Past exam of the systems biology course of the University of Cambridge / 2024 / Paper 2 / 6 / ii

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.