In vivo imaging has been central to biology from its beginning, with optical imaging being the best compromise between damage and resolution.
Through the application of multi-photon excitation, high-resolution imaging has become possible in scattering tissue such as the brain.
This has enabled the imaging of cellular activity in the intact cerebral cortex both in single-cell compartments and populations of neurons.
Careful attention to experimental parameters is needed to reliably detect action potentials.
In addition to the detection of neural activation on the millisecond scale, multi-photon microscopy can be used to measure the morphological stability and plasticity of neuronal processes on the time scale of months right down to the single-spine and single-synaptic-bouton level.
In the future, multi-photon imaging should enable the detection of signalling in neuron populations distributed in all three spatial dimensions and in behaving animals.
The appeal of in vivo cellular imaging to any neuroscientist is not hard to understand: it is almost impossible to isolate individual neurons while keeping them and their complex interactions with surrounding tissue intact. These interactions lead to the complex network dynamics that underlie neural computation which, in turn, forms the basis of cognition, perception and consciousness. In vivo imaging allows the study of both form and function in reasonably intact preparations, often with subcellular spatial resolution, a time resolution of milliseconds and a purview of months. Recently, the limits of what can be achieved in vivo have been pushed into terrain that was previously only accessible in vitro, due to advances in both physical-imaging technology and the design of molecular contrast agents.
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W.D. holds a patent on two-photon microscopy and receives royalties.
The deflection of light by particles with a deviating refractive index.
- Refractive index
A property of materials that governs the speed of light as it travels through the material and the deflection of light as it crosses boundaries between materials.
- Molecular absorption
A process by which the energy of a photon is used to elevate a molecule to a higher internal energy level. The photon itself is eliminated.
The emission of a photon by a molecule while the molecule undergoes a transition from an elevated energy state to a lower energy state.
The alteration or destruction of biological molecules as a result of photo-oxidative side effects of chromophore excitation.
- Signal-to-noise ratio
The ratio between signal size and measurement noise.
- High-resistance (sharp) micro electrode
A glass pipette with a tip diameter of less than 100 nm that is filled with saline and used to penetrate cells to gain electrical access to the cell interior.
- Tight-seal electrodes
Patch pipettes that are sealed to the plasma membrane and used to carry out electrical recording of the intra-cellular voltage.
- Functional MRI
(fMRI). The detection of changes in regional brain activity through their effects on blood flow and blood oxygenation which, in turn, affect the brightness of magnetic-resonance images.
- Cell-attached recording
Extracellular electrical recording of a neuron's spiking using a glass patch pipette sealed to the outside of the plasma membrane, without gaining access to the cell interior.
- Patch recordings
Electrical recordings made using a glass pipette with a tip diameter of approximately 1 micrometre that is filled with saline and used to form gigaohm seals on the cell membrane. By removing the membrane patch inside the pipette, electrical access to the cell interior can be gained.
- Numerical aperture
A measure of the angular spread of the light rays that emerge from or are able to enter an objective lens.
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