長庚大學共軛焦顯微鏡課程 長庚大學共軛焦顯微鏡課程. Spot light 長庚大學

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1 長庚大學共軛焦顯微鏡課程 Spot light 長庚大學共軛焦顯微鏡課程 長庚大學 Basic principle of Laser Scanning Confocal Microscopy The application of LSM 510 META detector Multiphoton microscopy basic principle and introduction 台灣儀器行徐華蔓 Page 1 Page 2 30/10/2007 Anita Hsu Where does the out-of-focus light come from? The Point-Spread-Function of a Microscope Where does the out-of-focus light come from? The Point-Spread-Function of a Microscope Point-Spread-Function fluorescent point source Point-Spread-Function fluorescent point source image The image of a point is not a point. y z x The image of a point is not a point. It s a complex 3- dimensional diffraction pattern. y z x y x z Page 3 30/10/2007 Anita Hsu Page 4 30/10/2007 Anita Hsu Conventional/Widefield Fluorescence Background emission from deeper image planes Mouse Intestine Section Conventional/Widefield Fluorescence Structures which are out-of-focus become visible in conventional widefield-fluorescence. Because of the focal depth inherent in all objectives, they are visible as an image blur (haze, image fog). Page 5 30/10/2007 Anita Hsu Page 6 30/10/2007 Anita Hsu

2 Why do we need Optical Sections? The Fundamental Problem Optical Sectioning Methods in Light Microscopy Conventional Images Conventional images from 3-dimensional objects consists of light from structures, which are in focus and the light from structures which are not in focus. conventional image in-focus structures out-of-focus structures + x Laser Scanning Microscopy Optical Sectioning Methods Pure Optics Optics & Mathematics Pure Mathematics Total Internal Reflection Confocal Scanner Multi-Photon Nipkow-Systems STED-Microscopy Structured Illumination ApoTome 3D-Deconvolution Page 7 30/10/2007 Anita Hsu Page 8 30/10/2007 Anita Hsu 長庚大學 TIRF TIRF Static imaging Phalloidin F-actine binding proteins coupled to Alexa 546, localization actin filaments, cytoskeletal submembran cortex Dr. Klingauf, MPI for Biophysical Chemistry, Göttingen Page 9 30/10/2007 Anita Hsu Conventional Fluorescence TIRF Chromaffine Cells Page 10 30/10/2007 Anita Hsu TIRF Dynamic Imaging Movement of vesicles within a cell Optical Sectioning Methods in Light Microscopy Adhesion, Movement, Cell-Cell Communication, Transport processes Optical Sectioning Methods Pure Optics Optics & Mathematics Pure Mathematics Channel 1 Epi Laser Scanning Microscopy Total Internal Reflection Structured Illumination ApoTome 3D-Deconvolution Confocal Scanner Multi-Photon Channel 2 TIRF Nipkow-Systems STED-Microscopy Page 11 30/10/2007 Anita Hsu Page 12 30/10/2007 Anita Hsu

3 長庚大學共軛焦顯微鏡 LSM 510 META System Overview Motorized Microscope LSM Laser module Scan Head T-PMT T-Light FL-Light 1 Scan detection module 2 Upright or Inverted Microscope 3 LSM Laser module 4 Electronic Control Unit (ECU) 5 LSM Software 6 Computer hardware 1 Scan Module 2 5 diff.time:0.030 µs AOTF User PC HeNe 633 DPSS 561 AOTF 4 Ar+ 458/ 477/488/ AOTF 3 Electronic Control Unit 6 Page 13 30/10/2007 Anita Hsu Page 14 30/10/2007 Anita Hsu Confocal Microscopy Optical path of a high-end laser scanning microscope What s confocal? The confocal principle LSM: Laser Scanning Microscope LSM 510 META A minute diaphragm, situated in a conjugated focal plane, prevents out of focus light to be detected. PMT Θ detector pinhole Confocal Plane Laser Beam main dichroic beamsplitter The pinhole diameter directly controls the thickness of the optical section. Θ specimen Page 15 30/10/2007 Anita Hsu Page 16 30/10/2007 Anita Hsu Non-Confocal / Confocal image Non-Confocal / Confocal image Widefield Microscope Confocal Microscope Wide Field Confocal Page 17 30/10/2007 Anita Hsu Page 18 30/10/2007 Anita Hsu

