Super-Resolution Imaging System. Professionals serve professionals. Three-Dimensional Simultaneous Multi-Channel Super Resolution Imaging

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1 Professionals serve professionals Super-Resolution Imaging System Three-Dimensional Simultaneous Multi-Channel Super Resolution Imaging NanoBioImaging Ltd.

2 Powerful Tool of Scientists Super-resolution microscopy has become a powerful imaging tool for biology and material science. To obtain reliable and high-resolution imaging results, it requires not only the knowledge of biology, but also the knowledge of physics, chemistry, computer science, and engineering. Yet, it has been challenging for most scientists. In collaboration with HKUST Super-Resolution Imaging Center, NanoBioImaging (NBI) has built an interdisciplined expert team covering optics, computer science, biology and chemistry. NBI offers customized products to our clients and comprehensive support including hardware maintenance, software update, protocol design, data analysis, and on-site and on-line troubleshooting services in all aspects. Stable and efficient hardware Professional analysis software Comprehensive technical support Your Best Choice User-friendly software Easy and seamless workflow Running in general lab conditions, even at anexhibition Advantages 20 nm resolution 10 times greater than conventional optical microscopes Simultaneous multi-channel imaging Active locking system with nanometer precision Optimized imaging buffer No warm-up time required Time-efficient, minimizing photobleaching Highly stable and efficient Balanced photochemical properties for different channels Time-saving 01

3 Stable and Efficient Hardware Most stable and reliable NBI developed active locking system to correct the sample drifting in real-time during data acquisition. The samples can be locked with nanometer precision and no post-acquisition correction is required. This not only guarantees reliable results without artifacts caused by software correction, but also saves users' time waiting for system stabilization and thermal equilibrium after each sample loading. Highly efficient NBI featured simultaneous multi-channel imaging delivers super resolution images of multiple targets at the same time, shortening the data acquisition time to 1/3 of other commercial products. What's remarkable, it minimizes photobleaching which is severe in sequential super-resolution imaging of multiple channels. 02

4 Intelligent and User-friendly Software Rohdea is a intelligent and user-friendly super-resolution imaging software developed by NBI. It produces reliable data and provides users with the best operation experience. Simplified imaging process Real-time and transparent parameter adjustment Up-to-date algorithms and real-time data reconstruction Convenient user data management Strong data analysis features 1 Simplified imaging process "What You See Is What You Get", simplifies operation process, and is user-friendly. We summarize the imaging process into four steps of the super-resolution imaging: "choosing region"- "widefield capture"-"parameter adjustment"-"super-resolution capture". 2 Real-time and transparent parameter adjustment The imaging results are subject to the parameters used in the data acquisition process. Rohdea provides a real-time parameter adjustment module that allows users to visually compare the effects of parameter changes on imaging results. 3 Up-to-date algorithms and real-time data reconstruction Rohdea integrates multiple up-to-date algorithms and functions for data acquisition and data processing. In particular, the results and parameters of SR image reconstruction are presented in real time, which is convenient for users to adjust in time. 4 Convenient user data management Rohdea allows the generation, modification, and automatic saving of parameter settings and imaging results. Besides, it provides parameter template for similar experiments, which is convenient for users to select and optimize in subsequent experiments. 5 Strong data analysis features Rohdea implements algorithms including clustering analysis, co-localization analysis, distance measurement, image processing, etc., delivering results that can be directly used for publications. 03

