Confocal Microscopy. Kristin Jensen
|
|
|
- Stephany Francis
- 5 years ago
- Views:
Transcription
1 Confocal Microscopy Kristin Jensen
2 References Cell Biological Applications of Confocal Microscopy, Brian Matsumoto, chapter 1 Studying protein dynamics in living cells,, Jennifer Lippincott-Schwartz Schwartz,, Erik Snapp and Anne Kenworthy (Can be found on PubMed)
3 History Invented by Marvin Minsky in 1957 First photomicrographs of recognizable cells in s: advances in -computer technology -laser technology -digital imaging software 1980 s: commercially available 1990 s: - advances in optics and electronics more stable and powerful lasers more efficient mirrors lower noise photodetectors brighter fluorophores better matched to laser lines higher resolution monitors and printers storage of images
4 Conventional Fluorescense versus Confocal Microscope Conventional: Whole object illuminated at the same time No z-resolution Excitation filters Confocal: One point illuminated Better lateral resolution Z-resolution => optical sections => 3D Lasers
5 Airy disk Dependent on sample features and detection size
6 Resolution Rayleigh criterion Points resolved when maximum of one airy disk coincides with first minimum of the other airy disk Lateral resolution = 0.46 λem / NA Axial resolution = 1.4 λem η/ / NA²
7 What do you see? Fluorescense Speed of light: c = λf λ = wavelength of light f = frequency of light Energy of light: E = hf h = Planck Molecule absorbs energy => excited Molecule emits energy => de-excited excited Planck s constant,, 6.63e Js
8 Absorption and emission spectra
9 Fluorophores
10 Confocal Laser Scanning Microscopy (CLSM) Microscope: upright/inverted inverted Light source (lasers) Scan Head Detectors Computer
11 Lasers Light amplification by stimulated emission of radiation Excitation source Advantages: High brightness Low noise Focused into a very small spot (spatial and temporal) Monochromatic (only one wavelength) Variations: Excited medium (solid state/ / gas) Available laser lines Cooling system Output power Operational lifetime
12 Path of the laser Laser Acustooptical tunable filter Aperture (pinhole) Dichromatic mirror Scanning mirrors Objectiv Excitation of specimen -> emission Objectiv Scanning mirrors Dichromatic mirror Aperture (pinhole) Detector
13 Path of the laser Laser Acustooptical tunable filter Aperture (pinhole) Dichromatic mirror Scanning mirrors Objectiv Excitation of specimen -> emission Objectiv Scanning mirrors Dichromatic mirror Aperture (pinhole) Detector
14 Acustooptical Tunable Filter Laser attenuation Selection of excitation wavelength (do not need line selection filters) Individually attenuation of lines from multiline laser Fast switching between imaging modes Sequential scanning => preventing crosstalk Selection of regions of interest (ROI( ROI s)
15 Path of the laser Laser Acustooptical tunable filter Aperture (pinhole) Dichromatic mirror Scanning mirrors Objectiv Excitation of specimen -> emission Objectiv Scanning mirrors Dichromatic mirror Aperture (pinhole) Detector
16 Scan Head
17 Scanning Stage scanning Beam scanning Point scanning Line scanning Spinning disk (Nipkow( disk) Raster scanning Raster rotation => fast Line scan Frame scan
18 Path of the laser Laser Acustooptical tunable filter Aperture (pinhole) Dichromatic mirror Scanning mirrors Objectiv Excitation of specimen -> emission Objectiv Scanning mirrors Dichromatic mirror Aperture (pinhole) Detector
19 Conjugate points
20 Pinhole or slit Light from focus plane focused onto pinhole => large signal Light from out-of of-focusfocus planes blocked by pinhole => small signal Increasing pinhole => decreasing resolution but higher signal Lateral resolution more dependent on pinhole than axial resolution Determines the thickness of the layer Aperture
21
22 Path of the laser Laser Acustooptical tunable filter Aperture (pinhole) Dichromatic mirror Scanning mirrors Objectiv Excitation of specimen -> emission Objectiv Scanning mirrors Dichromatic mirror Aperture (pinhole) Detector
23 Photodetector Filters Photomultiplier tube (PMT) Converts light energy into electrical energy Photons interact with material in detector making free electrons Electrons interact with dynodes making more electrons One PMT for each fluorochrome
24 Spectral Detector Prism or grating separates wavelengths Adjustable slits PMT
25 Computer Runs the hole system Intensity of lasers Pinhole Gain,, offset, sensitivity of detector Focusing Scanning and ROI s Z-direction Filters and shutters Resolution Image saving, processing,, display
