Fundamentals of Light Microscopy II: Fluorescence, Deconvolution, Confocal, Multiphoton, Spectral microscopy. Integrated Microscopy Course
|
|
- Barry Dennis
- 5 years ago
- Views:
Transcription
1 Fundamentals of Light Microscopy II: Fluorescence, Deconvolution, Confocal, Multiphoton, Spectral microscopy Integrated Microscopy Course
2 Review Lecture 1: Microscopy Basics Light train Kohler illumination* Getting an image Diffraction and refraction Objectives Special techniques Dark field Phase Contrast Differential Interference Contrast
3 Lecture Objectives 1. Understand the Principles of Florescence Microscopy 2. Understand how Deconvolution Improves Resolution 3. Understand How Laser Scanning Confocals work 4. Understand the Principles of Spectral Unmixing
4 Light Spectrum Visible Spectrum = 400nm to 750nm Planck s Law: E=hc/l where h is Planck s constant, c is the speed of light, and l is the wavelength of a photon Shorter Wavelength 1. Higher energy 2. More Harmful to cells 3. More easily scattered Longer Wavelength Lower energy Less harmful to cells Less easily scattered
5 Fluorescence First described in the 1800 s by Sir George Stokes (fluorspar) Light emission that continues only during the absorption of the excitation light Stokes Shift: the phenomenon in which the emitted light has longer wavelength than the excitation light
6 Jablonski Diagram 3 S Vibrational relaxation 0 3 Internal conversion Excited Singlet States S Vibrational relaxation 0 Emission light Heat Quenching 3 Excitation light S Ground state energy levels 2
7 Characteristics of Fluorescence 1. Because of the differing rotational, vibrational and even electronic states a molecule can have, peaks are BROAD. 2. Each spectrum is characteristic of a given molecule. 3. Before returning to its normal state, the electron remain at the excited level for approximately seconds. 4. Because the excited state is HIGHLY REACTIVE it can bleach (react with other molecules). 5. Everything is a point of light. Light goes in all directions.
8 Fluorescent Microscope (Epi-fluorescence) Excitation Wavelength Emission Filter Emission Wavelength Dichroic Beam splitter Excitation Filter 3 Major Parts 1. Excitation Filter (Ex) 2. Dichroic Beam splitter 3. Emission Filter (Em) Objective = Condenser Sample
9 Filter Spectrum FITC TxRed FITC Filter Set Texas Red Filter Set Ex Filter Dichroic Em Filter Ex Filter Dichroic Em Filter
10 Problem: Fluorophore Spectral Overlap With broad fluorescent peaks and broad collection parameters, fluorescence from one fluorophore can appear in a second channel Alexa Dye Excitations with FITC Filter Alexa Dye Emissions with FITC Filter You can mistake yellow or orange for green fluorophore! How to correct for this? One way is to do image subtraction Label with 1 fluorophore and see how bad the bleed through is in second channel. Get intensity ratio then subtract out contribution from other fluorophore Example: Corrected Intensity Green = Measured Intensity Green X% Intensity Orange Confocal allows better ways!
11 Resolution of Fluorescence Microscopy Bright field Max NA of a Oil Objective n (oil) = NA (oil) = (1.515) (1.00) = 1.5 Epi-Fluorescence Max NA of a Oil Objective n (oil) = NA (oil) = (1.515) (1.00) = 1.5 Max resolution for a oil objective under Bright Field d = 1.22λ / (NA ob + NA cond) λ = 546nm (green) d = 1.22 (546nm) / ( ) d = 266nm Max resolution for a oil objective under Epi-Florescence d = 1.22λ / (2 * NA obj) λ = 546nm (green) d = 1.22 (546nm) / (2* 1.5) d = 222nm * It is possible to detect fluorescence from smaller structures but it will be impossible to resolve them*
12 More pixels equals larger field of view Field of View (CCD)
13 What affects resolution? Size of pixel System Magnification & NA Camera Coupler Size of pixel determines the resolution Smaller pixels provide for better resolution Size of pixel also relates to sensitivity of the camera Larger pixels provide higher sensitivity Higher Magnification and Higher NA allows for use of larger pixels. Low magnification requires smaller pixels
14 Why does pixels size matter? Our ability to distinguish fine detail is determined by pixel size Resolution! Nyquist theory states that we must sample at 2.3 times the highest spatial frequency we wan to resolve. We can satisfy this requirement by varying pixel size.
