MICROSCOPY COURSE 2012

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1 MICROSCOPY COURSE 2012 INTRODUCTION TO BASIC LIGHT MICROSCOPY

2 AIM OF THE COURSE Teach basic principles of light microscopy Theoretical background Practical aspects Faced towards applications of light microscopy in life science Which information can we obtain with light microscopy? What are the limitations? Prerequisites Curiosity to learn microscopy

3 OPTICS HISTORY Ancient world Reflection 1621 W. Snellius Optical Refraction 1637 R. Descartes 1657 P. de Fermat Fermat s principle 1665 R. Hooke Light as a wave C. Huygens 1804 T. Young Interference 1818 Fresnel Diffraction/ Dispersion 1821 Frauenhofer Diffraction Patterns 1876 J. C. Maxwell Electromagnetic wave

4 OPTICS WHAT S ALL THE FUZZ ABOUT Why do we care about optics? Fundamental working principle of a light microscope Limitations of light microscopy Interaction/communication Scientists (coming from life science) are using microscopes Scientists/engineers (with a strong physics background) are building microscopes

5 BASICS IN OPTICS What is light? How can we describe light? Is the velocity of light finite or infinite? Behaviour of light Interaction of light with matter Influence the way light travels Optical instruments Microscopes Telescopes

6 REFLECTION/REFRACTION Reflection

7 OPTICAL REFRACTION

8 OPTICAL REFRACTION SNELL S LAW Ibn Sahl of Baghdad: On burning Mirrors and lenses (984)

9 FERMATS S PRINCIPLE Principle of least time Path taken between two points by a ray of light is the path that can be transversed in the least time Enables a description of optics by means of rays in isotropic media L ( AB) nds AB A B

10 GEOMETRICAL OPTICS THIN LENS Principal plane Focal length f optical axis Focal point Characterization of a lens: Focal length: f=50 mm=0.05 m Power: 1/f =20 m -1 = 20 dioptre

11 GEOMETRICAL OPTICS THIN LENS Principal plane Focal length f optical axis Focal point Working principle of lenses: Refraction Curvature

12 LENS MAKER FORMULAE curvature r 2 1 f = n 1 n m 1 1 r 1 1 r 2 curvature r 1 focal length focal point Factors that determine the focal length of a lens index of refraction index of refraction of the medium radius of the front surface radius of the back surface Material Index Vacuum Air at STP Water at 20 C 1.33 Fluorite Fused quartz 1.46 Glycerine Typical crown glass 1.52 Crown glasses Spectacle crown, C Material Index Flint glasses Heavy flint glass 1.65 Sapphire 1.77 Rare earth flint Lanthanum flint Arsenic trisulfide glass 2.04 Diamond hbase/tables/indrf.html#c1

13 SIMPLE LENS TYPES

14 GEOMETRICAL OPTICS THIN LENS- IMAGE FORMATION Principal plane -f f 1 f = 1 s s 1 s 1 object optical axis s 2 image s 0 = 5.35 cm s 1 = 14.1 cm f = 3.8 cm 1 s0 = 0.19 cm 1 1 s1 = 0.07 cm 1 1 f = 0.26 cm 1 1 cm

15 im GEOMETRICAL OPTICS THIN LENS- VIRTUAL IMAGE FORMATION Principal plane -f f optical axis object

16 GEOMETRICAL OPTICS TELESCOPE -f 1 l 1 f 1 =-f 2 l 2 f 2 optical axis d=f 1 +f 2 M = f 2 f 1

17 GEOMETRICAL OPTICS Paraxial approximation rays have fixed wavelength rays are almost parallel to the optical axis (small angle) sintan also known as Gaussian optics Ray tracing for simple systems powerful tool (simple and intuitive)

18 GEOMETRICAL OPTICS Advanced topics Thick lenses, stops Matrix formalism (ABCD) Straightforward calculation of complicated systems (multiple elements) Convenient for numerical calculations No paraxial approximation Disadvantages: No Dispersion

19 DISPERSION Phase velocity depends on the frequency of the wave. Rate at which the phase of a wave propagates in space Example of Dispersion: Rainbow

20 WAVES SIMPLE MODEL T V(t)=A cos (t ) A: Amplitude : angular frequency : Phase shift T=2/ =1/T (frequency) Phase shift time t

21 WAVES INTERFERENCE time t 1) 2) 3) 1 2 3

22 WAVE THEORY t f v x f Non-dispersing wave equation k v t kx i a t x f k t x i a t x f / )] ( exp[ ), ( exp ), ( phasevelocity angular frequency -number wave k v t kx i a t x f / )] ( exp[ ), ( phasevelocity

23 ATTENUATED HARMONIC WAVE k k1 ik 2 f a exp[ i( k1 ik 2) x a exp( k x)exp( i( k 2 it] 1 x t) x 1 x 2 x 3 x t

24 HUYGENS-FRESNEL PRINCIPLE Refraction Diffraction

25 WAVES PLANE WAVE wave vector k wave fronts x

26 LIGHT-WAVE THEORY YOUNG: DOUBLE SLIT EXPERIMENT

27 LIGHT-WAVE THEORY FAR FIELD DIFFRACTION Parallel light distance R R>1.3 m aperture 100 µm Observation plane J. Frauenhofer ( )

$in calcit ) Unequal indices of refraction (n e$ $of refraction depends on the polarization of$

28 BIREFRINGENCE DOUBLE REFRACTION Discovered by Rasmus Bartholin (1669) Unaxial anisotropy of material (e.g. in calcit ) Unequal indices of refraction (n e and n o ) e: extraordanary; o: ordanary Degree of refraction depends on the polarization of light (reference: optical axis of material)

29 ELECTROMAGNETIC WAVES Electromagnetic waves were first postulated by J.C. Maxwell They were experimentally proven by H. Hertz

Young interference 1871 Maxwell electromagnetic

30 WHAT IS LIGHT PARTICLE/WAVE 1669 Newton Emanations theory (particle) 1677 Huygens wave theory 1802 Young interference 1871 Maxwell electromagnetic light theory 1886 Hertz experimental proof of Maxwells theory

31 WHAT IS LIGHT PHOTOELECTRIC EFFECT Albert Einstein Nobel price 1921 Experiments by Lennart 1902 Maximal energy of the photo electrons is independent of the intensity of the light Slope is the same for different cathode materials Light can be described as particles called photons or light quants. particle-wave duality Detection of Photons

32 OPTICS SUMMARY Basic Definitions Reflection Refraction Diffraction Dispersion Models to describe optical phenomena Geometrical Optics Wave optics Light Particle-wave duality

33 More about light microscopy/optics 1. Lecture Biomicroscopy I + II, Prof. Theo Lasser, EPFL 2. Books a) Digital microscopy, Sluder, G; Wolf, D.E., eds, Elsevier, 2003 b) Optics, 4th ed., Eugene Hecht, Addison-Wesley, Internet a) b) b) Web sites of microscope manufactures Leica Nikon Olympus Zeiss 4. BIOp EPFL, SV-AI 0241, Sv-AI

PY3101 Optics. Overview. A short history of optics Optical applications Course outline. Introduction: Overview. M.P. Vaughan

PY3101 Optics. Overview. A short history of optics Optical applications Course outline. Introduction: Overview. M.P. Vaughan Introduction: Overview M.P. Vaughan Overview A short history of optics Optical applications Course outline 1 A Short History of Optics A short history of optics Optics: historically the study of visible