5 Full Service Professional training-more than operating the instrument Experts from NBI team cover physics computer science, biology and chemistry. NBI is ready to share our knowledge of principles, mechanisms and practices in all aspects of super-resolution microscopy with our customers. Comprehensive support 1 Super resolution imaging solution With our varied technical background and extensive imaging experiences, NBI experts help users imaging kick off right away by providing project consultation, feasibility analysis, protocol design etc.. 2 Sharing our knowledge base of sample preparation and imaging solutions We share our optimized sample preparation protocols and imaging configuration templates with our users, with the aim of helping they obtain the best imaging results within the shortest time. 3 Online and on-site support beyond maintenance Our technical support not only includes hardware maintenance, but also on-line and on-site consultation on rational of super-resolution fluorescent imaging, experimental protocols, data analysis and modeling, manuscript drafting, etc.. 4 Long-term collaboration-growing up together with users NBI is willing to collaborate with customers in a long-term manner. We will provide customers our latest techniques. We are also willing to meet your individualized technical demand. 5 Imaging service Consultation and sample delivery For free trails, please contact imagingservice@nbi.hk Clients Results delivered within one week NBI 200 nm Best images and reports mitochondrial inner and outer membranes synapse 200 nm (Images acquired with NBI SRiS 2.0, in cooperation with Herrup Group in HKUST) 04

6 Examples of Application Fields of application Cell biology Neuroscience Microbiology Immunology Nanomaterials Subcellular structures Protein localization Protein-protein interaction Chromatin organization Neuron Synapse Widefield Super-resolution 200 nm 200 nm Figure 1. Super-resolution imaging of neuron synapse in mouse brain slices. (Fig. a, c & e) Widefield images of the synapse structures. (Fig. b, d & f) Super-resolution images showing the synapse and well-separated pre-synapse & post-synapse structures. 200 nm 200 nm (Images acquired with NBI SRiS 2.0, in cooperation with Herrup group in Hong Kong University of Science and Technology) Nucleic acid-fish (a) Widefield Super-resolution (b) (c) Figure 2. Three-dimentional super-resolution images of telomere DNA. (Fig. a) Widefield image of single focus formed by telomere DNA. (Fig. b & c) Super-resolution images of two telomere foci, corresponding to the foci within the white boxes in the widefield image. Super resolution images show detailed structures, of which the changes within each focus could be easily identified. (Images acquired with NBI SRiS 2.0, in cooperation with Yong Zhao group from Sun Yatsen University) Organelles (a) (b) Figure 3. Three-channel super-resolution images of COS7 cell organelles. (Fig. a) Mitochondrial outer membrane protein TOM20 (magenta), mitochondrial matrix protein COX IV (blue), and Golgi protein TGN46 (green). (Fig. b) Mitochondrial outer membrane TOM20 (red), nuclear membrane protein lamin (blue), and Golgi protein TGN46 (green). (Images acquired with NBI SRiS 3.0 by NBI) 05

7 Examples Of Application Golgi Apparatus Figure 4. Super-resolution images of cis-golgi network protein GM 130 and trans-golgi network protein Golgin 97. (a) Widefield image shows the overlapping of these two proteins. (b) Super-resolution image clearly shows that the two proteins locate at two layers of golgi apparatus. (Images acquired with NBI SRiS 2.0, in cooperation with Guo Lab in Hong Kong University of Science and Technology) Yeast Organelle Figure 5. Super-resolution images of tubulin proteins Tub1 and Tub4 in yeast spindle-pole body. GFP and RFP tagged tubulin proteins were transfected into the yeast cell, and further stained with GFP nanobody and RFP nanobody, respectively. (Fig. a & b) widefield images. (Fig. c & d) Corresponding super-resolution images, two-layer structure of γ-tubulin complex was resolved. (Images acquired with NBI SRiS 2.0, in cooperation with Dr Yuanliang Zhai in Hong Kong University of Science and Technology) Mitochondria Figure 6. 3D Three-dimensional super-resolution images of mitochondria in COS7 cell. The mitochondrial outer membrane TOM20 was labelled indirectly. The mitochondria showed hollow tube structure. (Images acquired with NBI SRiS 2.0 by NBI) 06

