Bioimage Informatics

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "Bioimage Informatics"

Transcription

1 Bioimage Informatics Lecture 5, Spring 01 Fundamentals of Fluorescence Microscopy (II) Bioimage Data Analysis (I): Basic Operations Lecture 5 January 5, 01 1

2 Outline Performance metrics of a microscope Basic image analysis: open sources of images Basic image analysis: image filtering Basic image analysis: image intensity derivative calculation Project assignment 1

3 Performance metrics of a microscope Basic image analysis: open sources of images Basic image analysis: image filtering Basic image analysis: image intensity derivative calculation Project assignment 1 3

4 Performance Metrics of a Light Microscope Resolution: the smallest feature distance that can be resolved. Field of view: the area of a specimen that can be observed and recorded in an image. Depth-of-field: the axial distance (depth) range in the specimen that appears in focus in an image. Light collection power: determines image brightness. 4

5 Basic Concept of a Linear System A system is said to be linear if it satisfies the following two conditions - Homogeneity - Additivity r(t) S y(t) A linear system can be characterized in the time domain by its impulse response. A properly built and aligned microscope can be accurately modeled as a linear system. 5

6 Microscope as a Linear System A light microscope is a linear system whose impulse response is an Airy disk. 6

7 Airy Disk Airy (after George Biddell Airy) disk is the diffraction pattern of a point feature under a circular aperture. It has the following form I I J r 1 0 r J 1 (x) is a Bessel function of the first kind. Detailed derivation is given in Born & Wolf, Principles of Optics, 7th ed., pp

8 Microscope Image Formation: PSF & OTF The impulse response of the microscope is called its point spread function (PSF). The transfer function of a microscope is called its optical transfer function (OTF). The PSF of a properly built and aligned microscopy is an Airy Disk. 8

9 Numerical aperture (NA) determines microscope resolution and light collection power. Numerical Aperture NA n sin n: refractive index of the medium between the lens and the specimen : half of the angular aperture 9

10 Microscope Image Formation Microscope image formation can be modeled as a convolution with the PSF. I x,y O x,y psf x,y F I x,y F O x,y F psf x,y 10

11 Different Definition of Light Microscopy Resolution Limit (Demo) Rayleigh limit D 061. NA Sparrow limit D 047. NA 11

12 Field of View (Demo) Field of view: the region that is visible under a microscope If characterized in diameter D If characterized in area S Field diaphragm diameter M Field diaphragm diameter M 1

13 Depth-of-Field Depth-of-field: the axial distance (depth) in the specimen that appears in focus in the image. d tot n NA n M NA e n: refractive index of the medium between the lens and the specimen : emission wavelength M: magnification NA: numerical aperture e: smallest resolvable distance in the image plane 13

14 Example: Depth-of-Field Smaug1 mrna-silencing foci respond to NMDA and modulate synapse formation, M. Baez, et al, JCB, 195: ,

15 Image Intensity: Light Collecting Power For transmitted light I NA M For epi-fluorescence I NA M

16 The distance between the objective lens and the specimen. Working Distance Working distance does not directly influence imaging but may determine how images can be collected. 16

17 Summary: High Resolution Microscopy Size of cellular features are typically on the scale of a micron or smaller. To resolve such features require - Shorter wavelength (e.g. electron microscopy) - High numerical aperture (for resolution) - High magnification (for spatial sampling) D 061. NA 17

18 Summary: High Resolution Microscopy Higher magnification and higher numerical aperture mean - Smaller field of view S Field diaphragm diameter M - Smaller depth of field d tot n NA n e M NA - Lower light collection power I NA M - Smaller working distance 18

19 Performance metrics of a microscope Basic image analysis: open sources of images Basic image analysis: image filtering Basic image analysis: image intensity derivative calculation Project assignment 1 19

20 A Few Words about MATLAB There are many excellent tutorials online. There are many excellent reference books. It is worthwhile to invest some time on learning MATLAB. Please bring your questions to our teaching assistant. Anuparma Kuruvilla Office: C119 Hamerschlag Hall 0

21 Where & How to Get Image Data The number of open image repositories is constantly increasing. OME: open microscopy environment JCB DataViewer ASCB Cell Image Library 1

22 Performance metrics of a microscope Basic image analysis: open sources of images Basic image analysis: image filtering Basic image analysis: image intensity derivative calculation Project assignment 1

