Two-photon calcium imaging is a method used in neuroscience to observe the activity of brain cells within living animals. This technique enables researchers to study neural communication as it unfolds, providing insights into the dynamic processes of the brain. It allows direct visualization of how neurons function and interact in real-time. The primary objective of this approach is to capture the intricate patterns of neural firing that underlie various brain functions.
The Principles of Calcium Imaging
Calcium ions serve as a reliable indicator of neuronal activity because their concentration inside a neuron rapidly changes when the cell fires an action potential. When a neuron becomes active, voltage-gated calcium channels open, allowing calcium ions to flow into the cell. This influx of calcium acts as an intracellular messenger, triggering a cascade of biochemical events within the neuron. The increase in intracellular calcium is a direct consequence of the neuron’s electrical excitation, making it a proxy for neural firing.
To visualize these calcium changes, scientists employ genetically encoded calcium indicators (GECIs), with GCaMP being a prominent example. GCaMP is an engineered protein. These GECIs are introduced into specific neurons, often through viral transfection or by using transgenic animal lines, ensuring their expression only in the target cells.
Upon binding to calcium ions, the GCaMP protein undergoes a conformational change that alters its fluorescence properties. This structural shift causes the GCaMP to emit light, typically in the green spectrum, allowing researchers to observe a bright flash where calcium levels have risen. Therefore, when a neuron fires, the associated increase in intracellular calcium causes the GECI within it to fluoresce. This optical signal provides a direct readout of the neuron’s activity.
The Mechanics of Two-Photon Excitation
Two-photon excitation microscopy relies on a physical principle to achieve fluorescence. Unlike traditional single-photon microscopy, which uses one higher-energy photon to excite a fluorescent molecule, two-photon excitation requires the simultaneous absorption of two lower-energy photons. These two photons, from a long-wavelength laser, must strike the fluorescent molecule at the same instant for excitation to occur. The combined energy of these two longer-wavelength photons is sufficient to excite the fluorophore to a higher energy state, after which it emits a single, higher-energy photon as fluorescence.
This process is inherently non-linear, meaning excitation is extremely unlikely unless the light intensity is very high. Therefore, excitation is effectively confined to a tiny focal volume where the laser beam is most concentrated. Outside this precise focal point, the photon density drops rapidly, making two-photon absorption negligible and preventing fluorescence.
The lasers used for two-photon excitation are femtosecond-pulsed lasers, emitting very short bursts of light, often in the near-infrared range. This contrasts with single-photon (confocal) microscopy, which often uses continuous wave lasers emitting visible or ultraviolet light. The longer wavelength of the excitation light is a defining characteristic, differentiating it from the shorter wavelengths used in one-photon approaches.
Advantages for Studying the Living Brain
The synergy of calcium imaging with two-photon excitation provides an approach for neuroscience research. One advantage is the ability to image deep within biological tissues, such as the living brain. The longer-wavelength infrared light used in two-photon microscopy scatters less as it passes through tissue compared to the shorter, visible light wavelengths used in confocal microscopy. This reduced scattering allows the excitation light to penetrate deeper, enabling visualization of neurons up to 1 millimeter or more below the brain’s surface.
A second benefit is reduced phototoxicity and photobleaching. Because two-photon excitation only occurs at the precise focal point, cells and tissues outside this plane are not subjected to damaging excitation light. This localized excitation minimizes damage to surrounding tissue, allowing for longer imaging sessions on living animals without compromising cell viability. Such gentle imaging conditions are valuable for longitudinal studies, where the same neurons need to be observed over extended periods.
Two-photon microscopy offers high spatial resolution, making it possible to visualize individual neurons and even their sub-cellular components. The precise confinement of excitation to the focal volume means that researchers can resolve fine structures like dendritic spines, which are tiny protrusions on neurons that receive synaptic inputs. This enables detailed studies of how these microscopic structures change during learning or disease processes, providing a subcellular window into neural function.
Visualizing Neural Activity in Real-Time
Two-photon calcium imaging allows scientists to directly observe the dynamic activity of neural circuits in living organisms. Researchers can watch as hundreds or even thousands of individual neurons within an animal’s brain flash with light, indicating their electrical activity. This real-time visualization can occur while the animal is engaged in tasks, such as navigating a maze or responding to sensory stimuli.
By monitoring these patterns of neuronal activation, scientists can decode the neural circuits underlying complex behaviors. For instance, they can identify populations of neurons that become active when an animal forms a memory or makes a decision. The technique has been applied to study brain regions involved in memory and planning.
This technology is also instrumental in investigating neurological and psychiatric disorders. Researchers use two-photon calcium imaging to observe how neural activity is altered in conditions like Alzheimer’s disease, autism, or stroke. By visualizing these changes at the cellular level, the technique helps in understanding disease mechanisms and evaluating potential therapeutic interventions.