Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets

We demonstrate a simple and efficient method for producing ultrathin Bessel (‘non-diffracting’) light sheets of any color using a line-shaped beam and an annulus filter. With this robust and cost-effective technology, we obtained two-color, 3D images of biological samples with lateral/axial resolution of 250 nm/400 nm, and high-speed, 4D volume imaging of 20 μm sized live sample at 1 Hz temporal resolution.


Supplementary Note 1: LBS1 and LBS2 designs
The input line-shape light can in principle be formed using a cylindrical lens or prism pair compressor without a slit, particularly for single color imaging. For multicolour application, however, the slit has the essential role to define the position of the line, independent of color. For this reason, a 200 micron width slit is used throughout this paper. LBS1 is designed to minimize photo-bleaching by reducing the number and intensities of sidebands, thus maximizing the portion of energy in the center peak, which can be achieved by increasing the width of the annulus, i.e., (NA max -NA min ). On the other hand, longer light sheet is obtained with smaller NA max , albeit at the expense of a thicker sheet [1]. With this in mind, for LBS1 we used annulus with NA max =0.3 and NA min =0.112, as shown in Figure 1b. The resulting LBS was 15 µm long and having only two weak side-bands, which can be suppressed by the axial detection point spread function, yielding an overall single band PSF of 600 nm (FWHM) axial resolution with exceptionally low phototoxicity (See Supplementary Figure 4 for more detail.) This 15μm long LBS is sufficient for most cell imaging applications; if needed longer LBS can be crafted by scaling the annulus to smaller dimensions. It is also possible to use phase elements to create multi-foci thus double or triple the LBS length without any change in the cross-section of LBS.
LBS2 aims to achieve higher axial resolution, using an annulus with NA max =0.4 and NA min =0.225, as shown in Figure 1c. The light sheet can propagate ~12 µm with a 400 nm thick central band, but flanked by multiple progressively weaker bands. The overall axial PSF consists of this ultrathin central band, plus two weaker side bands which can be removed by deconvolution (Supplementary Figure 2

.)
While the light sheet thickness is determined by the annulus, the width of the light-sheet (the dimension of LBS in x) is in proportion to the length of the single slit. The18mm long slit length used resulted in a 40μm wide light sheet that is sufficient for most cases.
We found that a slit width of 200 µm appear to be optimal for our particular setup allowing over 50% of exit-fiber laser power to reach the sample for LBS1, or 38% for LBS2 at 488 nm (47% and 35% at 560nm). Multicolor imaging can be done with a fixed set of slit and annulus.
We note that, non-diffracting patterns in any shape can be mathematically expressed in circular coordinates 2,3 (i.e. Bessel beams) or elliptical coordinates (i.e. Mathieu beams 4 ), and it is possible to extend the depth of any pattern by producing angular spectrum in the k-space with an annular mask 5 . LBS is one of the examples to produce extend light sheet with a pattern (a slit in this case) and an annulus and is thus given the name line Bessel beam. Figure 1 | System setups and control sequence. (a) System schematics of a LBS microscope. The fiber output was collimated and shaped by a pair of cylindrical lenses to maximize output through a 200 µm slit. An f=500 mm lens is used to perform Fourier transform after the single silt, and project the diffraction pattern onto an annulus. The image of the annulus is zoomed by 2.5 times via a telescope system before projected onto the back focal plane of excitation objective. The LBS is produced at sample plane 30° to the plane of coverslip, and right in the focal plane of the detection objective. With the relative position between the emission and detection objectives fixed, the coverslip is driven by a piezo stage, allowing specimen to be scanned step by step. The fluorescent emission is collected by the detection objective and captured by a sCMOS camera after the multi-band emission filter. (b) The triggering sequence used in LBS imaging. Camera running in synchronous readout mode generates high output during the global exposure and low during data transfer. Laser controlled by an AOTF is turned on only during global exposure. For multicolor imaging, the wavelength is switched for adjacent frames. The piezo stage triggered by the falling of global exposure moves a step forward after both colors are imaged.