccine study,” Dustin said. “We were essentially vaccinating the mice and seeing what happened. So, from here, we need to develop better quantitative models of how vaccination works.” They also intend to explore the dynamics of host-pathogen interaction — such as with anthrax. Currently, little is known about these interactions; however, understanding how pathogens create suppression, for instance, would be beneficial.

Finally, use of two-photon microscopy could help generate new questions and new hypotheses about what is happening in the immune system. “Even in [the present] study,” Dustin said, “once we could see what was going on, we could develop hypotheses that we could test very quickly with conventional methods. We wouldn’t have known to ask the questions if we hadn’t seen the dynamics.” In this way, he added, use of the technique could provide insights enabling development of important new vaccines — a malaria vaccine, for example, or an effective HIV vaccine.

antibodiesBiophotonicsImmunologistsMicroscopyResearch & Technologytwo-photon microscopy

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