26 Spinning Disk Stationary light source and stage White, noncoherent light or lasers Thousands of apertures, µm m in diameter ~ 3600 rpm => image seen in real time Less z-resolution because of bigger pinhole
27 Pixelation Spatial resolution Optical Microscope (objective) => resel xy - direction = 0.46 λemem / NA Digital Computer => 2D pixel => 3D - voxel
28 Sampling Nyquist sampling criterion = two samples for every resolvable point (in each direction) Mismatch between sampling and resolution => the high frequencies disguise in the low frequencies => aliasing If microscope resolves 100 points => do not need 1000 pixels Optical resolution Detector characteristics Display pixelation
29 Spoiling resolution
30 Gray level Intensity level Detector => Just detectable difference (jdd) Computer => Gray level One gray level / jdd => good image reconstruction 8-bit converter => 256 gray levels Sensitivity,, offset, gain
31 Sensitivity,, offset, gain
32 Bleaching Laser bleaches the fluorophore => signal gets weaker Some fluorophores more sensitive than others Reduce intensity of laser Limit the time the specimen is exposed to the laser
33 Crosstalk
34 Fluorescense recovery after photobleaching (FRAP)
35 FRAP- kurver
36 Fluorescense recovery after photobleaching (FRAP)
37 Fluorescense recovery after photobleaching (FRAP) Diffusion constant: D = ω²γ / 4TD ω = radius of bleach circle γ = correction factor for amount of bleaching TD = diffusion time Mobile fraction: Mf = (I - I0) ) / (Ii( - I0) I = intensity at full recovery I0 = intensity at first picture after bleaching Ii = intensity before bleaching Increase in D Non diffusive behaviour Decrease in environment viscosity Decrease in D Formation of large aggregates or complexes Increase in environment viscosity Transient interaction with large or fixed molecules 100 % mobile Freely diffusion in Mf Protein is released from restricted compartment or fixed macromolecular complex in Mf Protein binds or form aggregates that are restricted in movement Protein is confined to another compartment Increase in M Decrease in M
38 Fluorescense loss in photobleaching (FLIP) Loss of fluorescense monitored instead of recovery The same area is repeatedly bleached Area around will be bleached because of moving molecules Compartments which are not getting darker are restricted areas for tagged molecules
39 Fluorescence resonance energy transfere (FRET) Detects close proximity of interacting proteins Certain pairs of fluorophores donor and acceptor F.ex.. CFP and YFP, FITC and Rhodamine,, and Cy3 and Cy5 When the pairs are in very close proximity Excitation of CFP => transfere of energy from CFP to YFP => sensitized emission from YFP Dependent on distance Förster distance, RO => energy transfere with efficiency of 50% Depends on spectral overlap, quanum yield of donor and relative orientation between the pair Measure: Quenching of donor fluorescense Sensitized acceptor fluorescense Acceptor photobleaching Anisotropy Decrease in the rate of donor photobleaching Decrease in excited lifetime of donor
40 Fluorescence correlation spectroscopy (FCS) Measures the fluctuation of photons from molecules moving in and out of a defined volume deflects average number of molecules => concentration time of diffusion in and out of compartment => diffusion constant Low concentrations of fluorophores (nm) When diffusion suddenly slows down or increase => protein protein interactions
41 Fluorescense localization after photobleaching (FLAP) Molecule to be located carries two fluorophores One to bleach, one as reference label Image differencing Can follow both bleached and unbleached molecules
Shreyash Tandon M.S. III Year
Shreyash Tandon M.S. III Year 20091015 Confocal microscopy is a powerful tool for generating high-resolution images and 3-D reconstructions of a specimen by using point illumination and a spatial pinhole
Why and How? Daniel Gitler Dept. of Physiology Ben-Gurion University of the Negev. Microscopy course, Michmoret Dec 2005
Why and How? Daniel Gitler Dept. of Physiology Ben-Gurion University of the Negev Why use confocal microscopy? Principles of the laser scanning confocal microscope. Image resolution. Manipulating the
Basics of confocal imaging (part I)
Basics of confocal imaging (part I) Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP arne.seitz@epfl.ch Lateral resolution BioImaging &Optics Platform Light
Point Spread Function. Confocal Laser Scanning Microscopy. Confocal Aperture. Optical aberrations. Alternative Scanning Microscopy
