OPTICAL PRINCIPLES OF MICROSCOPY Interuniversity Course 28 December 2003 Aryeh M. Weiss Bar Ilan University
FOREWORD This slide set was originally presented at the ISM Workshop on 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 W avelength (period T) Axis of Magnetic Field Axis of Propaga tion
SOME PROPERTIES OF LIGHT Index of refraction (n) Speed of light (c/n) Polarization Frequency (ν = 10 14-10 15 Hz) Wavelength (λ = 300-1100nm)
SOME OPTICAL PHENOMENA Reflection Refraction Interference Diffraction Polarization
REFLECTION AND REFRACTION θ i θ r θt Snell s Law: The angle of reflection (Ø r ) is equal to the angle of incidence (Ø i ) regardless of the surface material The angle of the transmitted beam (Ø t ) is dependent upon the composition of the material n 1 sin Ø i = n 2 sin Ø t The velocity of light in a material of refractive index n is c/n
TOTAL INTERNAL REFLECTION
INTERFERENCE 0 o 90 o 180 o 270 o 360 o Wavelength A A+B Amplitude The frequency does not change, but the amplitude is doubled B C C+D Constructive Interference Here we have a phase difference of 180 o (2π radians) so the waves cancel each other out D Destructive Interference Figure modified from Shapiro Practical Flow Cytometry 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 LENSES f f p q 1 1 1 + = p q f Magnification = q 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 light by the object. Minimal requirement for resolution is ability to collect at least one diffracted order 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 1 b 1 f 1 a 1 1 f 1 f
OBJECTIVES
WHY SO EXPENSIVE?
WAVELENGTH RANGE OF OBJECTIVES
OCULARS
IMPORTANT PART OF SYSTEM
ILLUMINATION Critical or Kohler
CONDENSER
RAYLEIGH CRITERION FOR TRAN-ILLUMINATED SYSTEMS r = 1.22 λ/(na obj + NA cond ) 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 about 500-1000 x NA.
DEPTH OF FIELD
GENERATION OF CONTRAST Darkfield Rheinberg illumination Phase contrast microscopy DIC (Nomarski)
DARKFIELD IMAGING
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
REFERENCES OPTICAL MICROSCOPY Michael W.Davidson and Mortimer Abramowitz The Florida State University www.microscopy.fsu.edu/primer/index.html FROM LENSES TO OPTICAL INSTRUMENTS Giorgio Carboni, Fun Science Gallery funsci.com/fun3_en/lens/lens.htm VIDEO MICROSCOPY 2 nd Ed. S. Inoue and K.R. Spring Plenum Press, NY 1997
REFERENCES Introduction to Confocal Microscopy and Image Analysis (Powerpoint slide sets) Dr. J. Paul Robinson Purdue University Cytometry Laboratories www.cyto.purdue.edu/flowcyt/educate/pptslide.htm Handbook of Optical Filters Chroma Technology Corp. http://www.chroma.com/handbook.html