23 Basic Concept of Image Filtering (I) Application I: noise suppression original noise added σ= σ=10 σ=0 3

24 Basic Concept of Image Filtering (II) Application II: image conditioning Canny, J., A Computational Approach To Edge Detection, IEEE Trans. Pattern Analysis and Machine Intelligence, 8(6): , Gonzalez & Woods, DIP /e 4

25 Basic Concept of Image Filtering (III) Gonzalez & Woods, DIP 3/e

26 Basic Concept of Image Filtering (IV) Image filtering in the spatial domain a b a b,,,,,, w s t f x s y t w s t f x s y t w x y f x y sa tb sa tb f(x,y) w(x,y) g(x,y) g x,y w x,y f x,y G u,v W u,v F u,v 6

27 Gaussian kernel in 1D x 1 Gx; e First order derivative x Gx; e 3 Second order derivative Gaussian Filter (I) x x x x G x; e Gx,y ; x, y e x y x y x y 7

28 Gaussian Filters (II) Some basic properties of a Gaussian filter - It is a low pass filter - It is separable x 1 F e e x y y x Gx,y ; x, y e e e x y x y x y x y 8

29 Performance metrics of a microscope Basic image analysis: open sources of images Basic image analysis: image filtering Basic image analysis: image intensity derivative calculation Project assignment 1 9

30 Combination of Noise Suppression and Gradient Estimation (I) Implementation I x i,j 1 1 I i,j I i,j Ii,j1Ii,j1 Iy i,j Notation: J: raw image; I: filtered image after convolution with Gaussian kernel G. A basic property of convolution G J G J I G I G Ix J Iy J x x x y y y 30

31 Performance metrics of a microscope Basic image analysis: open sources of images Basic image analysis: image filtering Basic image analysis: image intensity derivative calculation Project assignment 1 31

32 Basic Image Operations Reading an imaging Accessing individual pixels Setting a region of interest (ROI) Writing an image 3

33 Questions? 33

VISUAL PHYSICS ONLINE DEPTH STUDY: ELECTRON MICROSCOPES

VISUAL PHYSICS ONLINE DEPTH STUDY: ELECTRON MICROSCOPES VISUAL PHYSICS ONLINE DEPTH STUDY: ELECTRON MICROSCOPES Shortly after the experimental confirmation of the wave properties of the electron, it was suggested that the electron could be used to examine objects

More information

Lecture 8. Lecture 8. r 1

Lecture 8. Lecture 8. r 1 Lecture 8 Achromat Design Design starts with desired Next choose your glass materials, i.e. Find P D P D, then get f D P D K K Choose radii (still some freedom left in choice of radii for minimization

More information

Education in Microscopy and Digital Imaging

Education in Microscopy and Digital Imaging Contact Us Carl Zeiss Education in Microscopy and Digital Imaging ZEISS Home Products Solutions Support Online Shop ZEISS International ZEISS Campus Home Interactive Tutorials Basic Microscopy Spectral

More information

Point Spread Function. Confocal Laser Scanning Microscopy. Confocal Aperture. Optical aberrations. Alternative Scanning Microscopy

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

More information

Katarina Logg, Kristofer Bodvard, Mikael Käll. Dept. of Applied Physics. 12 September Optical Microscopy. Supervisor s signature:...

Katarina Logg, Kristofer Bodvard, Mikael Käll. Dept. of Applied Physics. 12 September Optical Microscopy. Supervisor s signature:... Katarina Logg, Kristofer Bodvard, Mikael Käll Dept. of Applied Physics 12 September 2007 O1 Optical Microscopy Name:.. Date:... Supervisor s signature:... Introduction Over the past decades, the number

More information

Microscopy: Fundamental Principles and Practical Approaches

Microscopy: Fundamental Principles and Practical Approaches Microscopy: Fundamental Principles and Practical Approaches Simon Atkinson Online Resource: http://micro.magnet.fsu.edu/primer/index.html Book: Murphy, D.B. Fundamentals of Light Microscopy and Electronic

More information

CCAM s Selection of. Zeiss Microscope Objectives

CCAM s Selection of. Zeiss Microscope Objectives CCAM s Selection of Zeiss Microscope Objectives 1. Magnification Image scale 2. Resolution The minimum separation distance between two points that are clearly resolved. The resolution of an objective is

More information

OPTICAL IMAGE FORMATION

OPTICAL IMAGE FORMATION GEOMETRICAL IMAGING First-order image is perfect object (input) scaled (by magnification) version of object optical system magnification = image distance/object distance no blurring object distance image

