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Comparison of diSPIM With Confocal

The following comparison was written with diSPIM in mind, but the main points apply to all types of SPIM/LSFM.

Spinning disk confocal (SDCM) is just a massively parallel confocal implementation so it's faster but otherwise has the same characteristics as laser scanning confocal.

  • Light dose: diSPIM has much lower dose and hence less photobleaching/phototoxicity than confocal. Reducing light dose is the main motivation for most SPIM users.
    • A rule of thumb is that you need X times as much light for confocal as you do for SPIM where X is the number of slices in your stack.
    • Figure 3 and Supplementary Video 2 of Wu et al. offer bleaching comparisons of imaging with diSPIM and SDCM. Other papers such as this one offer comparisons of light sheet with confocal which should apply to diSPIM.
  • Z resolution: diSPIM after registration/fusion has best Z-resolution (same as XY resolution). Next best is confocal, which in turn has better Z resolution than single-view SPIM.
    • Using Bessel beams to create the sheet can improve Z-resolution for single-view SPIM (“optical sectioning”), but won't meaningfully change dual view resolution after registration/fusion.
  • XY resolution: confocal and SPIM are comparable.
  • Speed: SPIM (per view) and SDCM have comparable speed if SDCM laser intensity is increased to compensate for the ~3% open area of the disks.
    • Camera readout speed bounds the maximum achievable frame rate. For example, 512 pixels high ROI is 2.5 ms readout time for sCMOS. Allowing 2.5 ms illumination time results in 5 ms total per image or 200 frames per second. Using a cylindrical lens instead of a scanned light sheet can reduce camera overhead.