15 Resolution and Pixel Size Small pixels Large pixels
16 Sampling and Pixels
17 When should I be concerned about Bit Depth? -When there are very bright and very dim areas in the sample you need to image -When you are making intensity measurements
18 Bit Depth Bit depth describes the range of intensity that the CCD can capture Cameras that have high bit depth usually are more sensitive to light Our eyes and computer monitors can only process ~ 8 bits or 256 shades of gray. Why do we have 12bit, 14bit and 16bit cameras?
19 Bit Depth What is Bit Depth? The number of different intensities that the camera can capture Usually we think of it as number of shades of gray Shades of gray is like a length ruler, but for intensity
20 Bit Depth Bit Depth is usually a number of 2 n 8 bit = 2 8, or 256 gray tones 12 bit = 2 12, or 4096 gray tones 16 bit = 2 16, or 65,536 grays tones More bits equals more contrast More bits equals better measurement of brightness = MORE DATA!
21
22 Fluorescence Microscopy Pros and Cons of Fluorescence Microscopy Pros Allows for multiple labels Techniques: Immunofluorescence, FISH Sensitive Relatively inexpensive Cons Out of focus light blurs image. Must have fluorescent reporter Fluorescent bleed through Difficult with thick sections
23 Challenges Spectral Overlap: Broad peaks cross over into one another. One fluorophore comes off in 2 channels Blur: Out of focus light decreases resolution Bleaching: Excited fluorophores react to become non-fluorescent Phototoxicity: Light can harm cells Background/Autofluorescence: Cells have fluorophores too. May look like the ones you want to examine.
24 Deconvolution Microscopy Computationally Increases Image Resolution
25 Deconvolution Microscopy and 3D Reconstruction X-Y X-Z Definitions 1. PSF (Point spread function) how a point of light reacts when it is in focus and out of focus, determined experimentally or back calculated from the collected images. 2. Voxel (volume element) 3- dimensional pixel Key points All fluorescent molecules emit light, both in focus and out of focus an image always blurred by the contribution of light from structures which are out of focus phenomenon can be relatively well defined by the optical properties of the image formation in the microscope imaging system Z X Y voxel
26 What does Deconvolution Do? Deconvolution looks at each voxel (3D pixel) and determines its relationship with the voxels around it. Then it either subtracts out what it calculates is blur from nearby bright voxels (easy way) or it moves out of focus light back to its voxel of origin (hard way). Raw Data Deconvolved Data
27 Methods for Deconvolution 1. Non-Restorative Algorithms (unsharp mask, nearest neighbors, no neighbors, multiple neighbors) Subtract a given amount of light from nearby voxels to account for blur effect. A. Relatively cheap, very simple to do, quick, computationally undemanding. Doable with Metamorph. B. Subtractive Trade higher contrast for decreased signal. UNUSABLE for quantitation.
28 2. Restorative Algorithms (inverse filter, constrained iterative deconvolution, maximum likelihood estimation, blind deconvolution and many others) A mathematical method for applying the point-spread function (PSF) to remove and/or reassign the "out-of-focus" blur in an image specimen. - NOT subtractive Premises a. Since the image has been acted on by the PSF, a set of weighted sines and cosines will represent this. Use Fourier Transformation to calculate these. c. Divide fourier transformation of measured image by fourier transformation of the PSF (the OTF) and you should get closer to true image. Perfect if all values were right. d. Reblur with original PSF and you should get back to observed image.
29 Types of Restorative Algorithm Inverse Filter does this once and stops. Can intensify noise and produce artifacts. Quicker. Constrained iterative deconvolution keeps trying to improve image by decreasing difference between reblurred and original. Certain constraints applied to solution. Slower and computationally intense. Maximum likelihood estimation further applies statistical calculations to noise and is still more computationally intense. May be better for noisy data Blind deconvolution can refer to either using an empirically determined PSF or determining the exact PSF as you go.