More information

CCAM Microscope Objectives

CCAM Microscope Objectives CCAM Microscope Objectives Things to consider when selecting an objective Magnification Numerical Aperture (NA) resolving power and light intensity of the objective Working Distance distance between the

More information

Heisenberg) relation applied to space and transverse wavevector

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

More information

Digital Camera Technologies for Scientific Bio-Imaging. Part 2: Sampling and Signal

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

More information

Confocal and 2-photon Imaging. October 15, 2010

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

More information

Very short introduction to light microscopy and digital imaging

Very 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 information

Lecture 23 MNS 102: Techniques for Materials and Nano Sciences

Lecture 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 information

Fourier transforms, SIM

Fourier transforms, SIM Fourier transforms, SIM Last class More STED Minflux Fourier transforms This class More FTs 2D FTs SIM 1 Intensity.5 -.5 FT -1.5 1 1.5 2 2.5 3 3.5 4 4.5 5 6 Time (s) IFT 4 2 5 1 15 Frequency (Hz) ff tt

More information

Diffraction. modern investigations date from Augustin Fresnel

Diffraction. modern investigations date from Augustin Fresnel Diffraction Diffraction controls the detail you can see in optical instruments, makes holograms, diffraction gratings and much else possible, explains some natural phenomena Diffraction was discovered

More information

Modulation Transfer Function

Modulation Transfer Function Modulation Transfer Function The Modulation Transfer Function (MTF) is a useful tool in system evaluation. t describes if, and how well, different spatial frequencies are transferred from object to image.

More information

DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS. GUI Simulation Diffraction: Focused Beams and Resolution for a lens system

DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS. GUI Simulation Diffraction: Focused Beams and Resolution for a lens system DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS GUI Simulation Diffraction: Focused Beams and Resolution for a lens system Ian Cooper School of Physics University of Sydney ian.cooper@sydney.edu.au DOWNLOAD

More information

Tissue Preparation ORGANISM IMAGE TISSUE PREPARATION. 1) Fixation: halts cell metabolism, preserves cell/tissue structure

Tissue Preparation ORGANISM IMAGE TISSUE PREPARATION. 1) Fixation: halts cell metabolism, preserves cell/tissue structure Lab starts this week! ANNOUNCEMENTS - Tuesday or Wednesday 1:25 ISB 264 - Read Lab 1: Microscopy and Imaging (see Web Page) - Getting started on Lab Group project - Organ for investigation - Lab project

More information

Imaging Introduction. September 24, 2010

Imaging 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 information

SETTING UP OF A TOTAL INTERNAL REFLECTION FLUORESCENT MICROSCOPE (TIRFM) SYSTEM: A DETAILED OVERVIEW

SETTING UP OF A TOTAL INTERNAL REFLECTION FLUORESCENT MICROSCOPE (TIRFM) SYSTEM: A DETAILED OVERVIEW PK ISSN 0022-2941; CODEN JNSMAC Vol. 51, (2011) PP 31-45 SETTING UP OF A TOTAL INTERNAL REFLECTION FLUORESCENT MICROSCOPE (TIRFM) SYSTEM: A DETAILED OVERVIEW A. R. KHAN 1 *, S. AKHLAQ 1, M. N. B. ABID

More information

Lecture 23 MNS 102: Techniques for Materials and Nano Sciences

Lecture 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 information

Εισαγωγική στην Οπτική Απεικόνιση

Εισαγωγική 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 information

Optical Performance of Nikon F-Mount Lenses. Landon Carter May 11, Measurement and Instrumentation

Optical Performance of Nikon F-Mount Lenses. Landon Carter May 11, Measurement and Instrumentation Optical Performance of Nikon F-Mount Lenses Landon Carter May 11, 2016 2.671 Measurement and Instrumentation Abstract In photographic systems, lenses are one of the most important pieces of the system

More information

Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light sheets

Multicolor 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 information

Lecture 15: Fraunhofer diffraction by a circular aperture

Lecture 15: Fraunhofer diffraction by a circular aperture Lecture 15: Fraunhofer diffraction by a circular aperture Lecture aims to explain: 1. Diffraction problem for a circular aperture 2. Diffraction pattern produced by a circular aperture, Airy rings 3. Importance

