Fluorescent Indicators. Martin Thomas, Cairn Research Ltd
|
|
- Hillary Briggs
- 5 years ago
- Views:
Transcription
1 Fluorescent Indicators Martin Thomas, Cairn Research Ltd
2 ptical Measurements Are Sensitive! Electric current 1A = 6.25 x electrons/sec Squid axon voltage clamp 1mA charges/sec Microelectrode voltage clamp 10uA charges/sec, down to approx 1nA charges/sec Patch clamp 1pA 10 7 charges/sec ptical Recording Photomultipliers and electron-multiplying CCD cameras can detect single photons, but we need a lot to get good images - tradeoff between spatial and time resolution
3 ptical Units Radiant Power Watt (W) Lumen (lm) Radiant Intensity Watts / Steradian (W/sr) Lumens / Steradian (candela) Illumination Intensity Lux (lm m -2 ) Footcandle (lm ft -2 ) At 555nm: 1W 680 lm 2.8x10 18 photons sec -1
4 Fluorescence Fluorescence is is the absorption of light by a molecule, to form a short-lived excited state, followed by re-emission of light at a longer wavelength. A crib for becoming an instant expert: FLIM Fluorescence Lifetime IMaging FRET Fluorescence Resonant Energy Transfer FISH Fluorescence In Situ Hybridisation TIRF Total Internal Reflection Fluorescence FRAP Fluorescence Recovery After Photobleaching
5 Fluorescence Fluorescence is is the absorption of light by a molecule, to form a short-lived excited state, followed by re-emission of light at a longer wavelength. A crib for becoming an instant expert: FLIM Fluorescence Lifetime IMaging FRET Fluorescence Resonant Energy Transfer FISH Fluorescence In Situ Hybridisation TIRF Total Internal Reflection Fluorescence FRAP Fluorescence Recovery After Photobleaching FLAF Four Letter Acronym Fluorescence
6 - C CŌ CŌ CŌ CŌ - C - C - C - C CŌ CŌ - C CŌ CŌ - C EGTA indo-1 fura-2 fluo-3 BAPTA - BCECF ph indicator - C - C H CŌ a + indicator: CŌ CŌ - C CŌ SBFI Indicator structures Ca 2+ buffers and indicators CŌ - C - C S Donor Cl H CŌ CŌ S CŌ - C - C Cl - CCF2: gene expression reporter Flox-6: voltage sensor S Accept or Lectin Fl CŌ (stolen from Roger Tsien)
7 Crystal structure of S65T GFP
8 Ca Indicator Spectra Fura2 EX spectra measured at 510nm EM, EM spectra measured at 340nm EX Ca bound Ca free Indo 1 Absorption (=EX?) spectra, EM spectra measured at 338nm EX Ca bound Ca free EX spectra dotted, EM spectra continuous, data from Invitro
9 SBFI a and ph Indicator Spectra EX spectra measured at 505nm EM, EM spectra measured at 340nm EX a bound a free BCECF EX spectra measured at 535nm EM, EM spectra measured at 480nm EX ph 5.2 ph 9.0 EX spectra dotted, EM spectra continuous, data from Invitrogen website
10 Fura2 excitation spectrum
11 Indo1 emission spectrum
12 Fura8 from AAT-Bioquest Excitation spectrum shifted to longer wavelengths So no need for UV-transmitting optics But seems more susceptible to photobleaching than Fura2
13 But the affinity of most indicators is too high to record transients properly! Slow Timescale Fast Timescale Endothelial cells plus ATP Hepatocytes plus flash-released IP3 gden et al (1995), Eur J Physiol 429,
14 Camelions
15 Yellow Cameleon Emission Spectra
16 Fluorescence Quantification Ca + I FREE = CaI (at equilibrium) K D = [Ca][I FREE ]/[CaI], so [Ca] = K D [CaI]/[I FREE ], or [Ca] = K D [CaI]/([I TTAL ] - [CaI]) If only CaI is fluorescent, then we can write: [Ca] = K D F/(F MAX - F) where F MAX is the fluorescence at saturating [Ca], i.e. [CaI] = [I TTAL ] If I FREE is also fluorescent, the relation becomes: [Ca] = K D (F - F MI )/(F MAX - F) where F MI is the fluorescence at zero [Ca], i.e. [I FREE ] = [I TTAL ] A similar derivation for ratiometric quantification gives: [Ca] = K D x C(R - R MI )/(R MAX - R) where C is is the relative change in fluorescence between zero and saturating [Ca] at the denominator wavelength of the ratio (see Grynkiewicz, Poenie and Tsien, 1985, J. Biol. Chem 260 p3440 for full derivation) For both quantification methods, BEWARE of buffering and saturation effects, which can significantly alter the amplitude and time course of the recorded signals.
