Figure 1
Comparison of SPIM, mSPIM, DSLM, and mDSLM architectures. (a) The optical layout of selective plane illumination microscopy (SPIM) is shown9. A cylindrical lens is used to create a focal line at the back focal plane (BFP) of an illumination objective, generating a static 2D light sheet within the sample. (b) A zoomed-in view of the illumination path within the sample. Due to a lack of angular diversity in the 2D light sheet, strong shadowing artifacts are visible due to occlusions. Widefield camera detection is used (dashed green box in panel b) to image the static 2D light sheet. (c) Multidirectional selective plane illumination microscopy (mSPIM) uses a pivoting mirror, located at a conjugate front focal plane (FFP*), to translate the line focus across the BFP of the illumination objective18. (d) This results in a 2D light sheet that rotates within the sample (in the yz plane) to average out the shadowing artifacts over time. (e) Digital scanned light-sheet microscopy (DSLM) uses a circular Gaussian pencil beam at the BFP of the illumination objective, and a scanning mirror located at a conjugate back focal plane (BFP*), to translate the pencil beam within the sample in the y direction, creating a 2D light sheet over time5. (f) To achieve a long Rayleigh range (a long depth of focus) a relatively low NA is used, resulting in minimal angular diversity in the x and y directions and similar (though slightly reduced) shadowing artifacts as SPIM. As the pencil beam in DSLM is scanned in y, it is synchronized with the rolling shutter of a sCMOS camera (dashed green lines), which acts as a confocal slit to reject background light and improve image contrast19,20. (g) Multidirectional digital scanned light-sheet microscopy (mDSLM) is similar to DSLM, with the addition of a cylindrical telescope to generate an elliptical Gaussian beam at the BFP of the illumination objective such that NAy is increased while NAx is unaltered. This results in a long Rayleigh range in the x direction (a light sheet that maintains its thickness over a relatively long propagation distance), but with increased angular diversity in the y direction to mitigate shadowing artifacts. The illumination and detection characteristics of the SPIM, mSPIM, DSLM, and mDSLM architectures are symbolically depicted at the bottom of panels (b,d,f and h).