More information

Point Spread Function Estimation Tool, Alpha Version. A Plugin for ImageJ

Point Spread Function Estimation Tool, Alpha Version. A Plugin for ImageJ Tutorial Point Spread Function Estimation Tool, Alpha Version A Plugin for ImageJ Benedikt Baumgartner Jo Helmuth jo.helmuth@inf.ethz.ch MOSAIC Lab, ETH Zurich www.mosaic.ethz.ch This tutorial explains

More information

MICROSCOPY MICROSCOPE TERMINOLOGY

MICROSCOPY MICROSCOPE TERMINOLOGY 1 MICROSCOPY Most of the microorganisms that we talk about in this class are too small to be seen with the naked eye. The instruments we will use to visualize these microbes are microscopes. The laboratory

More information

3D light microscopy techniques

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

More information

Chapter 3 Op+cal Instrumenta+on

Chapter 3 Op+cal Instrumenta+on Chapter 3 Op+cal Instrumenta+on 3-1 Stops, Pupils, and Windows 3-4 The Camera 3-5 Simple Magnifiers and Eyepieces 3-6 Microscopes 3-7 Telescopes Today (2011-09-22) 1. Magnifiers 2. Camera 3. Resolution

More information

Chapter 2 Fourier Integral Representation of an Optical Image

Chapter 2 Fourier Integral Representation of an Optical Image Chapter 2 Fourier Integral Representation of an Optical This chapter describes optical transfer functions. The concepts of linearity and shift invariance were introduced in Chapter 1. This chapter continues

More information

Basics of confocal imaging (part I)

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

More information

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. 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 information

Microscopy http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html 2005, Dr. Jack Ikeda & Dr. Gail Grabner 9 Nikon Labophot (Question

More information

FLUORESCENCE MICROSCOPY. Matyas Molnar and Dirk Pacholsky

FLUORESCENCE 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 information

Light Microscopy. Upon completion of this lecture, the student should be able to:

Light 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 information

Chapter 3 Op,cal Instrumenta,on

Chapter 3 Op,cal Instrumenta,on Imaging by an Op,cal System Change in curvature of wavefronts by a thin lens Chapter 3 Op,cal Instrumenta,on 3-1 Stops, Pupils, and Windows 3-4 The Camera 3-5 Simple Magnifiers and Eyepieces 1. Magnifiers

More information

ELEC Dr Reji Mathew Electrical Engineering UNSW

ELEC Dr Reji Mathew Electrical Engineering UNSW ELEC 4622 Dr Reji Mathew Electrical Engineering UNSW Filter Design Circularly symmetric 2-D low-pass filter Pass-band radial frequency: ω p Stop-band radial frequency: ω s 1 δ p Pass-band tolerances: δ

More information

Be aware that there is no universal notation for the various quantities.

Be aware that there is no universal notation for the various quantities. Fourier Optics v2.4 Ray tracing is limited in its ability to describe optics because it ignores the wave properties of light. Diffraction is needed to explain image spatial resolution and contrast and

More information

a) How big will that physical image of the cells be your camera sensor?

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

More information

Exercise 8: Interference and diffraction

Exercise 8: Interference and diffraction Physics 223 Name: Exercise 8: Interference and diffraction 1. In a two-slit Young s interference experiment, the aperture (the mask with the two slits) to screen distance is 2.0 m, and a red light of wavelength

More information

BASICS OF CONFOCAL IMAGING (PART I)

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

More information

High resolution extended depth of field microscopy using wavefront coding

High 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 information

Circular Dichroism Microscopy Free from Commingling Linear Dichroism via Discretely Modulated Circular Polarization

Circular Dichroism Microscopy Free from Commingling Linear Dichroism via Discretely Modulated Circular Polarization Supplementary information Circular Dichroism Microscopy Free from Commingling Linear Dichroism via Discretely Modulated Circular Polarization Tetsuya Narushima AB and Hiromi Okamoto A* A Institute for

More information

Introduction to Electron Microscopy

Introduction to Electron Microscopy Introduction to Electron Microscopy Prof. David Muller, dm24@cornell.edu Rm 274 Clark Hall, 255-4065 Ernst Ruska and Max Knoll built the first electron microscope in 1931 (Nobel Prize to Ruska in 1986)

More information

USE OF FT IN IMAGE PROCESSING IMAGE PROCESSING (RRY025)

