Journal: Scientific Reports

Article Title: Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets

doi: 10.1038/srep26159

Figure Lengend Snippet: ( 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): NA max and NA min ; 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.

Article Snippet: A multiband emission filter designed for GFP and mCherry (Semrock) is placed in front of the tube lens to block unwanted illumination from the excitation laser.

Techniques: CRAfT Assay, Generated