17 Ratiometric Measurement There must be a change in the shape (not just the amplitude) of the spectrum, so not possible for all indicators Corrects for changes in indicator concentration and excitation light intensity Easier quantification, as R MAX will be a known constant on a given experimental setup, whereas for nonratio indicators F MAX needs to be determined for each experiment Can utilise changes in either excitation spectrum or emission spectrum Detection of changes in emission spectrum is somewhat easier (use two detectors) Detection of of changes in excitation spectrum requires sequential measurements at different wavelengths (e.g. use a monochromator) A versatile measurement system should be able to handle both methods May be possible with either method to use more than one indicator at the same time (if their spectra are sufficiently different)
18 Ratio change on K+ perfusion: movement artefacts on the 340nm and 380nm traces don t show up on the ratio (vertical location of 340 and 380nm traces is arbitrary)
19 Loading Indicators Into Cells Indicator needs to be membrane-impermeant in order to remain in the cell, but how do we put it there? Microinjection can work well for large cells, but increasingly difficult for smaller cells. Shotgun approach using coated gold beads can also work well (e.g. for tissue slices). ther membrane disruptive techniques such as electroporation may also be possible. Can introduce polymeric (dextran linked) forms for cells that tend to expel small foreign molecules (e.g. plants). Ester loading can work well for smaller cells; esterified forms are membrane permeant, and so can enter cell, then are hydrolysed to the active free (membraneimpermeant) forms by internal esterases. But internal compartments other than cytosol may also be loaded, depending on location of the esterases. Genetic expression of a protein-based indicator is potentially very attractive, as expression can be targeted to specific cell compartments and the cells come readyloaded. But problems here include time and effort to make the constructs, and generally more limited signal range than for chemically synthesised indicators. In ALL CASES the presence of the indicator itself (e.g. buffering effects), as well as the means of its introduction, may disturb what you are trying to measure, so do remember this when planning experiments (e.g. use several different concentrations).
20 A fluorescence microscope (lympus BX series)
21 Filters and Dichroic Mirrors Transmission spectra for FM-143 filter set Specialist coatings permit filters with almost any optical profile to be created
22 High pressure Arc Lamps Arc Profile: Luminous intensity variation along lamp axis 75W lamp < 1mm Luminous Intensity distribution curve
23 Arc Lamp Emission Spectra Xenon Arc : Mercury Arc :
24 High Intensity LEDs Blue wavelengths now especially powerful, but more power in the green would still be useful. Available wavelengths now down to 340nm (Fura2 excitation!) Lower spectrum is of a white lamp actually blue with a phosphor.
25 Diffraction Grating Diffractive maxima occur at angles where the path length differences are an integral number of wavelengths. Thus longer wavelengths are diffracted at greater angles.
26 Monochromator peration Image of input slit formed at exit slit Triangular bandpass profile ptics must be matched to rest of optical system to maximise throughput
27 Detection Systems PHTMULTIPLIERS: Cheap, fast, sensitive, can use adjustable diaphragm to determine field of view, can easily record data in same acquisition system as e.g. electrical data. Why not buy one or two to use alongside your expensive CCD camera? PHTDIDES: More background noise than photomultipliers, so not suitable for weak signals. Avalanche photodiodes (APDs) are better in this respect, but still inferior. However, photodiodes do have a higher quantum (i.e. detection) efficiency, so may be better for strong signals, where photon noise dominates. CCD CAMERAS: More expensive, generally slower and more limited signal range, but give nice pictures for journal covers. 3D deconvolution in software can give reasonable depth resolution, but you need to record a vertical stack of images in order to to this. Cameras and software now much improved over earlier systems. LASER SCAIG CFCAL: Unlike the above methods, out-of-focus light is rejected, allowing true vertical sectioning. Potentially much more detail, but tradeoff is longer acquisition time for a full image, although e.g. linescan mode can be fast. Range of usable dyes restricted by availability/cost of appropriate lasers. Two-photon systems potentially even better, but at a price. IPKW DISC CFCAL: Scans multiple points on the specimen simultaneously, and images onto a CCD camera. ice idea in principle, also gives nice pictures, but depth resolution is poorer. ne problem seems to be that the optimum hole pattern in the spinning disc depends on the microscope objective, so a compromise here.