USE OF FT IN IMAGE PROCESSING IMAGE PROCESSING (RRY025) IMAGE PROCESSIG (RRY25) USE OF FT I IMAGE PROCESSIG Optics- originofimperfectionsinimagingsystems(limited resolution/blurring related to 2D FTs)- need to understand using Continuous FT. Sampling -Capturecontinuousimageontoasetofdiscrete

More information

INTRODUCTION THIN LENSES. Introduction. given by the paraxial refraction equation derived last lecture: Thin lenses (19.1) = 1. Double-lens systems

INTRODUCTION THIN LENSES. Introduction. given by the paraxial refraction equation derived last lecture: Thin lenses (19.1) = 1. Double-lens systems Chapter 9 OPTICAL INSTRUMENTS Introduction Thin lenses Double-lens systems Aberrations Camera Human eye Compound microscope Summary INTRODUCTION Knowledge of geometrical optics, diffraction and interference,

More information

TRAINING MANUAL. Multiphoton Microscopy LSM 510 META-NLO

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

More information

Optics of Wavefront. Austin Roorda, Ph.D. University of Houston College of Optometry

Optics of Wavefront. Austin Roorda, Ph.D. University of Houston College of Optometry Optics of Wavefront Austin Roorda, Ph.D. University of Houston College of Optometry Geometrical Optics Relationships between pupil size, refractive error and blur Optics of the eye: Depth of Focus 2 mm

More information

Vocabulary: Description: Materials: Objectives: Safety: Two 45-minute class periods (one for background and one for activity) Schedule:

Vocabulary: Description: Materials: Objectives: Safety: Two 45-minute class periods (one for background and one for activity) Schedule: Resolution Not just for the New Year Author(s): Alia Jackson Date Created: 07/31/2013 Subject: Physics Grade Level: 11-12 Standards: Standard 1: M1.1 Use algebraic and geometric representations to describe

More information

Sensitive measurement of partial coherence using a pinhole array

Sensitive measurement of partial coherence using a pinhole array 1.3 Sensitive measurement of partial coherence using a pinhole array Paul Petruck 1, Rainer Riesenberg 1, Richard Kowarschik 2 1 Institute of Photonic Technology, Albert-Einstein-Strasse 9, 07747 Jena,

More information

Supplementary information, Figure S1A-S1H The thickness and the uniformity of the light sheet at different DOFs. By

Supplementary information, Figure S1A-S1H The thickness and the uniformity of the light sheet at different DOFs. By Supplementary information, Figure S1A-S1H The thickness and the uniformity of the light sheet at different DOFs. By imaging FITC-containing solution, the thickness of the light sheet generated by the P3A-DSLM

More information

S200 Course LECTURE 1 TEM

S200 Course LECTURE 1 TEM S200 Course LECTURE 1 TEM Development of Electron Microscopy 1897 Discovery of the electron (J.J. Thompson) 1924 Particle and wave theory (L. de Broglie) 1926 Electromagnetic Lens (H. Busch) 1932 Construction

More information

Observing Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens. Compound Light Micros

Observing 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 information

Introduction to Light Microscopy. (Image: T. Wittman, Scripps)

Introduction to Light Microscopy. (Image: T. Wittman, Scripps) Introduction to Light Microscopy (Image: T. Wittman, Scripps) The Light Microscope Four centuries of history Vibrant current development One of the most widely used research tools A. Khodjakov et al. Major

More information

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE Prior to lab: 1) Read these instructions (p 1-6) 2) Go through the online tutorial, the microscopy pre-lab

More information

LEICA TCS SP5 AOBS TANDEM USER MANUAL

LEICA TCS SP5 AOBS TANDEM USER MANUAL LEICA TCS SP5 AOBS TANDEM USER MANUAL STARTING THE SYSTEM...2 THE LAS AF SOFTWARE...3 THE «ACQUIRE» MENU...5 CHOOSE AND CREATE A SETTING...6 THE CONTROL PANEL...8 THE DMI6000B MICROSCOPE...10 ACQUIRE ONE

More information

Optical Design of. Microscopes. George H. Seward. Tutorial Texts in Optical Engineering Volume TT88. SPIE PRESS Bellingham, Washington USA

Optical Design of. Microscopes. George H. Seward. Tutorial Texts in Optical Engineering Volume TT88. SPIE PRESS Bellingham, Washington USA Optical Design of Microscopes George H. Seward Tutorial Texts in Optical Engineering Volume TT88 SPIE PRESS Bellingham, Washington USA Preface xiii Chapter 1 Optical Design Concepts /1 1.1 A Value Proposition

