LIGHT SHEET BASED FLUORESCENCE CORRELATION AND CROSS-CORRELATION SPECTROSCOPY FOR QUANTITATIVE MEASUREMENTS OF BIO-MOLECULES IN LIVE CELLS ANAND PRATAP SINGH A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 LIGHT SHEET BASED FLUORESCENCE CORRELATION AND CROSS-CORRELATION SPECTROSCOPY FOR QUANTITATIVE MEASUREMENTS OF BIO-MOLECULES IN LIVE CELLS ANAND PRATAP SINGH (M.Sc Chemistry, BHU, INDIA) NATIONAL UNIVERSITY OF SINGAPORE 2014 is Th pa ge in io nt te lly na ft le nk bl a Declaration The work for this thesis was conducted under a collaboration between Assoc. Prof. Dr. Thorsten Wohland, NUS, Singapore and Prof. Dr. Jörg Langowski, DKFZ, Germany. All the research work has been performed at CBIS NUS, unless its mentioned. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Anand Pratap Singh Signature The results have been partially published in 1- Anand Pratap Singh† , Jan Wolfgang Krieger† , Jan Buchholz, Edoardo Charbon, Jörg Langowski, and Thorsten Wohland; The Performance of 2D Array Detectors for Light Sheet Based Fluorescence Correlation Spectroscopy, Opt. Express. 2013. 2- Jan Wolfgang Krieger† , Anand Pratap Singh† , Christoph S. Garbe, Thorsten Wohland, and Jörg Langowski; Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS), Opt. Express. 2014. 3- Anand Pratap Singh and Thorsten Wohland; Applications of Imaging FCS, Curr. Opin. Chem. Biol., 2014. † A. P. Singh and J. W. Krieger contributed equally to this work. iv Acknowledgements Foremost and above all, my sincerest gratitude to my Ph.D supervisor Assoc. Prof. Dr. Thorsten Wohland for his constant support, patience, and encouragement to make this thesis successful. Although I had my training in experimental physical chemistry, his constant thoughtful ideas improved my understanding of optics and microscope building. It is a great pleasure and experience to work with you and your team. I really appreciate, your positive attitude towards students and academic profession. Besides my supervisor, I would like to thanks Prof. Dr. Jörg Langowski for his constant support, valuable discussions, and thanks for hosting me for two weeks lab visit, it was a great experience to meet your lab members. I would also like to thank Dr. Katalin Tóth for her help during my DKFZ visit. I am particularly grateful for the assistance by Jan Krieger. I enjoyed and learned by all long conversation on building microscope, discussions on measurements and thanks for arranging everything during DKFZ trip. I thank Ms. Agata Pernus for sharing protocols and the discussions on cell measurements. I thank Dipanjan Bhattachrya for helpful discussions and support during the last four years. All TW lab members Ma Xiaoxiao, Wang Xi, Nirmalya Bag, Sun Guangyu, Angela Koh, Huang Shuangru, Sibel Yavas, Andreas Karampatzakis, Shi Hua Teo, Shiying Lim, Patrick Kramer, Kaijie Herbert Fan, and Kumaravel Kandaswamy for their critical comments, support and fun time throughout the last four year. I also thank to my senior lab members, Jagadish Sankaran for imaging FCS data fitting, Foo Yong Hwee for teaching confocal FCS, and Tapan Kumar Mistiri for teaching me cell culture. Radek Machánˇ for his lunch time chat on interesting v scientific, social and cultural issues, I really enjoy it. And special thanks to SPIM users Antonija, Adam, Angela, Xuewen, Andreas, and Kumar. I would like to acknowledge financial, academic and technical support from department of chemistry and center for bio-imaging sciences, particularly Madam Suriawati Bte Sa’Ad for all official matters. This graduate study would not have been possible without the NUS Graduate Fellowship during last four years. I would like to thank for all supports from Hamamatsu Photonics, Keybond technology for loan of ORCA-Flash4.0and Dynamic Analysis System Pte Ltd, Singapore for loan of SA-05commercial CMOS. I thank Prof. Dr. Sudipta Maiti, Dr. Ming Cherk Lee, Dr. A S Rama Koti, Dr. Jyotishman Dasgupta, Dr. Biswajit Maiti and my school teacher Ronald Rodrigues, they all have been a wonderful teacher and mentor at different levels of my academic career, thanks a lot for your constant help and support. To all my friends, Ajay, Anuradha, Debanjan, Jasmine, Kuldeep, Rahul, Subha, Veer Bhadur, Zubair and many more for their company and great fun together. This would not have been possible without your love, support, and friendship. You guys are simply awesome! Last but not least, I would like to thank my family members; my parents, sisters and brother for their all constant