28 Dotted lines show how response can be extended into the UV if the device has a quartz instead of glass window. Photodetector QE
29 ipkow Disc Microscope
30 The Cairnfocal DMD-Based Confocal Microscope Attaches to the sideport of a fluorescence microscope The nonconjugate side images from the DMD's off pixels Light from DMD collected at 24 degrees focusses normally onto the cameras 62 is the DMD 66 and 66' are the conjugate and nonconjugate cameras 86 and 86' are the excitation light sources (generally just use 86)
31 The Cairnfocal DMD-Based Confocal Microscope
32 Cairnfocal Data! From Ashley Cadby Lab, Sheffield
33 oise on ptical Signals Detector will contribute noise, but this will be a constant amount, although it can vary widely between different types of detector. ptical signals are inherently noisy because of statistical variation in arrival times of individual photons this is termed shot noise. Shot noise increases with the square root of the signal level. Therefore stronger signals are also noisier, and the signal-to-noise ratio increases only with the square root of the signal level, rather than linearly as one might have expected. But there may be considerable scope for increasing the optical signal, as collection efficiency increases with the square of the numerical aperture (A) of the system. Can also increase illumination intensity of course, but there are practical limits (e.g. photobleaching). Since noise sources are uncorrelated, the overall noise level is the square root of the sum of the squares of the individual components, so the total noise level is determined primarily by the largest one (more so than if they added linearly). For weak signals, detector noise may dominate, so low noise here is important, e.g. photomultipliers, image intensifiers. For stronger signals, shot noise is more likely to dominate, favouring use of more efficient (higher QE) detectors, e.g. avalanche photodiodes, back-thinned CCD chips. Latest CCD cameras with on chip multiplication may offer high QE AD low detector noise, but the statistical nature of the multiplication is expected to increase the effective shot noise by 2, so even this is not a perfect solution!
Ratio Imaging. Dividing one image by another to detect changing conditions. Images collected at different times, wavelengths, polarities, etc
Ratio Imaging Dividing one image by another to detect changing conditions Images collected at different times, wavelengths, polarities, etc Most common use of ratio imaging is to provide a quick spectral
More informationADVANCED 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
More informationWhy 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
More informationShreyash 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
More informationLSM 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
More informationExamination, 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
More informationApplications of Steady-state Multichannel Spectroscopy in the Visible and NIR Spectral Region
Feature Article JY Division I nformation Optical Spectroscopy Applications of Steady-state Multichannel Spectroscopy in the Visible and NIR Spectral Region Raymond Pini, Salvatore Atzeni Abstract Multichannel
More informationLight 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
More informationConfocal Microscopy. Kristin Jensen
Confocal Microscopy Kristin Jensen 17.11.05 References Cell Biological Applications of Confocal Microscopy, Brian Matsumoto, chapter 1 Studying protein dynamics in living cells,, Jennifer Lippincott-Schwartz
More informationArc Lamps and Monochromators for Fluorescence Microscopy
Topic Introduction Arc Lamps and Monochromators for Fluorescence Microscopy Rainer Uhl Fluorescence microscopy requires high photon-flux densities in the specimen plane. These intensities are only achieved
More informationConfocal, 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
More informationWelcome to: LMBR Imaging Workshop. Imaging Fundamentals Mike Meade, Photometrics
Welcome to: LMBR Imaging Workshop Imaging Fundamentals Mike Meade, Photometrics Introduction CCD Fundamentals Typical Cooled CCD Camera Configuration Shutter Optic Sealed Window DC Voltage Serial Clock
More informationChemistry 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.
More informationInvitation 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
More informationMultifluorescence 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
More information3D 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
More information3D 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
More informationImaging 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
More informationPrecision-tracking of individual particles By Fluorescence Photo activation Localization Microscopy(FPALM) Presented by Aung K.
Precision-tracking of individual particles By Fluorescence Photo activation Localization Microscopy(FPALM) Presented by Aung K. Soe This FPALM research was done by Assistant Professor Sam Hess, physics
More informationImaging 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
More informationNature Methods: doi: /nmeth Supplementary Figure 1. Schematic of 2P-ISIM AO optical setup.