More information

CHAPTER TWO METALLOGRAPHY & MICROSCOPY

CHAPTER TWO METALLOGRAPHY & MICROSCOPY CHAPTER TWO METALLOGRAPHY & MICROSCOPY 1. INTRODUCTION: Materials characterisation has two main aspects: Accurately measuring the physical, mechanical and chemical properties of materials Accurately measuring

More information

There is a range of distances over which objects will be in focus; this is called the depth of field of the lens. Objects closer or farther are

There is a range of distances over which objects will be in focus; this is called the depth of field of the lens. Objects closer or farther are Chapter 25 Optical Instruments Some Topics in Chapter 25 Cameras The Human Eye; Corrective Lenses Magnifying Glass Telescopes Compound Microscope Aberrations of Lenses and Mirrors Limits of Resolution

More information

Modulation Transfer Function

Modulation Transfer Function Modulation Transfer Function The resolution and performance of an optical microscope can be characterized by a quantity known as the modulation transfer function (MTF), which is a measurement of the microscope's

More information

Light microscopy BMB 173, Lecture 14, Feb. 21, 2018

Light microscopy BMB 173, Lecture 14, Feb. 21, 2018 Light microscopy The Structural Biology Continuum Next two lectures: Light microscopy Many slides taken from Scott Fraser, Murphy s Fundamentals of light microscopy, Alberts Molecular Biology of the Cell,

More information

Rates of excitation, emission, ISC

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,

More information

Microscope anatomy, image formation and resolution

Microscope 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 information

Bi/BE 227 Winter Assignment #3. Adding the third dimension: 3D Confocal Imaging

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

More information

Microscopy. Lecture 2: Optical System of the Microscopy II Herbert Gross. Winter term

Microscopy. Lecture 2: Optical System of the Microscopy II Herbert Gross. Winter term Microscopy Lecture 2: Optical System of the Microscopy II 212-1-22 Herbert Gross Winter term 212 www.iap.uni-jena.de Preliminary time schedule 2 No Date Main subject Detailed topics Lecturer 1 15.1. Optical

More information

Bio 407. Applied microscopy. Introduction into light microscopy. José María Mateos. Center for Microscopy and Image Analysis

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,

More information

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 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 information

ECEN. Spectroscopy. Lab 8. copy. constituents HOMEWORK PR. Figure. 1. Layout of. of the

ECEN. Spectroscopy. Lab 8. copy. constituents HOMEWORK PR. Figure. 1. Layout of. of the ECEN 4606 Lab 8 Spectroscopy SUMMARY: ROBLEM 1: Pedrotti 3 12-10. In this lab, you will design, build and test an optical spectrum analyzer and use it for both absorption and emission spectroscopy. The

More information

Chapter 1. Basic Electron Optics (Lecture 2)

Chapter 1. Basic Electron Optics (Lecture 2) Chapter 1. Basic Electron Optics (Lecture 2) Basic concepts of microscope (Cont ) Fundamental properties of electrons Electron Scattering Instrumentation Basic conceptions of microscope (Cont ) Ray diagram

More information

Prof. Enrico Gratton - Lecture 6 Fluorescence Microscopy

Prof. Enrico Gratton - Lecture 6 Fluorescence Microscopy Prof. Enrico Gratton - Lecture 6 Fluorescence Microscopy Instrumentation Light Sources: One-photon and Multi-photon Excitation Applications in Cells Lifetime Imaging Figures acknowledgements: E.D. Salmon

More information

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 Fundamentals of Light Microscopy II: Fluorescence, Deconvolution, Confocal, Multiphoton, Spectral microscopy Integrated Microscopy Course Review Lecture 1: Microscopy Basics Light train Kohler illumination*

More information

Today. next week. MIT 2.71/ /04/09 wk13-a- 1

Today. next week. MIT 2.71/ /04/09 wk13-a- 1 Today Spatially coherent and incoherent imaging with a single lens re-derivation of the single-lens imaging condition ATF/OTF/PSF and the Numerical Aperture resolution in optical systems pupil engineering

More information

The Compound Microscope. Brightfield: Köhler Illumination

The Compound Microscope. Brightfield: Köhler Illumination Outline History of Microscopy The Magnifying Glass The Compound Microscope Brightfield: Köhler Illumination Microscopy µικροσ (mikros): small σκοπειν (skopein): to observe History of Microscopy Well :