4 The advantages of confocal microscope - Light source Full wavelengths of mercury lamp v.s. single wavelength of laser The advantages of confocal microscope - Emission filter Cascade Signals separation by single module v.s. Cascade separation by series filter set PMT More Than a CONFOCAL PINHOLE! Laser 405nm 458 / 477/ 488 / 514 nm 561nm 633nm Page 19 30/10/2007 Anita Hsu Page 20 30/10/2007 Anita Hsu The advantages of confocal microscope - Point probe scanning The advantages of confocal microscope -detector From Spot to Image To get a 2 dimensional image from the specimen, the excitation spot has to be moved over the specimen The scanning mirrors move the the excitation beam in a linewise fashion Wide Field Wild-field exposure CCD Confocal Scanner point scanning pinhole PMT detector X X/Y Image Focus Cone Y Specimen CCD chip Photomultiplier tubes (PMT) Page 21 30/10/2007 Anita Hsu Page 22 30/10/2007 Anita Hsu The Comparison Between the LSM and the Conventional Light Microscope Light Source Wide Field Microscope Mercury or Xenon Lamp Laser Scanning Microscope Laser 1) Confocal Imaging Optimal optical sectioning in multifluorescent samples Multiple Pinhole Concept Illuminated Field Image Acquisition Wide Field Parallel, Frame at Once Spot Sequential, Pixelwise 2) Laser and scanning mirror control Easy sample manipulation Flexible scanning strategies (1D to multid) FRAP, FLIP, photoactivation, acceptor bleach (FRET) Real-Time electronics real ROIs bleach ROIs Signal Separation Detector Dichroic Beam Splitter, Emission Filter Eye or CCD Camera Beam Splitter Cascade, Emission Filter Diffraction limited by pinhole Photomultiplier (PMT) 3) Multifluorescence Imaging Acquisition of several dye signals simultaneously (FPs etc.) Clear separation of fluorescent dyes without the danger of crosstalk Image channels that show only the signal from one fluorescent label Optimal quantitative imaging i.e. colocalization META Detector Emission Fingerprinting Page 23 30/10/2007 Anita Hsu Page 24 30/10/2007 Anita Hsu

5 1) Confocal Imaging Optimal optical sectioning in multifluorescent samples Optimal colocalization analysis (adaptable confocal volumes 0 µm 2 µm 4 µm 6 µm 8 µm 10 µm 12 µm 14 µm 16 µm 18 µm 20 µm 22 µm 24 µm 26 µm 28 µm Wide Field Confocal A series of of confocal images from different optical planes contains the image information from the whole specimen Page 25 30/10/2007 Anita Hsu Page 26 30/10/2007 Anita Hsu An overlay (maximum projection) of these single images results in an image with an enhanced depth of focus This image contains all information from the specimen Optimal optical sectioning in multifluorescent samples 1) Confocal Imaging Optimal optical sectioning in multifluorescent samples Every detail is in focus! Page 27 30/10/2007 Anita Hsu Page 28 30/10/2007 Anita Hsu Fluorescence Detection Strategies Detection of fluorescent and unlabeled structures Optimal colocalization analysis Different imaging modes: (A) Reflection (B) Fluorescence (C) Transmitted light (D) RGB overlay Unlabeled structures can be visualized in by detecting laser reflection or in transmission mode. Sample: Rhizome of Convallaria majalis, Ruscaceae A C B D 1)Confocal Imaging Optimal optical sectioning in multifluorescent samples unequal sections with single pinhole Detection volume Optical slice thickness equal sections with multiple pinhole concept Page 29 30/10/2007 Anita Hsu Page 30 30/10/2007 Anita Hsu