Supplementary Figure 1 Schematic of 2P-ISIM AO optical setup. Excitation from a femtosecond laser is passed through intensity control and shuttering optics (1/2 λ wave plate, polarizing beam splitting
More informationDual-FL. World's Fastest Fluorometer. Measure absorbance spectra and fluorescence simultaneously FLUORESCENCE
Dual-FL World's Fastest Fluorometer Measure absorbance spectra and fluorescence simultaneously FLUORESCENCE 100 Times Faster Data Collection The only simultaneous absorbance and fluorescence system available
More informationAn 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.
More informationImproving the Collection Efficiency of Raman Scattering
PERFORMANCE Unparalleled signal-to-noise ratio with diffraction-limited spectral and imaging resolution Deep-cooled CCD with excelon sensor technology Aberration-free optical design for uniform high resolution
More informationBio 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,
More information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
More informationMore 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
More informationFlatness of Dichroic Beamsplitters Affects Focus and Image Quality
Flatness of Dichroic Beamsplitters Affects Focus and Image Quality Flatness of Dichroic Beamsplitters Affects Focus and Image Quality 1. Introduction Even though fluorescence microscopy has become a routine
More informationGuide 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
More informationa) 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
More informationDynamic 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
More informationAdvanced 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
More informationOPERATING 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
More informationPoint 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
More information/* */ Applications Physical background Technical data References Downloads. XtalLight 100C
/* */ Applications Physical background Technical data References Downloads XtalLight 100C XtalLight 100C is a combined UV-light and a green light source (100 nm and 20 nm spectral width respectively).
More informationFundamentals 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*
More informationTraining 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
More informationBasics 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
More informationANSWER KEY Lab 2 (IGB): Bright Field and Fluorescence Optical Microscopy and Sectioning
Phys598BP Spring 2016 University of Illinois at Urbana-Champaign ANSWER KEY Lab 2 (IGB): Bright Field and Fluorescence Optical Microscopy and Sectioning Location: IGB Core Microscopy Facility Microscope:
More informationComponents 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
More informationZeiss 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?
More informationLab4 Hanbury Brown and Twiss Setup. Photon Antibunching
Lab4 Hanbury Brown and Twiss Setup. Photon Antibunching Shule Li Abstract Antibunching is a purely quantum effect and cannot be realized from the classical theory of light. By observing the antibunching
More informationPractical 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
More informationFinal 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
More informationQuick 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
More informationNature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
Supplementary Figure 1 Supplemental correlative nanomanipulation-fluorescence traces probing nascent RNA and fluorescent Mfd during TCR initiation. Supplemental correlative nanomanipulation-fluorescence
More informationTraining 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
More informationImage Capture TOTALLAB
1 Introduction In order for image analysis to be performed on a gel or Western blot, it must first be converted into digital data. Good image capture is critical to guarantee optimal performance of automated
More informationDetectors for microscopy - CCDs, APDs and PMTs. Antonia Göhler. Nov 2014
Detectors for microscopy - CCDs, APDs and PMTs Antonia Göhler Nov 2014 Detectors/Sensors in general are devices that detect events or changes in quantities (intensities) and provide a corresponding output,
More informationZeiss 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
More informationPCS-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
More informationMicroscope anatomy, image formation and resolution
Microscope anatomy, image formation and resolution Ian Dobbie Buy this book for your lab: D.B. Murphy, "Fundamentals of light microscopy and electronic imaging", ISBN 0-471-25391-X Visit these websites:
More informationTraining 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
More informationThings to check before start-up.
Byeong Cha Page 1 11/24/2009 Manual for Leica SP2 Confocal Microscope Enter you name, the date, the time, and the account number in the user log book. Things to check before start-up. Make sure that your
More informationDigital 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
More information1 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
More informationOperation 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
More informationCFIM MICROSCOPY COURSE PROGRAMME PRINCIPLES OF MICROSCOPY CONFOCAL AND FLUORESCENCE MICROSCOPY
CFIM MICROSCOPY COURSE PROGRAMME PRINCIPLES OF MICROSCOPY 11.01.16-15.01.2016 CONFOCAL AND FLUORESCENCE MICROSCOPY 25.01.16-29.01.2016 PhD Course - University of Copenhagen Department of Biomedical Sciences
More informationMicroscopy. The dichroic mirror is an important component of the fluorescent scope: it reflects blue light while transmitting green light.
