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Axial resolution describes the ability to distinguish features separated along an imaging system's axis. In optical microscopy, the three-dimensional point spread function is usually longer along that axis than across it, so two layers can overlap even when comparable sideways separations are distinguishable.

A small fluorescent bead moved through focus provides an experimental response curve: record the detected intensity at successive axial positions and measure a stated width, such as its full width at half maximum. The result depends on numerical aperture, wavelength, specimen refractive index and the detection method. Confocal microscopy can reject much of the signal from outside the focal plane, improving optical sectioning.

Optical sectioning selects signal from a thin depth range of a specimen without physically cutting it. Repeating the measurement at successive focal depths gives a stack of sections from which three-dimensional organization can be reconstructed.

In a scanning confocal microscope, illumination is focused into the specimen and a detection pinhole rejects much of the fluorescence originating away from focus. A thick fluorescent specimen imaged with a wide-open and then a reduced pinhole demonstrates the rejected background; reducing the pinhole also discards useful photons, so section thickness and signal-to-noise ratio must be considered together.^[ref]

Start with the biological quantity to measure: compartment location, molecular colocalization, fast cell dynamics, deep-tissue activity or nanometre-scale organization. Specify the required field, depth, lateral and axial resolution, temporal sampling and acceptable perturbation before choosing the instrument.

For a thin fixed specimen or a shallow live preparation, confocal microscopy provides flexible multicolour imaging and effective optical sectioning with widely available labels. Adjust the pinhole and dwell time against signal-to-noise ratio, bleaching and speed. Repeatedly scanning a large volume can become too slow, and out-of-focus excitation can damage regions that contribute little useful signal.

For a living scattering specimen, two-photon excitation microscopy often offers better access to depth and confinement of excitation. Longer excitation wavelengths scatter less strongly in many tissues, and broad collection can recover scattered emission without a detection pinhole. These benefits do not eliminate depth-dependent optical aberrations, absorption, heating or focal phototoxicity. Pulsed excitation can also make instrumentation and suitable multicolour excitation more demanding.

If the priority is rapid, gentle volumetric observation of a suitable specimen, consider light-sheet fluorescence microscopy rather than forcing either point-scanning method to cover every use case. If subdiffraction molecular organization is required, consider a suitable super-resolution method and assess its labeling, speed and dose costs.

Finally, calibrate the point spread function in a sample-relevant medium, sample finely enough in all three dimensions, and check registration between colour channels. Match photon collection and exposure to the question, with controls for label-induced changes and photobleaching. The best method meets the biological resolution and timing requirements with sufficient signal and acceptable perturbation; the smallest nominal resolution number alone does not decide.

Two-photon excitation microscopy excites fluorescence through the nearly simultaneous absorption of two photons. In the weak unsaturated regime, the excitation rate is proportional to the square of the local illumination intensity. Focusing a pulsed laser therefore strongly favors excitation near its focus rather than throughout the illuminated path.

Scanning that focus provides optical sectioning without relying on a detection pinhole to reject all out-of-focus emission. The photons can have lower individual energies than a single excitation photon, but their combined energy must match an allowed transition. High peak intensity, bleaching and phototoxicity remain relevant; longer excitation wavelength alone does not guarantee less damage.^[ref]