Bi177 Lecture 5 Adding the Third Dimension Wide-field Imaging Point Spread Function Deconvolution Confocal Laser Scanning Microscopy Confocal Aperture Optical aberrations Alternative Scanning Microscopy
Bio 407. Applied microscopy. Introduction into light microscopy. José María Mateos. Center for Microscopy and Image Analysis
Center for Microscopy and Image Analysis Bio 407 Applied Introduction into light José María Mateos Fundamentals of light Compound microscope Microscope composed of an objective and an additional lens (eyepiece,
BASICS OF CONFOCAL IMAGING (PART I)
BASICS OF CONFOCAL IMAGING (PART I) INTERNAL COURSE 2012 LIGHT MICROSCOPY Lateral resolution Transmission Fluorescence d min 1.22 NA obj NA cond 0 0 rairy 0.61 NAobj Ernst Abbe Lord Rayleigh Depth of field
1 Co Localization and Working flow with the lsm700
1 Co Localization and Working flow with the lsm700 Samples -1 slide = mousse intestine, Dapi / Ki 67 with Cy3/ BrDU with alexa 488. -1 slide = mousse intestine, Dapi / Ki 67 with Cy3/ no BrDU (but with
LSM 510 META in Chang Gung University
Content LSM 510 META in Chang ung University LSM 510 META 路 理 The features and applications of LSM 510 META 01-09 Introduction of the hardware 10-12 Fluorescence observation in conventional microscope
Zeiss 780 Training Notes
Zeiss 780 Training Notes Turn on Main Switch, System PC and Components Switches 780 Start up sequence Do you need the argon laser (458, 488, 514 nm lines)? Yes Turn on the laser s main power switch and
5/4/2015 INTRODUCTION TO LIGHT MICROSCOPY. Urs Ziegler MICROSCOPY WITH LIGHT. Image formation in a nutshell. Overview of techniques
INTRODUCTION TO LIGHT MICROSCOPY Urs Ziegler ziegler@zmb.uzh.ch MICROSCOPY WITH LIGHT INTRODUCTION TO LIGHT MICROSCOPY Image formation in a nutshell Overview of techniques Widefield microscopy Resolution
Boulevard du Temple Daguerrotype (Paris,1838) a busy street? Nyquist sampling for movement
Boulevard du Temple Daguerrotype (Paris,1838) a busy street? Nyquist sampling for movement CONFOCAL MICROSCOPY BioVis Uppsala, 2017 Jeremy Adler Matyas Molnar Dirk Pacholsky Widefield & Confocal Microscopy
3D light microscopy techniques
3D light microscopy techniques The image of a point is a 3D feature In-focus image Out-of-focus image The image of a point is not a point Point Spread Function (PSF) 1D imaging 1 1 2! NA = 0.5! NA 2D imaging
Final Exam, 150 points PMB 185: Techniques in Light Microscopy
Final Exam, 150 points Name PMB 185: Techniques in Light Microscopy Point value is in parentheses at the end of each question. Note: GFP = green fluorescent protein ; CFP = cyan fluorescent protein ; YFP
Advanced Live Cell Imaging
FRET Analysis in Laser Scanning Microscopy What is FRET? FRET (fluorescence resonance energy transfer) is the non-radiative transfer of photon energy from an excited fluorophore (the donor) to another
Components of confocal and two-photon microscopes
Components of confocal and two-photon microscopes Internal training 07/04/2016 A. GRICHINE Platform Optical microscopy Cell imaging, IAB, ISdV Plan Confocal laser scanning microscope o o o Principle Main
TRAINING MANUAL. Multiphoton Microscopy LSM 510 META-NLO
TRAINING MANUAL Multiphoton Microscopy LSM 510 META-NLO September 2010 Multiphoton Microscopy Training Manual Multiphoton microscopy is only available on the LSM 510 META-NLO system. This system is equipped
Multifluorescence The Crosstalk Problem and Its Solution
Multifluorescence The Crosstalk Problem and Its Solution If a specimen is labeled with more than one fluorochrome, each image channel should only show the emission signal of one of them. If, in a specimen
Confocal Microscopy. (Increasing contrast and resolu6on using op6cal sec6oning) Lecture 7. November 2017
Confocal Microscopy (Increasing contrast and resolu6on using op6cal sec6oning) Lecture 7 November 2017 3 Flavours of Microscope Confocal Laser Scanning Problem: Out of Focus Light Spinning disc 2-Photon
Zeiss 880 Training Notes Zen 2.3
Zeiss 880 Training Notes Zen 2.3 1 Turn on the HXP 120V Lamp 2 Turn on Main Power Switch Turn on the Systems PC Switch Turn on the Components Switch. 3 4 5 Turn on the PC and log into your account. Start
Operation Guide for the Leica SP2 Confocal Microscope Bio-Imaging Facility Hunter College October 2009
Operation Guide for the Leica SP2 Confocal Microscope Bio-Imaging Facility Hunter College October 2009 Introduction of Fluoresence Confocal Microscopy The first confocal microscope was invented by Princeton