support, love and encouragement throughout my life. vi Abstract Cellular processes occur over a wide range of spatial ( nm - mm) and temporal scales ( µs - min). However, most microscopy methods not provide sufficient spatio-temporal resolution to cover this range. Therefore, I introduce here the combination of light sheet microscopy (single plane illumination microscopy SPIM) and fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS). SPIM-FCS/SPIM-FCCS allows measuring concentration, diffusion, transport, and interactions maps in a true imaging mode with single molecule sensitivity. The method provides diffraction limited spatial resolution with subms temporal resolution which is sufficient to quantify the dynamics of membrane, cytosolic and nuclear proteins in living cells and organisms. In this work I provide guidelines on the building and characterization of microscope setups, on the suitability of different cameras, on sample preparation and mounting and on the data analysis of this novel imaging FCS and FCCS methods and present applications to demonstrate its suitability for biophysical and biomedical studies. vii is Th pa ge in io nt te lly na ft le nk bl a Contents Declaration iv Acknowledgements v Abstract vii List of Tables xii List of Figures xiii List of Abbreviations and Symbols xv Introduction and Outline Fluorescence Correlation Spectroscopy: FCS 2.1 Introduction and Historical Background . . . . . . . . . . . . . . 2.2 FCS: A Tool to Measure Dynamics and Concentrations. . . . . . 11 2.3 Principles and Theoretical Background . . . . . . . . . . . . . . . 11 2.3.1 Diffusion coefficient and concentration . . . . . . . . . . . 18 2.3.2 FCS experimental setup . . . . . . . . . . . . . . . . . . 20 2.4 FCCS: A Measure of Bio-Molecular Interaction . . . . . . . . . . 21 2.5 Principles and Theoretical Aspects of FCCS . . . . . . . . . . . . 22 2.5.1 Spectral cross-talk corrected Autocorrelation and CrossCorrelation Functions . . . . . . . . . . . . . . . . . . . . 26 2.5.2 FCCS experimental setup . . . . . . . . . . . . . . . . . . 28 Single Plane Illumination Microscopy: SPIM 30 3.1 The Past and Present of Light-Sheet Microscopy . . . . . . . . . 30 3.2 Illumination Schemes . . . . . . . . . . . . . . . . . . . . . . . . 32 3.3 Light-Sheet Generation . . . . . . . . . . . . . . . . . . . . . . . 34 ix 3.3.1 GRIN lens: Objective-coupled planar illumination microscopy (OCPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.3.2 Cylindrical lens: Ultramicroscopy and SPIM . . . . . . . . 35 3.3.3 Digital scanned laser light-sheet microscope: DSLM . . . 36 3.4 Building protocol of SPIM . . . . . . . . . . . . . . . . . . . . . . 37 3.4.1 Step 1: Setting up the base . . . . . . . . . . . . . . . . . 37 3.4.2 Step 2: Mounting optics and holders . . . . . . . . . . . . 38 3.4.3 Step 3: Mounting and alignment of illumination optics . . 39 3.4.4 Step 4: Sample mounting unit . . . . . . . . . . . . . . . 42 3.4.5 Step 5: Detection optics alignment . . . . . . . . . . . . . 42 3.5 System instability and sources of vibration . . . . . . . . . . . . . 44 Light Sheet Imaging FCS: SPIM-FCS 48 4.1 SPIM-FCS: A Quantitative Bio-Imaging Tool . . . . . . . . . . . . 48 4.2 Theoretical Principles of Camera Based Imaging SPIM-FCS . . . 50 4.2.1 Theoretical aspect of imaging SPIM-FCS . . . . . . . . . 52 4.2.2 Molecule detection efficiency . . . . . . . . . . . . . . . . 53 4.2.3 SPIM-FCS fitting model . . . . . . . . . . . . . . . . . . . 54 4.2.4 Effective observation volume . . . . . . . . . . . . . . . . 55 4.3 Material and Method . . . . . . . . . . . . . . . . . . . . . . . . 56 4.3.1 Description of light-sheet microscope setup . . . . . . . . 56 4.3.2 Sample preparation . . . . . . . . . . . . . . . . . . . . . 57 4.3.3 Microchannel fabrication . . . . . . . . . . . . . . . . . . 57 4.3.4 Giant unilamellar vesicles . . . . . . . . . . . . . . . . . . 58 4.3.5 Cell culture protocol . . . . . . . . . . . . . . . . . . . . . 58 4.3.6 Sample mounting for SPIM-FCS . . . . . . . . . . . . . . 58 4.3.7 Light-sheet characterization . . . . . . . . . . . . . . . . . 60 4.4 Calibration of SPIM . . . . . . . . . . . . . . . . . . . . . . . . . 61 4.4.1 Volume overlap . . . . . . . . . . . . . . . . . . . . . . . 61 4.4.2 PSF determination 63 . . . . . . . . . . . . . . . . . . . . . x [122] L. 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J. 104, 553–564 (2013). 