More information

Shaping light in microscopy:

Shaping light in microscopy: Shaping light in microscopy: Adaptive optical methods and nonconventional beam shapes for enhanced imaging Martí Duocastella planet detector detector sample sample Aberrated wavefront Beamsplitter Adaptive

More information

Vision. The eye. Image formation. Eye defects & corrective lenses. Visual acuity. Colour vision. Lecture 3.5

Vision. The eye. Image formation. Eye defects & corrective lenses. Visual acuity. Colour vision. Lecture 3.5 Lecture 3.5 Vision The eye Image formation Eye defects & corrective lenses Visual acuity Colour vision Vision http://www.wired.com/wiredscience/2009/04/schizoillusion/ Perception of light--- eye-brain

More information

Biomedical Imaging 生物醫學影像學

Biomedical Imaging 生物醫學影像學 Biomedical Imaging 生物醫學影像學 楊自森助理教授 牙體技術學系 2013/02/24 tsyang@tmu.edu.tw 1 Course Outline 1. Course Introduction 2. Basic Optics and Light Microscopes 3. Fluorescence/Confocal/TIRF Microscopes 4. FRET Techniques

More information

Resolving Power of a Diffraction Grating

Resolving Power of a Diffraction Grating Resolving Power of a Diffraction Grating When measuring wavelengths, it is important to distinguish slightly different s. The ability of a grating to resolve the difference in wavelengths is given by the

More information

Cardinal Points of an Optical System--and Other Basic Facts

Cardinal Points of an Optical System--and Other Basic Facts Cardinal Points of an Optical System--and Other Basic Facts The fundamental feature of any optical system is the aperture stop. Thus, the most fundamental optical system is the pinhole camera. The image

More information

2/7/14. Brightfield Microscopy! It s all about Magnification..! or is it?!

2/7/14. Brightfield Microscopy! It s all about Magnification..! or is it?! Brightfield Microscopy It s all about Magnification.. or is it? 1 What actually does go into chosing a microscope Choice depends on what you need the microscope to do. Do you want to magnify stained specimens?

More information

Chapter 34 The Wave Nature of Light; Interference. Copyright 2009 Pearson Education, Inc.

Chapter 34 The Wave Nature of Light; Interference. Copyright 2009 Pearson Education, Inc. Chapter 34 The Wave Nature of Light; Interference 34-7 Luminous Intensity The intensity of light as perceived depends not only on the actual intensity but also on the sensitivity of the eye at different

More information

Physics 1C. Lecture 25B

Physics 1C. Lecture 25B Physics 1C Lecture 25B "More than 50 years ago, Austrian researcher Ivo Kohler gave people goggles thats severely distorted their vision: The lenses turned the world upside down. After several weeks, subjects

More information

Systems Biology. Optical Train, Köhler Illumination

Systems Biology. Optical Train, Köhler Illumination McGill University Life Sciences Complex Imaging Facility Systems Biology Microscopy Workshop Tuesday December 7 th, 2010 Simple Lenses, Transmitted Light Optical Train, Köhler Illumination What Does a

More information

LECTURE 13 DIFFRACTION. Instructor: Kazumi Tolich

LECTURE 13 DIFFRACTION. Instructor: Kazumi Tolich LECTURE 13 DIFFRACTION Instructor: Kazumi Tolich Lecture 13 2 Reading chapter 33-4 & 33-6 to 33-7 Single slit diffraction Two slit interference-diffraction Fraunhofer and Fresnel diffraction Diffraction

More information

Lecture 5. Telescopes (part II) and Detectors

Lecture 5. Telescopes (part II) and Detectors Lecture 5 Telescopes (part II) and Detectors Please take a moment to remember the crew of STS-107, the space shuttle Columbia, as well as their families. Crew of the Space Shuttle Columbia Lost February

More information

Figure 3.4 Approximate size of various types of cells. ~10 um. Red Blood Cells = mm 1500 um. Width of penny Pearson Education, Inc.