Microscopy I. Before coming to lab Read this handout and the background. II. Learning Objectives In this lab, you'll investigate the physics of microscopes. The main idea is to understand the limitations
More informationConfocal 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
More informationLast 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
More informationBASICS 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
More informationCavity QED with quantum dots in semiconductor microcavities
Cavity QED with quantum dots in semiconductor microcavities M. T. Rakher*, S. Strauf, Y. Choi, N.G. Stolz, K.J. Hennessey, H. Kim, A. Badolato, L.A. Coldren, E.L. Hu, P.M. Petroff, D. Bouwmeester University
More informationComponents of Optical Instruments 1
Components of Optical Instruments 1 Optical phenomena used for spectroscopic methods: (1) absorption (2) fluorescence (3) phosphorescence (4) scattering (5) emission (6) chemiluminescence Spectroscopic
More informationCHAPTER 9 POSITION SENSITIVE PHOTOMULTIPLIER TUBES
CHAPTER 9 POSITION SENSITIVE PHOTOMULTIPLIER TUBES The current multiplication mechanism offered by dynodes makes photomultiplier tubes ideal for low-light-level measurement. As explained earlier, there
More informationLSM 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.
More informationTRAINING 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
More informationAqualog. Water Quality Measurements Made Easy FLUORESCENCE
Aqualog Water Quality Measurements Made Easy FLUORESCENCE Water quality measurements made easy The only simultaneous absorbance and fluorescence system for water quality analysis! The new Aqualog is the
More informationAqualog. Water Quality Measurements Made Easy PARTICLE CHARACTERIZATION ELEMENTAL ANALYSIS FLUORESCENCE
Aqualog Water Quality Measurements Made Easy ELEMENTAL ANALYSIS FLUORESCENCE GRATINGS & OEM SPECTROMETERS OPTICAL COMPONENTS PARTICLE CHARACTERIZATION RAMAN SPECTROSCOPIC ELLIPSOMETRY SPR IMAGING Water
More informationHow is the Digital Image Generated? Image Acquisition Devices
In order for image analysis to be performed on a 2D gel, it must first be converted into digital data. Good image capture is critical to guarantee optimal performance of automated image analysis packages
More informationPurchasing a Back-illuminated scmos for Microscopy? Seven Reasons To Choose Sona
Purchasing a Back-illuminated scmos for Microscopy? Seven Reasons To Choose Sona Dr. Colin Coates, Andor July 2018 Technical Note Purchasing a Back-illuminated scmos for Microscopy: 7 Reasons to Choose
More informationMulti-channel imaging cytometry with a single detector
Multi-channel imaging cytometry with a single detector Sarah Locknar 1, John Barton 1, Mark Entwistle 2, Gary Carver 1 and Robert Johnson 1 1 Omega Optical, Brattleboro, VT 05301 2 Philadelphia Lightwave,
More informationSpectroscopy 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
More informationYou 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
More informationSpatially Resolved Backscatter Ceilometer
Spatially Resolved Backscatter Ceilometer Design Team Hiba Fareed, Nicholas Paradiso, Evan Perillo, Michael Tahan Design Advisor Prof. Gregory Kowalski Sponsor, Spectral Sciences Inc. Steve Richstmeier,
More informationNon-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
More information3. are adherent cells (ie. cells in suspension are too far away from the coverslip)
Before you begin, make sure your sample... 1. is seeded on #1.5 coverglass (thickness = 0.17) 2. is an aqueous solution (ie. fixed samples mounted on a slide will not work - not enough difference in refractive
More informationSpectroscopy of Ruby Fluorescence Physics Advanced Physics Lab - Summer 2018 Don Heiman, Northeastern University, 1/12/2018
1 Spectroscopy of Ruby Fluorescence Physics 3600 - Advanced Physics Lab - Summer 2018 Don Heiman, Northeastern University, 1/12/2018 I. INTRODUCTION The laser was invented in May 1960 by Theodor Maiman.