6 Colocalization in Confocal Microscopy Occurrence of two fluorescent emission signals inside the same detection volume Identical size of detection volumes for different color channels required Acquisition of Crosstalk free images required Optimal colocalization analysis 1)Confocal Imaging Optimal optical sectioning in multifluorescent samples Detection volume Optical slice thickness Intensities and position of the signals inside the detection volume may vary unequal sections with single pinhole equal sections with multiple pinhole concept Page 31 30/10/2007 Anita Hsu Page 32 30/10/2007 Anita Hsu Optimal colocalization analysis Quantitative Colocalization - Prerequisite d ~ P n λ/ (N.A) 2 detector Equal optical sections are absolutely necessary for Quantitative Colocalization unequal sections with single pinhole The thickness of the confocal sections varies with: Size of the confocal pinhole Wavelength of the light pinhole Multi-Pinhole Concept from Zeiss unequal sections with single pinhole equal sections with multiple pinhole concept Optical slice thickness Dyes Pinhole Optical slice DAPI, Hoechst 137 µm 1 µm egfp, FITC, Alexa µm 1 µm DsRed (RFP), Cy3, Rhod 132 µm 1 µm Cy5 128 µm 1 µm Page 33 30/10/2007 Anita Hsu Page 34 30/10/2007 Anita Hsu Optimal colocalization analysis Multiple pinhole concept Laser and scanning mirror control 2) Laser and scanning mirror control Easy sample manipulation Flexible scanning strategies (1D to multid) FRAP, FLIP, photoactivation, acceptor bleach (FRET) unequal sections with single pinhole equal sections with multiple pinhole concept Scan Mode --- 1D, 2D, and free 2D Image Multiple pinhole concept is essential for: Quantitative colocalization analysis Realistic 3D reconstructions U n i-d ire c tio n a l-s c a n D D S R o ta te d -S c a n R o ta te d D D S R a n d o m W in d o w - S c a n A rb itr.-r O I-S c a n Absolut linear scanner movement: The same dwell time for every pixel in the images (essential for any quantitative measurements) Page 35 30/10/2007 Anita Hsu Page 36 30/10/2007 Anita Hsu

7 Laser and scanning mirror control 2) Laser and scanning mirror control Easy sample manipulation Laser and scanning mirror control Line and Spline scan - High speed flexibility Line Scan Spline Scan Real Regions of Interest (rroi) Irregular shaped areas Up to 99 areas simultaneously Sample irradiation only during data Acquisition (beam blanking) No photobleaching in surrounding areas free positioning free rotation (0-360 o ) x, y, z-cutline, t free definable line geometry free positioning x, y, z-cutline, t Ideal for fast physiological processes e.g. Ca-Imaging, fast z-cutline (with HRZ) Ideal to follow irregular shaped biological structures (free-hand definition) z Developing drosophila embryo Bovin endothelial cells Page 37 30/10/2007 Anita Hsu Page 38 30/10/2007 Anita Hsu Laser and scanning mirror control Scan Mode --- 1D, 2D, and free 2D Image Two independent scanning mirrors Free scan field rotation (0-360 o ) Free online zooming (crop) Any geometry: 1x x2048 Faster rectangular acquisition (e.g. video rate) 1) Confocal Imaging Optimal optical 3D sectioning in thick tissue Optimal optical 3D sectioning in multifluorescent samples 2) Laser and scanning mirror control Easy sample manipulation Flexible scanning strategies (1D to multid) FRAP, FLIP, photoactivation,, acceptor bleach (FRET) 3) Multifluorescence Imaging Acquisition of several dye signals simultaneously (FPs etc.) Clear separation of fluorescent dyes without the danger of crosstalk Image channels that show only the signal from one fluorescent label Optimal quantitative imaging i.e. colocalization Page 39 30/10/2007 Anita Hsu Page 40 30/10/2007 Anita Hsu Real regions of interest (rroi) From Spot to Image AOTF Pixel-precise regions of interest (ROIs) Irregular shaped areas Up to 99 areas simultaneously Sample irradiation only during data acquisition (beam blanking) No photobleaching in surrounding areas To get a 2 dimensional image from the specimen, the excitation spot has to be moved over the specimen The scanning mirrors move the the excitation beam in a linewise fashion X X/Y Image Focus Cone Y Specimen Page 41 30/10/2007 Anita Hsu Page 42 30/10/2007 Anita Hsu