Bi/BE 227 Winter Assignment #3. Adding the third dimension: 3D Confocal Imaging
Bi/BE 227 Winter 2016 Assignment #3 Adding the third dimension: 3D Confocal Imaging Schedule: Jan 20: Assignment Jan 20-Feb 8: Work on assignment Feb 10: Student PowerPoint presentations. Goals for this
Practical work no. 3: Confocal Live Cell Microscopy
Practical work no. 3: Confocal Live Cell Microscopy Course Instructor: Mikko Liljeström (MIU) 1 Background Confocal microscopy: The main idea behind confocality is that it suppresses the signal outside
Introduction to light microscopy
Center for Microscopy and Image Anaylsis Introduction to light microscopy Basic concepts of imaging with light Urs Ziegler ziegler@zmb.uzh.ch Light interacting with matter Absorbtion Refraction Diffraction
Quick Start Guide. Leica SP5 X
Quick Start Guide Leica SP5 X Please note: Some of the information in this guide was taken from Leica Microsystems Leica TCS SP5 LAS AF Guide for New Users. This work is licensed under the Creative Commons
Introduction to light microscopy
Center for Microscopy and Image Anaylsis Introduction to light Basic concepts of imaging with light Urs Ziegler ziegler@zmb.uzh.ch Microscopy with light 1 Light interacting with matter Absorbtion Refraction
Confocal, hyperspectral, spinning disk
Confocal, hyperspectral, spinning disk Administrative HW 6 due on Fri Midterm on Wed Covers everything since previous midterm 8.5 x 11 sheet allowed, 1 side Guest lecture by Joe Dragavon on Mon 10/30 Last
Confocal and 2-photon Imaging. October 15, 2010
Confocal and 2-photon Imaging October 15, 2010 Review Optical Elements Adapted from Sluder & Nordberg 2007 Review Optical Elements Collector Lens Adapted from Sluder & Nordberg 2007 Review Optical Elements
Fundamentals of Light Microscopy II: Fluorescence, Deconvolution, Confocal, Multiphoton, Spectral microscopy. Integrated Microscopy Course
Fundamentals of Light Microscopy II: Fluorescence, Deconvolution, Confocal, Multiphoton, Spectral microscopy Integrated Microscopy Course Review Lecture 1: Microscopy Basics Light train Kohler illumination*
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
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 20071030 長庚大學 Basic principle of Laser Scanning Confocal Microscopy The application of LSM 510 META detector Multiphoton microscopy basic principle and introduction
ADVANCED METHODS FOR CONFOCAL MICROSCOPY II. Jean-Yves Chatton Sept. 2006
ADVANCED METHODS FOR CONFOCAL MICROSCOPY II Jean-Yves Chatton Sept. 2006 Workshop outline Confocal microscopy of living cells and tissues X-Z scanning Time series Bleach: FRAP, photoactivation Emission
Leica TCS SP8 Quick Start Guide
Leica TCS SP8 Quick Start Guide Leica TCS SP8 System Overview Start-Up Procedure 1. Turn on the CTR Control Box, Fluorescent Light for the microscope stand. 2. Turn on the Scanner Power (1) on the front
Rates of excitation, emission, ISC
Bi177 Lecture 4 Fluorescence Microscopy Phenomenon of Fluorescence Energy Diagram Rates of excitation, emission, ISC Practical Issues Lighting, Filters More on diffraction Point Spread Functions Thus Far,
Microscope Confocal LSM510 META
Microscope Confocal LSM510 META Welcome to the Zeiss LSM 510 Meta Confocal tutorial. Before using the LSM 510 META, Log off any other computer that is open with your personal login. You will need to put
Maria Smedh, Centre for Cellular Imaging. Maria Smedh, Centre for Cellular Imaging
Nonlinear microscopy I: Two-photon fluorescence microscopy Multiphoton Microscopy What is multiphoton imaging? Applications Different imaging modes Advantages/disadvantages Scattering of light in thick
Introduction to light microscopy
Center for Microscopy and Image Anaylsis Introduction to light Imaging with light / Overview of techniques Urs Ziegler ziegler@zmb.uzh.ch Light interacting with matter Absorbtion Refraction Diffraction
Microscope Confocal Sp2 Upright.
Microscope Confocal Sp2 Upright. Welcome to the Leica Sp2 Confocal Upright tutorial. Before using the Sp2 Invert, You will need to put down your name on the reservation system = http://svintranet.epfl.ch/index.php?optio
Invitation for a walk through microscopy. Sebastian Schuchmann Jörg Rösner
Invitation for a walk through microscopy Sebastian Schuchmann Jörg Rösner joerg.roesner@charite.de Techniques in microscopy Conventional (light) microscopy bright & dark field, phase & interference contrast
You won t be able to measure the incident power precisely. The readout of the power would be lower than the real incident power.
