A technology that helps scientists spy on the inner workings of mice is empowering immunologists, researchers said in San Francisco, California, this week at the 13–17 December American Society for Cell Biology annual meeting.

Breast cancer cells (red) caught in the act of migrating. Credit: B. Gligorijevic

Multiphoton microscopy uses pulsed lasers to track single cells deep inside animal tissues, revealing how they interact with the cells around them. The technique uses lower-energy lasers than other light-microscopy methods, and so it is less toxic to cells — and less prone to the light scattering that can blur images.

'Intravital imaging' experiments that use multiphoton microscopy inside living animals were confined to a handful of labs until a couple of years ago. Now, says Jeffrey Segall of the Albert Einstein College of Medicine of Yeshiva University in New York, the technique "has reached a critical mass" — thanks to the renewed interest in cell environments, and the availability of cheaper, more reliable, multiphoton microscopes.

Look inside

At a 15 December workshop at the meeting, Jackson Egen of the National Institute of Allergy and Infectious Disease in Bethesda, Maryland, showed how his group, led by Ronald Germain, has used multiphoton intravital microscopy to discover how a particular gene mutation causes a human immunodeficiency disease. The team found that the mutation disrupts the interactions between T cells and B cells that are critical to the immune response1.

And Segall described how scientists at the Einstein biophotonics centre take intravital imaging deeper inside tissues. They construct a tiny glass window that is surgically grafted inside a mouse. They then inject the mice with fluorescently labelled cells, which change colour when hit by a laser light, and can be tracked through the mouse's body via the window. This allows researchers to follow the cells' movements for days.

On 15 December, in a symposium at the meeting, Bojana Gligorijevic, a research associate at the Albert Einstein College of Medicine, described how Einstein scientists used this technique to trace cancerous cells in a model of breast cancer2. They gained a real-time view of the events that lead to metastasis, in which cancer cells move away from primary tumours. They have found, for example, that tumour cells are more likely to metastasize if close to a major blood vessel. This underscores the need to study tissues in their natural context. "These tumour cells are all equal when we inject them," says Gligorijevic. "The only reason their behaviour is different in tumour, is because they are in different microenvironments."

Cells in motion

Early multiphoton intravital microscopy studies focused on simply observing the behaviour of cells, but the field is now shifting towards examining how cells respond to external and internal disruptions, such as infections or molecular modifications.

For example, Egen has used the technique to watch how certain immune cells help sandflies transmit through their bites the parasite that causes leishmaniasis3. And Ulrich von Andrian of Harvard Medical School in Boston, Massachusetts, has used multiphoton intravital microscopy to answer long-standing immunological questions, such as which specific cells recognize viruses that infect the lymph nodes and present them to cells that will fight the infection4.

"The prevailing view was a very static one of two cells interacting without much else going on," says von Andrian. "Now that we can actually watch these movies, we realize it is nothing like that — there's this orchestrated dance going on at all times that needs to be taken into account if you want to understand the immune response."