8 Selected Publications by NBI SRiS Users A. Cheng, T. Zhao, KH. Tse, HM. Chow, Y. Cui, L. Jiang, S. Du, MMT. Loy, K. Herrup, "ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations." Proc Natl Acad Sci U S A Jan 9;115(2):E292-E301. H. Huang, X. Lin, Z. Liang, T. Zhao, S. Du, M.M.T. Loy, K.-O. Lai, A.K.Y. Fu, & N. Ip, Cdk5-mediated phosphorylation of liprina1 is critical for neuronal activity-dependent synapse development, Proceedings of the National Academy of Sciences Aug 15;114(33):E6992-E7001. Au Franco K.C., Jia Y., Jiang K., Grigoriev I., Hau Bill K.T., Shen Y. H., Du S.W., Akhmanova A., Qi Robert Z.. GAS2L1 Is a Centriole-Associated Protein Required for Centrosome Dynamics and Disjunction. Developmental cell Jan 9;40(1): X. Gu, E. Zhao, T. Zhao, J.W.Y. Lam, S. Du, M.M.T. Loy, & B.Z. Tang, A mitochondrion-specific photoactivatable turn-on bioprobe for super-resolution imaging under physiological conditions without additives, Advanced Materials, 2016, 28 (25), H. Wang, X. Zhuang, X. Wang, A.H.Y. Law, T. Zhao, S. Du, M.M.T. Loy, & L. Jiang, A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation. Plant Physiol Oct;172(2): G. Vail, A. Cheng, Y.R. Han, T. Zhao, S. Du, M.M.T. Loy, K. Herrup, & M. R. Plummer, ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity," J Neurophysiology, 2016, 116 (1), Y. Du, H. Zhang, M. Zhao, D. Zou, and C. J. Xue, Faster super-resolution imaging of high density molecules via a cascading algorithm based on compressed sensing, Opt. Express 23, (2015). T. Zhao, Y. Wang, Y. Zhai, X. Qu, A. Cheng, S. Du, & M.M.T. Loy, A user-friendly two-color super-resolution localization microscope, Opt. Express 23, 1879 (2015). 07

9 System Configuration SRiS 2.0 Series Two-Channel Standard Two-Channel Advanced Two-Channel Professional scmos detector scmos detector scmos detector Microscope Microscope Microscope Nano XYZ stage (Manual) Nano XYZ stage (Auto) Nano XYZ stage (Auto) Opto-split ii Opto-split ii Opto-split ii Optical element Optical element Optical element 656 and 750 nm lasers 656 and 750 nm lasers 656 and 750 nm lasers (Advanced) Channel filter Channel filter Channel filter Workstation Workstation Workstation Optical table Optical table Optical table NBI Active Locking System element NBI Active Locking System element NBI Active Locking System element NBI outlook NBI outlook NBI outlook NBI automatic control system NBI automatic control system NBI automatic control system NBI Rohdea 3.0 (Standard) NBI Rohdea 3.0 (Standard) NBI Rohdea 3.0 (Advanced) SRiS 3.0 Series Three-Channel Standard Three-Channel Advanced Three-Channel Professional scmos detector scmos detector scmos detector Microscope Microscope Microscope Nano XYZ stage (Manual) Nano XYZ stage (Auto) Nano XYZ stage (Auto) Opto-split iii Opto-split iii Opto-split iii Optical element Optical element Optical element 561, 656, and 750 nm lasers 561, 656, and 750 nm lasers 561, 656, and 750 nm lasers Channel filter Channel filter Channel filter Workstation Workstation Workstation Optical table Optical table Optical table NBI Active Locking System element NBI Active Locking System element NBI Active Locking System element NBI outlook NBI outlook NBI outlook NBI automatic control system NBI automatic control system NBI automatic control system NBI Rohdea 3.0 (Standard) NBI Rohdea 3.0 (Standard) NBI Rohdea 3.0 (Advanced) 08

10 SEEING IS BELIEVING Tel Web Address (852) (86) Rm 505, Biotech Centre 1, No.9 Science Park West Avenue, Hong Kong Science Park, Shatin, N.T., Hong Kong