1. a) Given the transfer function of a detector (below), label and describe these terms: i. dynamic range ii. linear dynamic range iii. sensitivity iv. responsivity b) Imagine you are using an optical
Training Guide for Carl Zeiss LSM 510 META Confocal Microscope
Training Guide for Carl Zeiss LSM 510 META Confocal Microscope AIM 4.2 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Turn ON Components and System/PC switches
Training Guide for Leica SP8 Confocal/Multiphoton Microscope
Training Guide for Leica SP8 Confocal/Multiphoton Microscope LAS AF v3.3 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Turn ON power switch for epifluorescence
Digital Camera Technologies for Scientific Bio-Imaging. Part 2: Sampling and Signal
Digital Camera Technologies for Scientific Bio-Imaging. Part 2: Sampling and Signal Yashvinder Sabharwal, 1 James Joubert 2 and Deepak Sharma 2 1. Solexis Advisors LLC, Austin, TX, USA 2. Photometrics
BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP LASER SCANNING CONFOCAL MICROSCOPY PRACTICAL CONSIDERATIONS
LASER SCANNING CONFOCAL MICROSCOPY PRACTICAL CONSIDERATIONS IMPORTANT PARAMETERS Pixel dwell time Zoom and pixel number PIXEL DWELL TIME How much time signal is collected at every pixel Very small values,
Development of a High-speed Super-resolution Confocal Scanner
Development of a High-speed Super-resolution Confocal Scanner Takuya Azuma *1 Takayuki Kei *1 Super-resolution microscopy techniques that overcome the spatial resolution limit of conventional light microscopy
Imaging Retreat - UMASS Customized real-time confocal and 2-photon imaging
Imaging Retreat - UMASS 2012 Customized real-time confocal and 2-photon imaging Mike Sanderson Department of Microbiology and Physiological Systems University of Massachusetts Medical School Thanks for
ZEISS LSM510META confocal manual
ZEISS LSM510META confocal manual Switching on the system 1) Switch on the Remote Control button located on the table to the right of the microscope. This is the main switch for the whole system including
INTRODUCTION TO MICROSCOPY. Urs Ziegler THE PROBLEM
INTRODUCTION TO MICROSCOPY Urs Ziegler ziegler@zmb.uzh.ch THE PROBLEM 1 ORGANISMS ARE LARGE LIGHT AND ELECTRONS: ELECTROMAGNETIC WAVES v = Wavelength ( ) Speed (v) Frequency ( ) Amplitude (A) Propagation
Confocal Microscopy Confocal Microscopy Acousto-optic products
Confocal Microscopy Confocal Microscopy Acousto-optic products AA OPTO-ELECTRONIC QUANTA TECH Introduction Confocal microscopy is an imaging technique used to increase micrograph contrast and/or to reconstruct
Supplementary Figure S1: Schematic view of the confocal laser scanning STED microscope used for STED-RICS. For a detailed description of our
Supplementary Figure S1: Schematic view of the confocal laser scanning STED microscope used for STED-RICS. For a detailed description of our home-built STED microscope used for the STED-RICS experiments,
Leica TCS SL Confocal Training. Neuroscience Imaging Core Staff. Core Director. Facility Manager
Leica TCS SL Confocal Training Neuroscience Imaging Facility The Ohio State University Rightmire Hall 614-292-1367 Staff Core Director Anthony Brown, Ph. D. 060 Rightmire Hall 614-292-1205 brown.2302@osu.edu
Examination, TEN1, in courses SK2500/SK2501, Physics of Biomedical Microscopy,
KTH Applied Physics Examination, TEN1, in courses SK2500/SK2501, Physics of Biomedical Microscopy, 2009-06-05, 8-13, FB51 Allowed aids: Compendium Imaging Physics (handed out) Compendium Light Microscopy
Leica Sp5 II Confocal User Guide
Leica Sp5 II Confocal User Guide Turning on the Confocal System (instructions are posted in the room) 1. Turn on Laser Power Button 2. Turn Key to On position 3. Turn on Scanner Power Button 4. Turn on
The DCS-120 Confocal Scanning FLIM System
he DCS-120 Confocal Scanning FLIM System he bh DCS-120 confocal scanning FLIM system converts a conventional microscope into a high-performance fluorescence lifetime imaging system. he system is based
TRAINING MANUAL. Olympus FV1000
TRAINING MANUAL Olympus FV1000 September 2014 TABLE OF CONTENTS A. Start-Up Procedure... 1 B. Visual Observation under the Microscope... 1 C. Image Acquisition... 4 A brief Overview of the Settings...
