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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