We describe an important new application for an existing technology. Multi-photon microscopy (MPM) had previously been adopted in fields such as neuroscience and developmental biology, but our report was the first to apply this method to visualize the motility and behaviors of live immune system cells in intact lymphoid tissues.

By quantitatively analyzing single-cell dynamics in situ, we provided a fundamental description of T-cell and B-cell trafficking behavior and laid the groundwork for subsequent live tissue imaging studies. Since our report, the technique has grown increasingly popular with immunologists, and has been adapted to study diverse immunological models.

The cells of the immune system are distributed via the blood throughout the body. As these cells move from one tissue to another they rely on cues in the local tissue environment to instruct the cell’s behavior.

The complex tissue signals that control the immune response cannot be adequately replicated in test tubes and culture dishes; therefore, it is important to study the immune system in the natural environment of whole tissues and also in living animals.

MPM is a new technique that allowed us to look deeply into intact organs and visualize living cells over long periods of time without damaging them. Many other researchers have now adopted this technique to help discover how cells work together to protect the body from infection and cancer.

The research grew from a collaboration between Ian Parker (a neurobiologist) and Mike Cahalan (an immunologist). Ian had constructed a custom-designed multiphoton microscope designed for fast imaging of calcium signals from neurons deep within brain tissue. One day, Mike knocked on Ian’s door and asked "Hey, can we put a lymph node under your microscope?" The first trial proved a success, as we were able to visualize fluorescently-labeled T cells to depths of several hundred microns within intact mouse lymph nodes. However, it then took a great deal of work (in which Mark Miller played a major role) to establish the technique as a practicable means to visualize and analyze the real-time, in situ behavior of immune system cells. Key technical accomplishments included developing conditions to stabilize and maintain the viability of tissues for imaging, perfecting the technology to acquire 4-dimensional time-lapse movies over periods of hours, improving the system to increase its sensitivity and minimize laser-induced photodamage, and developing analytical tools to analyze and characterize cell motility.

The ability to track cells in lymphoid organs will likely be applied to improve vaccination methods and to gain insight into pathogenic mechanisms of bacterial and viral infections. In a more general way, it is likely that two-photon microscopy will be applied to image human cells and to improve diagnostic capabilities or optimize therapeutic approaches against cancer and autoimmune disorders.