OPERATING INSTRUCTIONS
Zeiss LSM 510 M eta Confocal M icroscope OPERATING INSTRUCTIONS Starting the System: 1. Turn the black knob on the laser box one-quarter turn from Off to On. You will hear the laser cooling mechanisms
3D light microscopy techniques
3D light microscopy techniques The image of a point is a 3D feature In-focus image Out-of-focus image The image of a point is not a point Point Spread Function (PSF) 1D imaging 2D imaging 3D imaging Resolution
LSM 780 Confocal Microscope Standard Operation Protocol
LSM 780 Confocal Microscope Standard Operation Protocol Basic Operation Turning on the system 1. Sign on log sheet according to Actual start time 2. Check Compressed Air supply for the air table 3. Switch
LSM 710 Confocal Microscope Standard Operation Protocol
LSM 710 Confocal Microscope Standard Operation Protocol Basic Operation Turning on the system 1. Switch on Main power switch 2. Switch on System / PC power button 3. Switch on Components power button 4.
Dynamic Confocal Imaging of Living Brain. Advantages and risks of multiphoton microscopy in physiology
Dynamic Confocal Imaging of Living Brain Advantages and risks of multiphoton microscopy in physiology Confocal laser scanning microscopy In conventional optical microscopy focused and out-offocus light
Opterra II Multipoint Scanning Confocal Microscope. Innovation with Integrity
Opterra II Multipoint Scanning Confocal Microscope Enabling 4D Live-Cell Fluorescence Imaging through Speed, Sensitivity, Viability and Simplicity Innovation with Integrity Fluorescence Microscopy The
Imaging Beyond the Basics: Optimizing Settings on the Leica SP8 Confocal
Imaging Beyond the Basics: Optimizing Settings on the Leica SP8 Confocal Todays Goal: Introduce some additional functionalities of the Leica SP8 confocal HyD vs. PMT detectors Dye Assistant Scanning By
PCS-150 / PCI-200 High Speed Boxcar Modules
Becker & Hickl GmbH Kolonnenstr. 29 10829 Berlin Tel. 030 / 787 56 32 Fax. 030 / 787 57 34 email: info@becker-hickl.de http://www.becker-hickl.de PCSAPP.DOC PCS-150 / PCI-200 High Speed Boxcar Modules
Comparing FCS and FRAP as methodologies for calculating diffusion
Bi/BE 227 Winter 2018 Assignment #4 Comparing FCS and FRAP as methodologies for calculating diffusion Schedule: Jan 29: Assignment Jan 29-Feb 14: Work on assignment Feb 14: Student PowerPoint presentations.
Bi Imaging. Multicolor Imaging: The Important Question of Co-Localization. Anna Smallcombe Bio-Rad Laboratories, Hemel Hempstead, UK
Multicolor Imaging: The Important Question of Co-Localization Anna Smallcombe Bio-Rad Laboratories, Hemel Hempstead, UK The use of specific fluorescent probes, combined with confocal or multiphoton microscopy
Training Guide for Carl Zeiss LSM 5 LIVE Confocal Microscope
Training Guide for Carl Zeiss LSM 5 LIVE Confocal Microscope AIM 4.2 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Verify that main power switches on the
Leica TCS SP8 Quick Start Guide
Leica TCS SP8 Quick Start Guide Leica TCS SP8 System Overview Start-Up Procedure 1. Turn on the CTR Control Box, EL6000 fluorescent light source for the microscope stand. 2. Turn on the Scanner Power
Rapid Adaptive Optical Recovery of Optimal Resolution over Large Volumes
SUPPLEMENTARY MATERIAL Rapid Adaptive Optical Recovery of Optimal Resolution over Large Volumes Kai Wang, Dan Milkie, Ankur Saxena, Peter Engerer, Thomas Misgeld, Marianne E. Bronner, Jeff Mumm, and Eric
The Nature of Light. Light and Energy
The Nature of Light Light and Energy - dependent on energy from the sun, directly and indirectly - solar energy intimately associated with existence of life -light absorption: dissipate as heat emitted
Guide to Confocal 5. Starting session
Guide to Confocal 5 Remember that when booking and before starting session you can check for any problems at https://www.bris.ac.uk/biochemistry/uobonly/cif/index.html Starting session Switch on microscope
Introduction to light microscopy
Center for Microscopy and Image Anaylsis Introduction to light microscopy (an overview) Microscopy with light Components of a light microscope 1. Light source 2. Objective 3. Sample or specimen holder
More fancy SPIM, Even fancier SPIM
More fancy SPIM, Even fancier SPIM Last class Light sheet microscopy Fancy SPIM (ispim, dspim, etc ) This class Multi camera SPIM SIM SPIM Bessels d x,y = λ em 2 NA d z = 2 NA λ ex + n(1 cosθ λ em 1 IsoView
Confocal Laser Scanning Microscopy
Name of the Core Facility: Confocal Laser Scanning Microscopy CORE Forschungszentrum Immunologie Mainz Welcome to the CSLM Core Facility: The CLSM Core Facility enables working groups to incorporate high
Training Guide for Carl Zeiss LSM 880 with AiryScan FAST
Training Guide for Carl Zeiss LSM 880 with AiryScan FAST ZEN 2.3 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2018) Power ON Routine 1 2 Turn ON Main Switch from the remote control
Light Microscopy for Biomedical Research
Light Microscopy for Biomedical Research Tuesday 4:30 PM Quantification & Digital Images Michael Hooker Microscopy Facility Michael Chua microscopy@unc.edu 843-3268 6007 Thurston Bowles http://microscopy.unc.edu/lmbr
a) How big will that physical image of the cells be your camera sensor?