30 Typical Constrained Iterative Deconvolution Output Standard Avg Counts Iteration Residual (R) Residual Normalized R The Normalized R is the best way to see if the image is still improving
31 Differences Between Methods: There is a reason to go through iterative solutions CID Original Nearest Neighbor
32 Deconvolution Microscopy Raw Deconvolved Pros and Cons of Deconvolution Microscopy Pros Allows for higher Resolution Allows collection of image plane stacks Hg Bulb allows for variety of filter combinations Can get superior sensitivity Cheaper than confocal Cons Computationally intense if iterative Minimal penetration of thick sections Sensitive to spherical aberrations Can amplify noise, produce artifacts Requires 3D data set
33 Confocal Microscopy Physically Increases Image Resolution
34 Laser Confocal Scanning Microscopy Laser 1980 s Dichroic Beam Splitter Photo- Multiplier Tube (PMT) Detector Objective Confocal Pin Hole Out of Focus Sample In Focus Out Of Focus
35 Laser Light Sources Lasers Bulb
36 Laser Confocal Scanning Microscopy The laser beam excites a point on the specimen. It also inadvertently excites other points on the specimen. Only the In-Focus emission light is allowed to be detected by the PMT. The Light detected by the PMT is associated to a pixel (picture element) on the monitor. The laser beam then moves to the the next point and another pixel is collected. Size Confocal Pinhole Result on the Intensity of the Image Result on the Resolution of the Image Increase opening Increase Intensity decrease resolution Decrease opening Decrease intensity increase resolution
37 Laser Confocal Scanning Microscopy No Pin Hole 50um Pin Hole Pros and Cons of Confocal Microscopy Pros Allows for higher resolution Allow collection of stacks of image planes and 3D reconstruction Laser penetrates somewhat thick sections Better control of bleed through/autofluorescence Faster than deconvolution Cons Limited EX peaks on lasers Phototoxicity (up to a 40º C temp jump at focal point) Loss of image intensity Fairly expensive Prone to Photobleaching Precise Laser Positioning (FRAP)
38 Multiphoton Microscopy Optically increases resolution
39 Multiphoton Microscopy ( nm) Based on phenomena described by Maria Goppert- Mayer (1931) Two photons of low energy (long wavelength) can excite a fluorophore resulting in emission of a photon of higher energy (short wavelength) Requires nearsimultaneous absorption of the two photons ( nm)
40 Multi-Photon Confocal Microscopy Characteristics 1. Attosecond pulses only properly strike at the In-Focus plane of the specimen. 2. Multiple short pulses of longer wavelength light have the same amount of energy as one long pulse of shorter wavelength. Pros and Cons of Multi-Photon Microscopy Pros More penetrating up to hundreds of micron No out of focus light Laser is tunable. Can get range of wavelengths including UV dyes. Better for Living cells if you re careful. Cons Best excitation wavelength is not always obvious Cost (Very Expensive) Laser is POWERFUL. Can damage cells, slides and even walls. Difficult to align and use (Less so now) Can also kill off specific cells/organelles.
41 Multi-Photon Images Mouse Liver Tissue Mouse Cardiac Tissue Grey = hepatocytes Green = collagen Green = collagen Red = myocytes
42 Spectral Unmixing Computationally Resolves Spectral Overlap
43 Spectral Unmixing Allows one to use and resolve multiple fluorophores with highly overlapping emission spectra Specific Cases: You need to use fluorophores with highly overlapping signal (GFP and YFP; Mitotracker red and dsred)
44 Emission Fingerprinting - the Method 1. *contains spectral information for each pixel 2. Instead of using an emission filter, spectral unmixing relies on obtaining intensity values for each pixel at a number of wavelengths, and then creating a spectrum. Next, the user compares this spectrum to defined spectra for different fluorophores. After mathematic comparison, you can determine the true value of each fluorophore present. 3.
