OPTICAL PRINCIPLES OFMICROSCOPY

Interuniversity Course – 28 December 2003Aryeh M. Weiss

Bar Ilan University

FOREWORD

This slide set was originally presented at the ISM Workshopon Theoretical and Experimental Microscopy Techniques,Eilat, 2 May 2002.

Most of the graphics in this presentation were taken from the references given at the end of this presentation.

TYPES OF MICROSCOPY

• Light (UV, visible, IR)

• Electron (SEM, TEM)

• Near-field scanning microscopy- scanning tunneling- atomic force- near field optical

OVERVIEW

• Properties of light

• Optical image formation

• Microscope basics

• Contrast generation

ELECTROMAGNETIC WAVE

avelength (period T)Axis o

f

Magnetic Field

Axis of Propaga

W

tion

SOME PROPERTIES OF LIGHT

• Index of refraction (n)• Speed of light (c/n)• Polarization• Frequency (ν = 1014-1015 Hz) • Wavelength (λ = 300-1100nm)

SOME OPTICAL PHENOMENA

• Interference

• Diffraction

• Polarization

• Reflection

• Refraction

REFLECTION AND REFRACTION• Snell’s Law: The angle of reflection (Ør) is equal to the

angle of incidence (Øi) regardless of the surface

material• The angle of the transmitted

beam (Øt) is dependent upon the composition of the

material

n1 sin Øi = n2 sin Øt

The velocity of light in a material of refractive index n is c/n

θi

θtθr

TOTAL INTERNAL REFLECTION

INTERFERENCE

ConstructiveInterference

DestructiveInterference

A

B

C

D

A+B

C+D

Am

plitude

0o 90o 180o 270o 360o Wavelength

The frequency does not change, but the amplitude is doubled

Here we have a phase difference of 180o (2π radians) so the waves cancel each other out

Figure modified from Shapiro “Practical FlowCytometry” Wiley-Liss, p79

INTERFERENCE FILTERS

DIFFRACTION

POLARIZATION

POLARIZERS

Glan-Thompson polarizer

POLARIZATION VECTORS

BIREFRINGENCE

PARTICLE CHARACTER OF LIGHT

• Light is made up of photons• Energy/photon (e) is proportional to

frequency e=hν or e=hc/λ• Important in:

-noise analysis-interaction with matter (eg absorption, fluorescence)-many commonly used detectors(eg, PMT, CCD, film, etc)

LIGHT SOURCES

• Black body sources (halogen lamps)-spectrum is continuous-spectrum peak depends on temp.(“color temperature”)

• Spectral sources (Hg, Xe, other arc lamps, lasers)-spectrum has structure (peaks)-spectrum is a function of the electronic properties of the gas

Halogen

Xenon

Hg

OPTICAL IMAGE FORMATION

• Imaging with one lens – lens equation• Magnification• Compound microscope • Diffraction limit• Abbe theory of image formation• Aberrations (chromatic, spherical)

PROPERTIES OF THIN LENSESf

p q

f

1 1 1=+p q f

qMagnification = p

VIRTUAL IMAGES

THE COMPOUND MICROSCOPE

DIFFRACTION LIMITED IMAGING

NUMERICAL APERTURE

High N.A.

θ

Low N.A.

θ

N.A.= n sin(θ)

RAYLEIGH CRITERION

r = 0.61λ/NA

NA = n sin(θ)

For high NA objectives (0.4-1.4)

1.5λ > r > 0.44λ

EFFECT OF NA ON RESOLUTION

ABBE THEORY OF IMAGE FORMATION

• Image is formed by diffraction of lightby the object.

• Minimal requirement for resolution is ability to collect at least one diffractedorder in addition to “zero” order.

ORDERS OF DIFFRACTION

DIFFRACTION LIMIT IN ABBE THEORY

ABERRATIONS

• CHROMATIC ABERRATION- due to n(λ)

• SPHERICAL ABERRATION- paraxial approx.

ANATOMY OF A MICROSCOPE

• Objectives

• Oculars

• Upright or Inverted

• Illumination

UPRIGHT MICROSCOPE

INVERTED MICROSCOPE

INFINITY OPTICS – WHAT AND WHY

1b

1f

1a

1 1f

1f

OBJECTIVES

WHY SO EXPENSIVE?

WAVELENGTH RANGE OF OBJECTIVES

OCULARS

IMPORTANT PART OF SYSTEM

ILLUMINATION

• Criticalor

• Kohler

CONDENSER

RAYLEIGH CRITERION FOR TRAN-ILLUMINATED SYSTEMS

r = 1.22 λ/(NAobj + NAcond)

• Methods which reduce the condenser NA reduce resolution.

• Worst case is a factor of two, for coherent illumination.

KOHLER ILLUMINATION

• Field diaphragm conjugate to object and determines area of illumination

• Condensor diaphragm conjugate to source and controls NA of condenser.

Wide Field Iris

Narrow Field Iris

OTHER ILLUMINATION ISSUES

• Halogen lamp color spectrum changes with temp.

• Use ND filters to adjust illumination,NOT diaphragms or lamp voltage.

• There are usually color filters to adjust illuminations for:- monochromatic light (green filters)- daylight correction (blue filter).

PUTTING IT ALL TOGETHER

• NA of objective and condenser determine resolution.

• Ocular must create an image suitable for viewing by eye. Requirements will differ for film, CCD, or other detectors.

EMPTY MAGNIFICATION

• Magnification greater than the resolution of the system is useless..

• Human eye resolves 1-2 minutes of arc..

• Maximum useful magnification is about500-1000 x NA.

DEPTH OF FIELD

GENERATION OF CONTRAST

• Darkfield

• Rheinberg illumination

• Phase contrast microscopy

• DIC (Nomarski)

DARKFIELDIMAGING

DARKFIELD IMAGES

RHEINBERG ILLUMINATION

RHEINBERG IMAGE

PHASE CONTRAST MICROSCOPY

PHASE CONTRAST ALLIGNMENT

PHASE CONTRAST IMAGE

APODIZED PHASE CONTRAST

Holzwarth, Webb, Kubinski, and Allen, J. Microscopy, p249-254 (1997)

DIC IMAGES

X-pol Y-pol

WHAT IS CONTRAST

MTF

MTF WITH CONTRAST GENERATION

OTHER CONTRAST GENERATION METHODS

• Polarization

• Hoffman modulation

• Interference

• Fluorescence

TAKEHOME MESSAGES

• Numerical aperture determines resolution

• Empty magnification is bad

• Contrast generation often lowers resolution,but it is usually worth it

• Keep dirt off of the image planes

• Use ND filters to adjust illumination intensity

REFERENCESOPTICAL MICROSCOPYMichael W.Davidson and Mortimer AbramowitzThe Florida State University www.microscopy.fsu.edu/primer/index.html

FROM LENSES TO OPTICAL INSTRUMENTSGiorgio Carboni, Fun Science Galleryfunsci.com/fun3_en/lens/lens.htm

VIDEO MICROSCOPY – 2nd Ed.S. Inoue and K.R. SpringPlenum Press, NY 1997

REFERENCES

Introduction to Confocal Microscopy and Image Analysis (Powerpoint slide sets)Dr. J. Paul RobinsonPurdue University Cytometry Laboratorieswww.cyto.purdue.edu/flowcyt/educate/pptslide.htm

Handbook of Optical FiltersChroma Technology Corp.http://www.chroma.com/handbook.html