1. Consider a regular wide-field microscope set up with a 60x, NA = 1.4 objective and a monochromatic digital camera with 8 um pixels, properly positioned in the primary image plane. This microscope is
(12) United States Patent (10) Patent No.: US 6,388,807 B1. Knebel et al. (45) Date of Patent: May 14, 2002
USOO6388807B1 (12) United States Patent (10) Patent No.: Knebel et al. () Date of Patent: May 14, 2002 (54) CONFOCAL LASER SCANNING (56) References Cited MICROSCOPE U.S. PATENT DOCUMENTS (75) Inventors:
Spectroscopy in the UV and Visible: Instrumentation. Spectroscopy in the UV and Visible: Instrumentation
Spectroscopy in the UV and Visible: Instrumentation Typical UV-VIS instrument 1 Source - Disperser Sample (Blank) Detector Readout Monitor the relative response of the sample signal to the blank Transmittance
Zeiss LSM 510 Confocor III Training Notes. Center for Cell Analysis & Modeling
Zeiss LSM 510 Confocor III Training Notes Center for Cell Analysis & Modeling Confocor 3 Start Up Go to System Module Turn on Main Switch, System/ PC, and Components Switches Do you need the arc lamp?
High-speed 1-frame ms scanning confocal microscope with a microlens and Nipkow disks
High-speed 1-framems scanning confocal microscope with a microlens and Nipkow disks Takeo Tanaami, Shinya Otsuki, Nobuhiro Tomosada, Yasuhito Kosugi, Mizuho Shimizu, and Hideyuki Ishida We have developed
Improving Image Quality on the UACC Leica SP5 confocal
Improving Image Quality on the UACC Leica SP5 confocal Douglas Cromey, MS (UACC TACMASR Microscopy) Some days it is difficult to come up with a brightly stained sample. This document contains some strategies
WHITE PAPER FAST PROTEIN INTERACTION BINDING CURVES WITH INO S F-HS CONFOCAL MICROSCOPE
WHITE PAPER FAST PROTEIN INTERACTION BINDING CURVES WITH INO S F-HS CONFOCAL MICROSCOPE Christian Tardif, Jean-Pierre Bouchard Pascal Gallant, Sebastien Roy, Ozzy Mermut September 2017 Introduction Protein-protein
Last updated: May 2014 Y.DeGraaf
FLINDERS MICROSCOPY BIOMEDICAL SERVICES AVAILABLE MICROSCOPES AND SPECIFICATIONS & INFORMATION REGARDING TRAINING FOR NEW USERS Last updated: May 2014 Y.DeGraaf If you have new staff or students (Honours/Masters
An 8-Channel Parallel Multispectral TCSPC FLIM System
An 8-Channel Parallel Multispectral TCSPC FLIM System Abstract. We describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 50 10 6 s -1.
Spectral Imaging with the Opterra Multipoint Scanning Confocal
Spectral Imaging with the Opterra Multipoint Scanning Confocal Outline Opterra design overview Scan Modes Light Path Spectral Imaging with Opterra Drosophila larva heart. Opterra Design Overview Supravideo
Travel to New Dimensions- LSM 880. The Resolution of a Microscope is limited. The Resolution of a Microscope is limited. Image. Image. Object.