111 Appendix A Photograph of SPIM and alignment tools Photograph of SPIM F IGURE A.1: Photograph of SPIM-FC(C)S setup at CBIS, NUS Beam expander Neutral density filter Dual view optics 561 nm laser EMCCD camera 488 nm laser Detection objective Dichroics A.1 Cylindrical lens Relay Gimbal telescope mirror Illumination and detection direction Y Z D n tio c e et Illum X inat ion Projection objective Sample mounting unit Detection Sample objective mounting stage Projection objective 112 Sample chamber A.2 Beam characterization F IGURE A.2: Photograph of mirror used for the characterizing of light sheet. F IGURE A.3: Photograph of mirror for characterizing and visualizing the light sheet. 113 F IGURE A.4: Photograph of light sheet from top and side view. Projection objective Detection objective Sample chamber (filled with buffer) Projection objective Detection objective Light sheet Sample chamber (filled with buffer) A.3 Dual Channel Alignment by a TEM Grid F IGURE A.5: Photograph of TEM grid for aligning two channels on same camera sensor: 114 Appendix B The list of SPIM components Serial and product no. Local supplier and contact person Manufacturing company Remark and Description 488 nm LX 40 mW Laser 21 Dennis Wee dennis.wee@laser-21.com Coherent, Inc, Santa Clara Modulation is possible 561S-25-COL -PP 25 mW Acexon Pt. Ltd Lawrence Chua lawrence.chua@acexon.com Oxxius S.A. France Modulation is not possible Lasers Optomechanical components System rail Sys 65 S/N: 16.011.0300 16.011.0500 Optical rail to mount optical components ( 4/2 pc.) Sys 65 slides S/N: 16.021.0020 16.021.0040 Rail slides ( 20 pc.) Holder S/N: 14.110.0025 Objective holders (2 pc.) EINSR D25/RMS S/N: 14.711.2501 RMS objective mount (2 pc.) Translation stage S/N: 14.341.1300 Lens holders S/N: 14.711.2513 Analytical Technologies Mary Yan mary@analytical-online .com OWIS GmbH Germany Objective µm positioner mount (2 pc.) Cylindrical lens holder (1 pc.) Rotary stage DT40-D25 S/N:33.040.2550 Cylindrical lens rotator XY Slit S/N: 27.160.1212 Adjustable slit aperture (1 pc.) 115 Lens mount S/N: 16.202.0045 Lens holders (8 pc.) Gimbal mount S/N: 26.306.0382 Mirror mount (1 pc.) Nanopositioner S/N: P-721 PIFOC Kinematic Mirror mount S/N: 5MBM24-1-2 XYZlinear stages: 8MT184-13DC and rotation stage: 8MR174-1-20 ✂ Optomechanical components Detection Obj. positioner 100 nm resolution PI Singapore Lim Melvin M.Lim@pi.ws Acexon Tech. Pte. Singapore Lee King Chua leeking.chua@acexon.com SPIM sample chamber Standa Ltd. Vilnius Lithuania Custom made sample chamber Custom made sample chamber mounting collar AceXon Tech. Pte. Singapore Achromatic lens ④ φ 25 50 mm Edmund Optics Inc. Singapore Kelly Lee sgsales@edmundoptics.com.sg FL=40,150 mm φ 12.5 mm 75mm FL S/N 68-162 Filters SN: LL01-488R-25 FF01-536/40-25 FF01-612/69-25 Dichroic SN: LM01-552-25 Acexon Tech. Pte. Singapore Notch filter SN: NF561–18 Optical components mir- motorized linear x-, y- and z stages together with a rotation stage Physics workshop NUS Sample chamber mounting collar Mounting dichroic rors Semrock Inc New York Visible coated achromatic lenses laser beam expansion Achromatic cylindrical lens Laser selection green channel red channel laser beam combiner Avoids scattered 561 nm laser Thorlabs Inc, USA SLMPLN 20X /0.25 WD= 25 mm Low NA air projection objective Olympus Singapore Ravichandran Palaniappan p_ravi.osp@olympus.com.sg LUMPLFLN-W 60X/1.0 WD = 2.0 mm LU074700 FL= 180 mm High NA water dipping detection objective Objective tube lens Dual View DV2 Micro lambda Singapore Larry Chug larry.cheung@microlambda. com.sg Photometrics Tucson USA Dual view optics Shearing interferometer SN:SI050 Laser 21 Singapore Thorlab USA Laser beam collimation tester Table continued to next page . 116 Imaging sensor Andor iXon X3 860 Custom holders and mounts Miscellaneous Zugo photonics Singapore Xiao Yong xiao.yong@zugophotonics.com JEG Eng. Supplies Eddie Lee jegeddielee@yahoo.com.sg 1)- LVL02 2)- PSX501 3)- SH6MS25 4)- WPH10M-488 5)- WPH10M-561 6)- WFS150-5C 7)- SM1A7 8)- SI100 9)- NDC-100C-4 10)- S130C, PM100D Thorlabs Inc, USA Andor Technology Belfast, UK EMCCD sensor Sample mounts and holders 1)- Bubble leveler 2)- LED light 3)- M screw kit 4,5)- Half-wave plates for 488 and 561 lasers 6)- Laser wavefront sensor 7)- Laser target 8)- Shear plate 9)- Neutral density filter 10)- Power meter Table B.1: List of the optical and opto-mechanical components used for building the light-sheet microscope at National University of Singapore . 117 [...]