Characterisation of Bessel and Gaussian beam illumination modes on LSM imaging quality of early chick embryo development Ricardo F Bango Da Cunha Correia1, Sascha L Reidt2, Antti I Karjalainen1, Manli Chuai1, Michael P MacDonald2, Cornelis J Weijer1 1School of Life Sciences & 2Dept. of Physics, University of Dundee, Dundee, UK Introduction Results & Discussion We have previously implemented a scanned light sheet microscope dedicated to imaging of large flat samples, in particular chick embryos [1]. The main results are that use of the camera LSSM slightly improves image quality in the case of Gaussian beam illumination, by suppression of out of focus light and scattered light. LSSM is absolutely necessary to obtain acceptable images using Bessel beam illumination. The images below illustrates the advantage of using the camera LSSM (B) compared to camera conventional mode of operation (A), for a Bessel light sheet. B z y x 45° ~2000 images ~2-3 minutes (12-14 frames/s) ~ 2GB C t ~20hours ~1TB Illumination axis Detection axis Light sheet Sample A A B During development the chick embryo gets progressively thicker and much more curved in 3D space. Currently the Gaussian illumination allows visualization of approximately 15µm deep into the embryo. Investigation of later developmental stages would greatly benefit from increased light penetration and an extended focus of the light sheet. Recent proof of principle studies have suggested that Bessel and Airy beams could provide superior depth of penetration and extension of the Rayleigh range of the light sheet [2, 3, 4]. We have now implemented a Spatial Light Modulator (SLM) generated Bessel beam and combined this with image acquisition using the light sheet scanning mode of the sCMOS camera to partially reject scattering and the contribution of the Bessel beam side lobes [4]. Our experiments also have highlighted principle difficulties in obtaining good synchrony between the camera light sheet scanning mode and the scanned light sheet at multiple positions along axial axis of the light sheet simultaneously, limiting effective range of the extended focus. The images below show a chick embryo cross-section imaged with Gaussian beam and camera LSSM (A) and Gaussian beam and conventional camera mode (B). The LSSM image (A) shows a reduction of scattered light and an improvement of contrast. A B 100 µm Bessel & Gaussian Beams – Light Sheet The Bessel beam is generated with an SLM as indicated in the diagram below. The images below show the image quality improvement using Bessel beam after implementation of LSSM (A) compared to conventional camera exposure mode (B). AOTF – Acousto Optic Tunable Filter FM – Flip-Mirror M – Mirror Li – Lens (i=1,2,3,4,5) ½ WP – Half Wave Plate SLM – Spatial Light Modulator P – Pinhole SL – Scan Lens TL – Tube Lens IO – Illumination Objective C – Chamber, to hold the embryo 3D-S – 3D Stage DO – Delivery Objective F - Filter A B 100 µm Images of the chick embryos surface using the camera LSSM (B and D) and the camera conventional mode (A and C) are shown below. Images A and B were acquired with Gaussian illumination. Images C and D were acquired with Bessel illumination. Bessel illumination images benefit extensively from using the camera LSSM. The images showing the light sheet and the beam profiles (A and B) generated with Gaussian beam illumination and the bottom images (C and D) are generated with Bessel beam illumination. The red rectangle illustrates the Rayleigh range. 5 µm 100 µm A B C D 50 µm A B C D sCMOS Light Sheet Scanning Mode (LSSM) In our current system the Scanner triggers the sCMOS Camera to acquire an image. The camera chip is exposed during the time the light sheet (blue line) is moving rapidly from left to right or vice-versa through the sample. Recent sCMOS cameras are provided with a new image acquisition mode suitable to scanned light sheet microscopy. The camera readout is synchronised and limited to the area illuminated by the scanned light sheet (A, B and C). Conclusion A B C Camera LSSM greatly improves images acquired using a scanned Bessel beam. The improvement of the image is immediate, without applying any post processing such as filtering or deconvolution. There are minor improvements in contrast but none in imaging depth in the images acquired with a scanned Gaussian light sheet and LSSM. Gaussian beams show better resolution and higher contrast but the extended focus of Bessel beams may be helpful in specific experimental conditions. Only the column of pixels synchronised with the light sheet is activated to capture the image while the remaining pixels are off. Acknowledgements References This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 608133. [1] Rozbicki, E., et al., Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation. Nat Cell Biol, 2015. 17(4): p. 397-408. [2] Vettenburg, et al., Light-sheet microscopy using an Airy beam. Nature Methods, 2014. 11: p. 541-544. [3] Gao, et al.,3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy Nature Protocols, 2014. 9: p. 1083-1101. [4] Fahrbach, et al., Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media. Nature Communications, 2012. 3(632).