Travel to New Dimensions- LSM 880 LSM 880: The Power of Sensitivity Our Latest Member of the LSM 880 with GaAsP Detectors Sensitivity, and Ease of Use Innovative High-End Laser Scanning Microscopes from
Supplemental Figure 1: Histogram of 63x Objective Lens z axis Calculated Resolutions. Results from the MetroloJ z axis fits for 5 beads from each
Supplemental Figure 1: Histogram of 63x Objective Lens z axis Calculated Resolutions. Results from the MetroloJ z axis fits for 5 beads from each lens with a 1 Airy unit pinhole setting. Many water lenses
Resolution. Diffraction from apertures limits resolution. Rayleigh criterion θ Rayleigh = 1.22 λ/d 1 peak at 2 nd minimum. θ f D
Microscopy Outline 1. Resolution and Simple Optical Microscope 2. Contrast enhancement: Dark field, Fluorescence (Chelsea & Peter), Phase Contrast, DIC 3. Newer Methods: Scanning Tunneling microscopy (STM),
Non-Descanned FLIM Detection in Multiphoton Microscopes
Non-Descanned FLIM Detection in Multiphoton Microscopes Abstract. Multiphoton microscopes use a femtosecond NIR laser to excite fluorescence in the sample. Excitation is performed via a multi-photon absorption
LSM 510 Meta Training Notes
LSM 510 Meta Training Notes Turning on the system Turn on X-Cite power supply. This supplies light for epifluorescence for viewing your samples through the microscope. Turn on the remote control switch.
Heisenberg) relation applied to space and transverse wavevector
2. Optical Microscopy 2.1 Principles A microscope is in principle nothing else than a simple lens system for magnifying small objects. The first lens, called the objective, has a short focal length (a
Chemistry 524--"Hour Exam"--Keiderling Mar. 19, pm SES
Chemistry 524--"Hour Exam"--Keiderling Mar. 19, 2013 -- 2-4 pm -- 170 SES Please answer all questions in the answer book provided. Calculators, rulers, pens and pencils permitted. No open books allowed.
MULTIPHOTON MICROSCOPY. Matyas Molnar Dirk Pacholsky
MULTIPHOTON MICROSCOPY Matyas Molnar Dirk Pacholsky Information Information given here about 2 Photon microscopy were mainly taken from these sources: Background information on 2-Photon microscopy: http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/
Microscopy from Carl Zeiss
Microscopy from Carl Zeiss Contents Page Contents... 1 Introduction... 1 Starting the System... 2 Introduction to ZEN Efficient Navigation... 5 Setting up the microscope... 10 Configuring the beam path
Spectrophotometer. An instrument used to make absorbance, transmittance or emission measurements is known as a spectrophotometer :
Spectrophotometer An instrument used to make absorbance, transmittance or emission measurements is known as a spectrophotometer : Spectrophotometer components Excitation sources Deuterium Lamp Tungsten
Advanced Optical Microscopy lecture. 03. December 2012 Kai Wicker
Advanced Optical Microscopy lecture 03. December 2012 Kai Wicker Today: Optical transfer functions (OTF) and point spread functions (PSF) in incoherent imaging. 1. Quick revision: the incoherent wide-field
Technology Note ZEISS LSM 880 with Airyscan
Technology Note ZEISS LSM 880 with Airyscan Introducing the Fast Acquisition Mode ZEISS LSM 880 with Airyscan Introducing the Fast Acquisition Mode Author: Dr. Annette Bergter Carl Zeiss Microscopy GmbH,
MOM#3: LIGHT SHEET MICROSCOPY (LSM) Stanley Cohen, MD
MOM#3: LIGHT SHEET MICROSCOPY (LSM) Stanley Cohen, MD Introduction. Although the technical details of light sheet imaging and its various permutations appear at first glance to be complex and require some
Reflecting optical system to increase signal intensity. in confocal microscopy
Reflecting optical system to increase signal intensity in confocal microscopy DongKyun Kang *, JungWoo Seo, DaeGab Gweon Nano Opto Mechatronics Laboratory, Dept. of Mechanical Engineering, Korea Advanced
Operation Of The Leica SP8 Multiphoton Confocal System Using Single Or Multiple Fluorochromes
University of South Carolina Scholar Commons Theses and Dissertations 2018 Operation Of The Leica SP8 Multiphoton Confocal System Using Single Or Multiple Fluorochromes Amy E. Rowley University of South
Alba v5 Laser Scanning Microscope
D E S C R I P T I O N Alba v5 Laser Scanning Microscope The instrument for quantitative cell biology at single-molecule detection Alba is a laser scanning microscope that incorporates several measurement
Opterra. Multipoint Scanning Confocal Microscope. Innovation with Integrity. Cell-Friendly, High-Speed, High-Resolution Imaging
Opterra Multipoint Scanning Confocal Microscope Cell-Friendly, High-Speed, High-Resolution Imaging Innovation with Integrity Fluorescence Microscopy Opterra Multipoint Scanning Confocal Microscope Superior