Figure 3.4 Approximate size of various types of cells. ~10 um. Red Blood Cells = mm 1500 um. Width of penny Pearson Education, Inc. Figure 3.4 Approximate size of various types of cells. ~10 um Red Blood Cells 1.5mm 1500 um Width of penny = 1500 Figure 4.3 The limits of resolution (and some representative objects within those ranges)

More information

Burton's Microbiology for the Health Sciences

Burton's Microbiology for the Health Sciences Burton's Microbiology for the Health Sciences Chapter 2. Viewing the Microbial World Chapter 2 Outline Introduction Using the metric system to express the sizes of microbes Microscopes Simple microscopes

More information

BASICS IN BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP LIGHT MICROSCOPY

BASICS IN BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP LIGHT MICROSCOPY BASICS IN LIGHT MICROSCOPY OVERVIEW 1. Motivation 2. Basic in optics 3. How microscope works 4. Illumination and resolution 5. Microscope optics 6. Contrasting methods -2- MOTIVATION Why do we need microscopy?

More information

Comparison of an Optical-Digital Restoration Technique with Digital Methods for Microscopy Defocused Images

Comparison of an Optical-Digital Restoration Technique with Digital Methods for Microscopy Defocused Images Comparison of an Optical-Digital Restoration Technique with Digital Methods for Microscopy Defocused Images R. Ortiz-Sosa, L.R. Berriel-Valdos, J. F. Aguilar Instituto Nacional de Astrofísica Óptica y

More information

CFIM MICROSCOPY COURSE PROGRAMME PRINCIPLES OF MICROSCOPY CONFOCAL AND FLUORESCENCE MICROSCOPY

CFIM 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 information

Introduction to light microscopy

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

More information

PHYS 202 OUTLINE FOR PART III LIGHT & OPTICS

PHYS 202 OUTLINE FOR PART III LIGHT & OPTICS PHYS 202 OUTLINE FOR PART III LIGHT & OPTICS Electromagnetic Waves A. Electromagnetic waves S-23,24 1. speed of waves = 1/( o o ) ½ = 3 x 10 8 m/s = c 2. waves and frequency: the spectrum (a) radio red

More information

DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS RAYLEIGH-SOMMERFELD DIFFRACTION INTEGRAL OF THE FIRST KIND CIRCULAR APERTURES

DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS RAYLEIGH-SOMMERFELD DIFFRACTION INTEGRAL OF THE FIRST KIND CIRCULAR APERTURES DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS RAYLEIGH-SOMMERFELD DIFFRACTION INTEGRAL OF THE FIRST KIND CIRCULAR APERTURES Ian Cooper School of Physics, University of Sydney ian.cooper@sydney.edu.au

More information

Physics 202, Lecture 28

Physics 202, Lecture 28 Physics 202, Lecture 28 Today s Topics Michelson Interferometer iffraction Single Slit iffraction Multi-Slit Interference iffraction on Circular Apertures The Rayleigh Criterion Wave Superposition Using

More information

microscopy A great online resource Molecular Expressions, a Microscope Primer Partha Roy

microscopy A great online resource Molecular Expressions, a Microscope Primer Partha Roy Fundamentals of optical microscopy A great online resource Molecular Expressions, a Microscope Primer http://micro.magnet.fsu.edu/primer/index.html Partha Roy 1 Why microscopy Topics Functions of a microscope

More information

INTRODUCTION TO OPTICAL MICROSCOPY

INTRODUCTION TO OPTICAL MICROSCOPY Experimental Biophysics TEK265, FYST23, TNF060, FAF010F Lab Exercise Supervisor: Oskar Ström Written by Peter Jönsson and Jason Beech Updated by Henrik Persson, Karl Adolfsson and Zhen Li oskar.strom@ftf.lth.se

More information

Microscopy. CS/CME/BioE/Biophys/BMI 279 Nov. 2, 2017 Ron Dror

Microscopy. CS/CME/BioE/Biophys/BMI 279 Nov. 2, 2017 Ron Dror Microscopy CS/CME/BioE/Biophys/BMI 279 Nov. 2, 2017 Ron Dror 1 Outline Microscopy: the basics Fluorescence microscopy Resolution limits The diffraction limit Beating the diffraction limit 2 Microscopy:

More information

ANSWER KEY Lab 2 (IGB): Bright Field and Fluorescence Optical Microscopy and Sectioning

ANSWER 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 information

Experiment 1: Fraunhofer Diffraction of Light by a Single Slit

Experiment 1: Fraunhofer Diffraction of Light by a Single Slit Experiment 1: Fraunhofer Diffraction of Light by a Single Slit Purpose 1. To understand the theory of Fraunhofer diffraction of light at a single slit and at a circular aperture; 2. To learn how to measure

More information