Figure 1
From: Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets
Generation of LBS with a single slit and an annulus.
(a) A simplified schematics illustrating LBS generation. L1: Collimating lens; CL1 and CL2: a pair of cylindrical lenses shaping the beam appropriately to maximize the energy passing through the single slit; L2: Fourier transform lens; EO: Excitation objective; DO: Detection objective. The beam is defined as propagating along y-axis. Using custom designed annuli one can craft LBS optimized for (b) ultralow phototoxicity (LBS1) or (c) high resolution (LBS2). In (b,c) from left to right: the annulus used and its numerical apertures (NA): NAmax and NAmin; the diffraction pattern after the annulus; the cross-section of the LBS generated at sample plane; the intensity plot (blue) along the dotted yellow line and the axial point spread function of the NA = 1.1 detection objective (red); and the axial plot of overall point spread function. (d,e) show the experimentally generated LBSs at different wavelengths (488 nm for GFP and 560 nm for mCherry). The point spread functions were measured with 20 nm fluorescent beads [left two columns in (d,e)], which are in good agreement with the theoretical plot in (b,c). (f) The measured FOV profile of Gaussian light-sheet, LBS1 and LBS2. The Gaussian light-sheet is generated by taking away the annulus and confining the maximum NA to 0.5 so that the beam waist has a thickness of 600 nm, which is comparable to the thickness of LBS1. Scale bars in (d,e): 5 μm.