8 From Spot to Image Plane The display type of 3D images This plane represents an optical section X/Y/Z Stack Display along z- axis Z-Drive 3 D information is acquired by moving the excitation focus not only in XY direction but also in Z direction The result is a 3 D data stack consisting of number of XY images representing different optical sections from the specimen Page 43 30/10/2007 Anita Hsu Page 44 30/10/2007 Anita Hsu The display type of 3D images The display type of 3D images Page 45 30/10/2007 Anita Hsu Page 46 30/10/2007 Anita Hsu Perform the dimensional measurement 1) Confocal Imaging Optimal optical 3D sectioning in thick tissue Optimal optical 3D sectioning in multifluorescent samples 2) Laser and scanning mirror control Easy sample manipulation Flexible scanning strategies (1D to multid) FRAP, FLIP, photoactivation, acceptor bleach (FRET) 3) Multifluorescence Imaging Acquisition of several dye signals simultaneously (FPs etc.) Clear separation of fluorescent dyes without the danger of crosstalk Image channels that show only the signal from one fluorescent label Optimal quantitative imaging i.e. colocalization Multiple Pinhole Concept Real-Time electronics real ROIs bleach ROIs META Detector Emission Fingerprinting Page 47 30/10/2007 Anita Hsu Page 48 30/10/2007 Anita Hsu

9 Multifluorescence imaging Multifluorescence imaging approaches are ideally suited to directly visualize relationships or interactions of molecules and other cell components in their natural context. Use multiple markers simultaneously 長庚大學共軛焦顯微鏡課程 Spot light Basic principle of laser scanning confocal microscopy The application of LSM 510 META detector Multiphoton microscopy basic principle and introduction Page 49 30/10/2007 Anita Hsu Page 50 30/10/2007 Anita Hsu LSM 510 META Problem: conditions of excitation and emission cross-talk LSM 510 META The Problem of excitation and emission cross-talk Example: Cross-talk problem when visualizing multiple fluorescent proteins The Solution LSM 510 META Note: it is often difficult to clearly separate two fluorescence markers exhibiting extensive cross-talk. With more markers, the problem grows increasingly complex. 1) Parallel spectral acquisition of emission signal 2) Use spectral characteristics to identify and separate different signals META detector Emission Fingerprinting Page 51 30/10/2007 Anita Hsu Page 52 30/10/2007 Anita Hsu The Solution LSM 510 META Innovative detector concept LSM 510 META Solution: Detection of the spectral characteristic together with the x/y information for every pixel using the META Detector Lambda scan META Detector: Array of 32 PMT elements Reflective grating for even dispersion Simultaneous acquisition of whole emission spectrum META detector PMT array with 32 elements Reflection grating for even dispersion Capture full emission spectra Page 53 30/10/2007 Anita Hsu Page 54 30/10/2007 Anita Hsu

10 Acquisition of Lamda Stack Emission Fingerprinting - The basic principle 1 - Acquisition of Lambda Stack(s) Acquire the complete emission spectrum (Lambda Stack: x([yzt]- ) 2 - Selection of Reference Spectra Selected in Lambda Stack or recalled from Spectra Database 3 - Linear Unmixing Separate the emission signals Page 55 30/10/2007 Anita Hsu Page 56 30/10/2007 Anita Hsu LSM 510 META: Emission Fingerprinting Emission Fingerprinting - new FPs Cell expressing GFP and YFP Lambda Stack (Experimental Data) Reference Reference CFP, CGFP, GFP and YFP Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, respectively GFP + YFP GFP YFP Pixel-by-pixel analysis 100 = Relative contribution of GFP and YFP Linear unmixing determines the relative contribution of each fluorochrome in every pixel of an image. Lambda Stack GFP YFP (raw data) (unmixed) (unmixed) Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan Page 57 30/10/2007 Anita Hsu Page 58 30/10/2007 Anita Hsu Emission Fingerprinting - new FPs Emission Fingerprinting - new FPs Record lambda stack of single labeled controls CFP, CGFP, GFP and YFP 1. Record lamda stack Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, respectively. CFP CGFP GFP YFP 2. Recall reference spectra CFP CGFP 3. Apply linear unmixing Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan GFP YFP Page 59 30/10/2007 Anita Hsu Page 60 30/10/2007 Anita Hsu

