The human brain contains billions of neurons connected in an intricate network. Observing the activity of these cells in a living brain was a long-standing scientific challenge, limiting our understanding of how neural processes create thoughts and behaviors. The development of two-photon calcium imaging marked a step forward, providing neuroscientists with a tool to visualize the dynamic conversations between neurons in real time. This technology allows researchers to peer into the working brain, revealing the patterns of cellular activity that underlie its capabilities.
The Mechanics of Two-Photon Calcium Imaging
Two-photon calcium imaging operates on a principle called two-photon excitation. Unlike conventional microscopy that uses a single high-energy photon, this technique uses a specialized laser that emits lower-energy photons in rapid pulses. Excitation of a fluorescent molecule occurs only when two lower-energy photons arrive at the same location at virtually the same instant, combining their energy.
To visualize neural activity, scientists introduce fluorescent calcium indicators into neurons. These indicators are often genetically encoded proteins, like GCaMP, that can be targeted to specific neuron types. These molecules are engineered to bind with calcium ions (Ca2+) and change their brightness in proportion to the local calcium concentration.
The functional link to brain activity is the influx of calcium during a neuron’s firing, known as an action potential. When a neuron becomes active, channels on its membrane open, allowing calcium ions to rush into the cell. This increase in intracellular calcium is detected by the indicator molecules, which begin to fluoresce. A specialized microscope then captures this emitted light, making the neuron’s activity visible as a flash.
Key Advantages for Studying the Living Brain
The physics of two-photon excitation provides advantages for imaging within the dense, light-scattering environment of the brain. The technique uses longer-wavelength infrared light, which penetrates deeper into biological tissue with less scattering than the light used in standard microscopy. This allows scientists to visualize neurons located hundreds of micrometers below the brain’s surface.
Because the simultaneous arrival of two photons is a rare event, fluorescence is confined to a minuscule focal point. This localized excitation means that tissue above and below the plane of focus is not exposed to intense laser light, reducing cell damage. This gentler process allows for prolonged imaging sessions, sometimes lasting hours, which is useful for studying processes like learning that unfold over time.
This localization also provides inherent optical sectioning, generating a sharp image from a single plane without the blur common in conventional fluorescence microscopy. By scanning the focal point, researchers can build a stack of these optical sections to create detailed three-dimensional reconstructions of neural structures and their activity.
Perhaps the most useful advantage is the ability to perform these imaging experiments in awake, behaving animals. By securing an animal’s head under the microscope, researchers can record the activity of specific neurons while the animal performs tasks or responds to stimuli. This allows for a direct correlation between patterns of neural firing and specific behaviors.
Scientific Breakthroughs and Applications
The application of two-photon calcium imaging has catalyzed breakthroughs across neuroscience. Its uses include:
- Mapping how neurons are functionally connected and how information flows through networks. This has been applied to deciphering the organization of circuits responsible for processing sensory information.
- Providing insights into sensory processing. By presenting stimuli like images or sounds, researchers can watch how neurons in the visual or auditory cortex respond, revealing how cells are tuned to specific features.
- Studying learning and memory. Scientists can track the activity of the same neurons over days as an animal learns a task, allowing them to observe neuroplasticity, where neuronal responses change with experience.
- Investigating neurological and psychiatric disorders. In animal models of conditions like Alzheimer’s or epilepsy, researchers can observe how neuronal activity becomes dysregulated, providing clues to disease mechanisms and a platform to test potential therapies.
Deciphering Neuronal Activity from Calcium Signals
The raw output of a two-photon imaging experiment is a time-lapse movie showing a field of flashing neurons. Each flash, a calcium transient, represents a burst of electrical activity. The brightness and duration of the fluorescence provide information about the intensity and timing of the underlying neural firing.
Translating these flashes into meaningful data requires computational processing. Algorithms first correct for movement artifacts, especially when imaging awake animals. The software then identifies individual cells and extracts a “trace” of fluorescence intensity for each neuron over the recording. This trace serves as a proxy for the cell’s activity over time.
While calcium transients are closely correlated with action potentials, the relationship is not always one-to-one, as a single transient may correspond to several action potentials in quick succession. Interpreting the data requires considering the properties of the specific calcium indicator used. Scientists then correlate these extracted activity patterns with specific events, such as a stimulus or a behavior, to draw conclusions about what the neurons are encoding.