More informationAkinori Mitani and Geoff Weiner BGGN 266 Spring 2013 Non-linear optics final report. Introduction and Background
Akinori Mitani and Geoff Weiner BGGN 266 Spring 2013 Non-linear optics final report Introduction and Background Two-photon microscopy is a type of fluorescence microscopy using two-photon excitation. It
More informationRenishaw InVia Raman microscope
Laser Spectroscopy Labs Renishaw InVia Raman microscope Operation instructions 1. Turn On the power switch, system power switch is located towards the back of the system on the right hand side. Wait ~10
More informationObservational Astronomy
Observational Astronomy Instruments The telescope- instruments combination forms a tightly coupled system: Telescope = collecting photons and forming an image Instruments = registering and analyzing the
More informationSpark Spectral Sensor Offers Advantages
04/08/2015 Spark Spectral Sensor Offers Advantages Spark is a small spectral sensor from Ocean Optics that bridges the spectral measurement gap between filter-based devices such as RGB color sensors and
More informationPowerful DMD-based light sources with a high throughput virtual slit Arsen R. Hajian* a, Ed Gooding a, Thomas Gunn a, Steven Bradbury a
Powerful DMD-based light sources with a high throughput virtual slit Arsen R. Hajian* a, Ed Gooding a, Thomas Gunn a, Steven Bradbury a a Hindsight Imaging Inc., 233 Harvard St. #316, Brookline MA 02446
More informationUniversity of Wisconsin Chemistry 524 Spectroscopic Components *
University of Wisconsin Chemistry 524 Spectroscopic Components * In journal articles, presentations, and textbooks, chemical instruments are often represented as block diagrams. These block diagrams highlight
More informationMulticolor 4D Fluorescence Microscopy using Ultrathin Bessel Light sheets
SUPPLEMENTARY MATERIAL Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light sheets Teng Zhao, Sze Cheung Lau, Ying Wang, Yumian Su, Hao Wang, Aifang Cheng, Karl Herrup, Nancy Y. Ip, Shengwang
More informationthe need for an intensifier
* The LLLCCD : Low Light Imaging without the need for an intensifier Paul Jerram, Peter Pool, Ray Bell, David Burt, Steve Bowring, Simon Spencer, Mike Hazelwood, Ian Moody, Neil Catlett, Philip Heyes Marconi
More informationTechnology 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,
More informationSupplementary Information. Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots
Supplementary Information Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots Bin Dong 1,, Xiaochen Yang 2,, Shaobin Zhu 1, Diane C.
More informationIntroduction 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
More informationExperimental Analysis of Luminescence in Printed Materials
Experimental Analysis of Luminescence in Printed Materials A. D. McGrath, S. M. Vaezi-Nejad Abstract - This paper is based on a printing industry research project nearing completion [1]. While luminescent
More informationplasmonic nanoblock pair
Nanostructured potential of optical trapping using a plasmonic nanoblock pair Yoshito Tanaka, Shogo Kaneda and Keiji Sasaki* Research Institute for Electronic Science, Hokkaido University, Sapporo 1-2,
More informationAqualog. CDOM Measurements Made Easy PARTICLE CHARACTERIZATION ELEMENTAL ANALYSIS FLUORESCENCE GRATINGS & OEM SPECTROMETERS OPTICAL COMPONENTS RAMAN
Aqualog CDOM Measurements Made Easy ELEMENTAL ANALYSIS FLUORESCENCE GRATINGS & OEM SPECTROMETERS OPTICAL COMPONENTS PARTICLE CHARACTERIZATION RAMAN SPECTROSCOPIC ELLIPSOMETRY SPR IMAGING CDOM measurements
More informationCell Biology and Bioimaging Core
Cell Biology and Bioimaging Core Leica TCS SP5 Operating Instructions Starting up the instrument 1. First, log in the log book located on the confocal desk. Include your name, your lab s PI, an account
More informationOpterra 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
More informationBoulevard 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
More informationNikon. King s College London. Imaging Centre. N-SIM guide NIKON IMAGING KING S COLLEGE LONDON
N-SIM guide NIKON IMAGING CENTRE @ KING S COLLEGE LONDON Starting-up / Shut-down The NSIM hardware is calibrated after system warm-up occurs. It is recommended that you turn-on the system for at least
More informationSupplementary Information for. Surface Waves. Angelo Angelini, Elsie Barakat, Peter Munzert, Luca Boarino, Natascia De Leo,
Supplementary Information for Focusing and Extraction of Light mediated by Bloch Surface Waves Angelo Angelini, Elsie Barakat, Peter Munzert, Luca Boarino, Natascia De Leo, Emanuele Enrico, Fabrizio Giorgis,
More information:... resolution is about 1.4 μm, assumed an excitation wavelength of 633 nm and a numerical aperture of 0.65 at 633 nm.
PAGE 30 & 2008 2007 PRODUCT CATALOG Confocal Microscopy - CFM fundamentals :... Over the years, confocal microscopy has become the method of choice for obtaining clear, three-dimensional optical images
More informationRates 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,
More informationLSM 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.
More information