... FCS and dual color FCCS measurements in vitro and in vivo More specifically, this particular research study begins with the building of a light- sheet microscope, followed by its characterization, calibration and point spread function (PSF) determination for both single FCS and dual color FCCS measurements In addition, its applications in solution and live cell measurements are discussed Light- sheet based. .. great interest, if a fluorescence imaging method can provide spatio-temporal dynamics and binding maps of proteins localized in membrane, cytosol and nucleus This particular study combines fast SPIM imaging (single plane illumination microscopy) and camera based FCS and FCCS (fluorescence cross -correlation spectroscopy) , which creates spatio-temporal diffusion and concentration maps of bio- molecules in. .. identify, and characterize the key players in biological systems to enable understanding in protein-protein interactions or protein-DNA interactions A number of techniques, including protein microarrays, pull down assays and chromatin immunoprecipitation have been used on a large scale, but often are error prone, require excessive experimental effort and provide no temporal information for living samples... are intrinsically restricted due to their technical complication in sample preparation Single molecule detection (SMD) and single particle tracking (SPT) offer the study of sparse particle trajectories, and rates of binding and dissociation in both in vitro and in vivo environments [74–77] They have been applied even to single small organic fluorophores [78] and fluorescent proteins [79] The advantage of. .. elucidate underlying mechanism at the molecular scale and its relation to the functions in organisms A variety of techniques have been developed to study protein dynamics and protein-protein interaction in vivo and in vitro [8] Fluorescence based methods provide high molecular specificity (chimeric fluorescent proteins), high signal to noise ratio, and usually can be performed in living cells and embryos... in vitro and in live cells Imaging SPIM-FC(C)S is a novel quantitative bio- imaging tool, it provides diffraction limited spatial resolution and the temporal resolution of 2, 500 fps for more than 3, 000 contiguous data points from a single experiment The main aim of the research was to build a light- sheet microscope, establish a calibration protocol and demonstrate its capabilities for both single color... challenge lying ahead for developmental biologists, cell biologists, molecular biologists and biophysicists is to link the information on the molecular scale to single cell responses to a functional level understanding in organs or small embryos (the intricate relationship between different scales of samples, embryo ðñ tissue ðñ single cell ðñ single protein) [3] No doubt, the genomewide ‘-omics’ are invaluable... protein dynamics play an important role in regulating various processes on the single cell to organism level [5, 6] The quantification of these physical parameters (esp dynamics, concentrations, and interactions) in a test-tube do not provide any insight into the spatio-temporal heterogeneity of the living system [7] Therefore, measuring protein dynamics and their interactions with other biomolecules in. .. rejects outof-focus fluorescence signal This made it possible to measure few molecules in the observation volume In the intervening four decades, several variants of FCS were developed and applied in chemistry, biology and medicine (see review on FCS and other fluctuation approach and their principles [95, 96] and recent application in various fields [97–100]) 2.3 Principles and Theoretical Background In FCS,... Schematic of illumination arm alignment 40 3.6 Sample mounting unit 43 3.7 Detection arm alignment 44 3.8 Typical correlation plots in presence and absence of vibration 45 3.9 Building and illumination unit of light- sheet microscope 47 4.1 Illustration of illumination and observation region 51 4.2 Principle of imaging FCS (SPIM-FCS) . LIGHT SHEET BASED FLUORESCENCE CORRELATION AND CROSS -CORRELATION SPECTROSCOPY FOR QUANTITATIVE MEASUREMENTS OF BIO- MOLECULES IN LIVE CELLS ANAND PRATAP SINGH A THESIS SUBMITTED FOR THE. DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 LIGHT SHEET BASED FLUORESCENCE CORRELATION AND CROSS -CORRELATION SPECTROSCOPY FOR QUANTITATIVE MEASUREMENTS. identify, and characterize the key players in biological systems to enable understanding in protein-protein interactions or protein-DNA interactions. A number of techniques, including protein microarrays,