45 Linear Unmixing How it works! Unmixing separates the total emission signal into weighted contributions of each dye based on the knowledge of their emission fingerprints pixel by pixel! Combined spectra = a x GFP + b x YFP
46 A final problem data management (Or: Where is my file?!! ) Answer save files with a fixed filename such as: Initial for scope 1ss1022ad Slide number Field of view Your initials The date Second answer Save in a fixed place on server
47 Imaging Ethics Don t believe anything you read and only half of what you see Assume accurate representation of reality Images = data Spatially arranged in xy matrix Each pixel represents image intensity Numerical sampling of the specimen as presented by the data acquisition system to the sensor
48 Imaging Ethics Always do manipulations on copy Original on CD/DVD/checksum Protects you from allegations of fraud Simple adjustments are OK Darkroom techniques contrast, gamma Cropping OK avoid acquisition bias Honesty best policy document in methods
49 Imaging Ethics Digital images that will be compared to one another should be acquired under identical conditions, and any post- acquisition image processing should also be identical. Use of software filters to improve image quality is usually not recommended for biological images. Collage/cloning image is very questionable Don t use lossy file types Resolution and magnification are important
50 References: 1. Molecular Probes 2. Chroma Filters 3. Omega Optical 4. Zeiss Microscopes 5. Applied Precision Deconvolution 6. Fluorescent Protein Spectra 7. Free Image Processing Program ` 8. Confocal List serve listserv.acsu.buffalo.edu/cgi-bin/wa?s1=confocal 9. Microscopy List serve Olympus Nikon
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
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 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 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 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 informationMaria 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
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 informationZeiss 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
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 informationMULTIPHOTON 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/
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 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 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 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 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 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 informationImaging Introduction. September 24, 2010
Imaging Introduction September 24, 2010 What is a microscope? Merriam-Webster: an optical instrument consisting of a lens or combination of lenses for making enlarged images of minute objects; especially:
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 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 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 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 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 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 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 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 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 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 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 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 informationLeica 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
More informationIntroduction 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
More informationSupplemental 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
More informationLight Microscopy. Upon completion of this lecture, the student should be able to:
Light Light microscopy is based on the interaction of light and tissue components and can be used to study tissue features. Upon completion of this lecture, the student should be able to: 1- Explain the
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 informationNikon Instruments Europe
Nikon Instruments Europe Recommendations for N-SIM sample preparation and image reconstruction Dear customer, We hope you find the following guidelines useful in order to get the best performance out of
More informationFLUORESCENCE MICROSCOPY. Matyas Molnar and Dirk Pacholsky
FLUORESCENCE MICROSCOPY Matyas Molnar and Dirk Pacholsky 1 The human eye perceives app. 400-700 nm; best at around 500 nm (green) Has a general resolution down to150-300 μm (human hair: 40-250 μm) We need
More informationResolution. 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),
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 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 information(Quantitative Imaging for) Colocalisation Analysis
(Quantitative Imaging for) Colocalisation Analysis or Why Colour Merge / Overlay Images are EVIL! Special course for DIGS-BB PhD program What is an Image anyway..? An image is a representation of reality
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 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 informationObserving Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens. Compound Light Micros
PHARMACEUTICAL MICROBIOLOGY JIGAR SHAH INSTITUTE OF PHARMACY NIRMA UNIVERSITY Observing Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens.
More informationTraining 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
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 informationBi/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
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 informationINTRODUCTION 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
More informationRatio 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 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 informationMULTIPHOTON MICROSCOPY
MULTIPHOTON MICROSCOPY Methods for Cell Analysis Course BioVis Uppsala, 2014 Matyas Molnar Dirk Pacholsky Information Information given here about 2 Photon microscopy were mainly taken from these sources:
More informationEUV microscopy - a user s perspective Dimitri Scholz EUV,
EUV microscopy - a user s perspective Dimitri Scholz EUV, 09.11.2011 Imaging technologies: available at UCD now and in the next future Begin ab ovo - Simple approaches direct to the goal - Standard methods
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 informationLeica 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
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 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 informationZEISS 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