11 Save Data : ***.lsm 1) Confocal Imaging Optimal optical 3D sectioning in thick tissue Optimal optical 3D sectioning in multifluorescent samples 2) Laser and scanning mirror control Easy sample manipulation Flexible scanning strategies (1D to multid) FRAP, FLIP, photoactivation, acceptor bleach (FRET) 3) Multifluorescence Imaging Acquisition of several dye signals simultaneously (FPs etc.) Clear separation of fluorescent dyes without the danger of crosstalk Image channels that show only the signal from one fluorescent label Optimal quantitative imaging i.e. colocalization Multiple Pinhole Concept Real-Time electronics real ROIs bleach ROIs META Detector Emission Fingerprinting All scan parameters stored Repeat experiments by one click For multi user environment More transparent information Keeps overview --- Image Database: ***.mdb Page 61 30/10/2007 Anita Hsu Page 62 30/10/2007 Anita Hsu Save Data Image Database -> Reusable Parameter Settings Save Data--- Export:.tif,.jpg,.avi, All scan parameters stored Repeat experiments by one click... Reproducibility!! Page 63 30/10/2007 Anita Hsu Page 64 30/10/2007 Anita Hsu The beam path in the scan head Page 66 Anita Hsu

12 Beam Path Setting -Tell the system what signal you want to get Main Dichroic Beamsplitter -- HFT Ch: detector To Detection From Laser HFT 488 To / From Specimen Function: separate excitation and emission light The coating of the beamsplitter reflects a small spectral band corresponding to the selected laser line. The light emitted from the specimen is allowed to pass through this element without being influenced HFT R/FL detector 1 transmitted light channel HFT is the German abbreviation for Haupt-Farb-Teiler, which means Main Dichroic Beamsplitter Page 67 30/10/2007 Anita Hsu Page 68 30/10/2007 Anita Hsu Second Beam Splitter-- NFT Beam Path Setting -Tell the system what signal you want to get Short wavelength Simultaneous 2 color detection NFT 490 Long wavelength Long wavelength exi emi Alexa Short wavelength The fluorescence emission from the sample is separated by dichroic beamsplitters and/or by filters (longpass, bandpass) Page 69 30/10/2007 Anita Hsu Page 70 30/10/2007 Anita Hsu Beam Path Setting -Tell the system what signal you want to get EMISSION CROSS TALK Single track: get fluorescent signals simultaneously get signals simultaneously exi. emi. Ar FITC Cy FITC 572 TRITC He-Ne LP560 Page 71 30/10/2007 Anita Hsu Page 72 30/10/2007 Anita Hsu

13 Single track: get fluorescent signals simultaneously The crosstalk problem Multiwavelength excite at the same time. Multiwavelength emit and detected at the same time. Multi emission light separate by beamspliter. Benefit: Acquire image fast Reduce illumination time Limit: Crosstalk problem! FITC / Rhod Simultaneous Sequential Multi-tracking FITC Rhod merged Rhod signal will affected by FITC signal ( ) Page 73 30/10/2007 Anita Hsu Page 74 30/10/2007 Anita Hsu Avoid cross talking by multitracking Multi track: get fluorescent signals sequentially Multitracking Effective elimination of emission crosstalk Improved signal/noise by using long pass - instead of band-detection First Track: FITC Second Track: Rhod Simultaneous Single track Sequential Multi track FITC Rhod merged Page 75 30/10/2007 Anita Hsu Page 76 30/10/2007 Anita Hsu