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What are the spatial (lateral and axional), and temporal resolutions that one is expected to get with the following optical imaging techniques?

  • Optical Bioluminiscence Imaging (BLI)
  • Optical Fluorescence Imaging (FLI)
  • Multiphoton Microscopy (e.g. 2-photon microscopy)

Are they any better than the others in providing higher resolutions?

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1 Answer 1

The lateral and axial resolution depends on the numerical aperture of your objective (lateral = lambda/2NA; axial = 2lambda/NA^2). You can improvee the axial resolution with FLI by using a confocal approach or with 2-photon microscopy (2PM). In 2PM you need absorption of two infrared photons to get emission. The probability of 2 infrared absorption depends quadratically on the excitation intensity. Therefore, fluorescence is restricted where the illumination beam is focused which give a better sectioning. An advantage of 2PM is the higher penetration depth and reduce phototoxicity. Indeed you can use 2 infrared photons to excite fluorophore which required visible excitation.

Temporal resolution depends on the area you want to measure and the integration time.

Resolution is limited by the diffraction. In the best case with the 3 imaging techniques you mentioned you can get ~200-300 nm in lateral resolution and 500 - 800 nm in axial resolution Higher resolution can be achieved by using super resolution techniques. They are separated in two groups. The first one exploid the photo-physical properties of the fluorophore to excite and de-excite it. This allows to resolve sub-diffraction areas. This is the case STED (Stimulated emission depletion) microscopy or SSIM (saturated structured illimination microscopy). The second group use the fluctuation of the fluorophore emission to localize it better. It includes techniques such as STORM (STochastic Optical Reconstruction Microscopy, PALM (Photo-Activated Localization Microscopy) and SOFI (super-resolution optical fluctuation imaging).

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