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 informationConfocal 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
More informationSupplemental Method Information Zeiss LSM710
Supplemental Method Information Zeiss LSM710 1 Under the Light Path window set up the confocal for imaging a green dye (Alexa488-EGFP). For example, set up the light path as shown here using the 488 nm
More informationDevelopment 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
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 information5/4/2015 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
More informationBi 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
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 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 informationΕισαγωγική στην Οπτική Απεικόνιση
Εισαγωγική στην Οπτική Απεικόνιση Δημήτριος Τζεράνης, Ph.D. Εμβιομηχανική και Βιοϊατρική Τεχνολογία Τμήμα Μηχανολόγων Μηχανικών Ε.Μ.Π. Χειμερινό Εξάμηνο 2015 Light: A type of EM Radiation EM radiation:
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 informationHigh resolution extended depth of field microscopy using wavefront coding
High resolution extended depth of field microscopy using wavefront coding Matthew R. Arnison *, Peter Török #, Colin J. R. Sheppard *, W. T. Cathey +, Edward R. Dowski, Jr. +, Carol J. Cogswell *+ * Physical
More informationLecture 23 MNS 102: Techniques for Materials and Nano Sciences
Lecture 23 MNS 102: Techniques for Materials and Nano Sciences Reference: #1 C. R. Brundle, C. A. Evans, S. Wilson, "Encyclopedia of Materials Characterization", Butterworth-Heinemann, Toronto (1992),
More informationMicroscope 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
More informationLeica 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
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 informationMicroscopy 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
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 informationUser manual for Olympus SD-OSR spinning disk confocal microscope
User manual for Olympus SD-OSR spinning disk confocal microscope Ved Prakash, PhD. Research imaging specialist Imaging & histology core University of Texas, Dallas ved.prakash@utdallas.edu Once you open
More informationConfocal Imaging Through Scattering Media with a Volume Holographic Filter
Confocal Imaging Through Scattering Media with a Volume Holographic Filter Michal Balberg +, George Barbastathis*, Sergio Fantini % and David J. Brady University of Illinois at Urbana-Champaign, Urbana,
More informationTravel 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
More informationINTRODUCTION TO OPTICAL MICROSCOPY
Experimental Biophysics TEK265, FYST23, TNF060, FAF010F Lab Exercise Supervisor: Karl Adolfsson Written by Peter Jönsson and Jason Beech Updated by Henrik Persson, Karl Adolfsson and Zhen Li karl.adolfsson@ftf.lth.se
More informationIntroduction 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
More informationMicroscopy Live Animal Imaging
Microscopy Live Animal Imaging A collaborative environment that provides the knowledge, instruments, and expertise needed to visualize life at scales ranging from single molecules to entire animals. Project
More informationOperating Instructions for Zeiss LSM 510
Operating Instructions for Zeiss LSM 510 Location: GNL 6.312q (BSL3) Questions? Contact: Maxim Ivannikov, maivanni@utmb.edu 1 Attend A Complementary Training Before Using The Microscope All future users
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 informationNikon AZ100. Laser Scanning Macro Confocal Microscope. Jordan Briscoe Adam Fries Kyle Marchuk Kaitlin Corbin. May 2017.
Nikon AZ100 Laser Scanning Macro Confocal Microscope Jordan Briscoe Adam Fries Kyle Marchuk Kaitlin Corbin May 2017 Contents 1 Introduction 2 2 Hardware - Startup 2 3 Software/Operation 4 3.1 Multidimensional
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 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 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 informationMIF ZEISS LSM510 CONFOCAL USER PROTOCOL
MIF ZEISS LSM510 CONFOCAL USER PROTOCOL START-UP Turn on the Mercury Bulb Power Supply (if needed). Power-on the Control Box. Turn on the computer. Open the LSM 510 software. Choose Scan New Images and
More informationLeica_Dye_Finder :53 Uhr Seite 6 Dye Finder LAS AF
Dye Finder LAS AF Dye Finder Multicolor live cell fluorescence microscopy is limited by the availability of spectrally separable fluorescent dyes. Fluorescent dyes (or spectral GFP variants) with incongruent
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 informationNature Methods: doi: /nmeth Supplementary Figure 1. Comparison of HySP and linear unmixing under different signal-to-noise ratios (SNRs).
Supplementary Figure 1 Comparison of HySP and linear unmixing under different signal-to-noise ratios (SNRs). (a) TrueColor images of 32 channel datasets of zebrafish labeled with H2B-Cerulean, kdrl:egfp,
More informationVery short introduction to light microscopy and digital imaging
Very short introduction to light microscopy and digital imaging Hernan G. Garcia August 1, 2005 1 Light Microscopy Basics In this section we will briefly describe the basic principles of operation and
More informationIntroduction 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 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 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 informationQuick Guide. LSM 5 MP, LSM 510 and LSM 510 META. Laser Scanning Microscopes. We make it visible. M i c r o s c o p y f r o m C a r l Z e i s s
LSM 5 MP, LSM 510 and LSM 510 META M i c r o s c o p y f r o m C a r l Z e i s s Quick Guide Laser Scanning Microscopes LSM Software ZEN 2007 August 2007 We make it visible. Contents Page Contents... 1
More informationConfocal 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
More informationSingle-photon excitation of morphology dependent resonance
Single-photon excitation of morphology dependent resonance 3.1 Introduction The examination of morphology dependent resonance (MDR) has been of considerable importance to many fields in optical science.
More informationNikon C1si Spectral Laser Scanning Confocal Microscope. User Guide
Nikon C1si Spectral Laser Scanning Confocal Microscope User Guide Contents: C1Si Turn-On/ShutDown Procedures... 2 Overview... 4 Setup for epi-illumination to view through the eyepieces:... 5 Setup for
More informationOperating Checklist for using the Laser Scanning Confocal Microscope. Leica TCS SP5.
Smith College August 2010 Operating Checklist for using the Laser Scanning Confocal Microscope Leica TCS SP5. CONTENT, page no. Startup, 1 Initial set-up, 1 Software, 2 Microscope Specimen observation
More information