The Generalized Microscope Colin Sheppard Nano-­‐Physics Department Italian Ins<tute of Technology (IIT) Genoa, Italy [email protected] The Microscope!
• microscope: instrument magnifying
objects by means of lenses so as to
reveal details invisible to the naked eye.!
(Oxford English Dictionary)!
Geometry of Imaging Light microscope •  Image from –  Amplitude –  Phase –  Polariza<on –  Chroma<c effects (colour, dispersion, etc.) Scanning Systems • Magnification of image is ratio of size of image to amplitude of scan
• Independent of probe diameter.
Scanning op<cal microscope (SOM) Advantages of scanning •  Image is an electronic signal –  Image enhancement, restora<on, reconstruc<on and interpreta<on •  Illumina<on by a focused probe –  New imaging modali<es •  Confocal •  Differen<al phase contrast (DPC) –  Probe causes physical effect that can be monitored to form an image •  Spectroscopic •  Op<cal beam induced current (OBIC) •  For e.g. fluorescence microscopy, resolu<on is determined by the wavelength of the excita'on light, not the fluorescent light (~5-­‐10% beUer) Equivalence of scanning and conven<onal microscopes • Based on Principle of Reciprocity!
• Holds even with loss or multiple scattering!
(but not inelastic scattering)!
Pogany & Turner, Acta Cryst. A24 103 (1968)!
Cowley, App. Phys. Lett. 15 58 (1969)!
Zeitler & Thomson, Optik 31 258 (1970)!
Welford, J. Microscopy 96 105 (1972)!
Barnett, Optik 38 585 (1973)!
Engel, Optik 41 117 (1974)!
Kermisch, J. Opt. Soc. Am. 67 1357 (1977)!
Sheppard, Optik 78, 39-43 (1986), J. Opt. Soc. Am. A 3, 755-756 (1986)!
Equivalence and Reciprocity!
•  The principle of reciprocity states that:!
–  The field measured by a detector at a point 2 due to a
particular source at 1 is equal to the field measured by
the detector when placed at point 1 due to a source
placed at point 2.!
•  The principle is generally true, even in the
presence of loss or multiple scattering, except if
there are magnetic or polarization effects. Reciprocity breaks down: • If there is energy loss, e.g. –  Fluorescence • Stokes’ shi^ is 5-­‐10% • Resolu<on beUer for scanning rather than conven<onal system –  Secondary electrons • Polariza<on or magne<c effects, e.g. –  Linear polarizer is non-­‐reciprocal • In general seems related to a quantum effect (collapse of wave func<on) Types of microscope Conventional
Conventional with image scanning
Equivalent
Scanning
Scanning
Confocal
Scanning microscopes of Type 1 (non-­‐confocal) Köhler detection
Critical detection
Amar Choudhury, Colin Sheppard, Pete Hale & Rudi Kompfner Oxford, Summer 1976 • First paper to use term “confocal microscope”
Confocal microscopy •  Improved resolu<on •  Op<cal sec<oning –  3D imaging –  Surface profiling •  Reduced scaUered light –  Imaging through scaUering media, e.g. <ssue Confocal depth discrimina<on confocal pinhole
Confocal microscopy – Reflectance •  Industrial applica<ons, surface profiling •  ScaUering media, <ssue – Fluorescence •  Autofluorescence or labelled •  Fixed or living Oxford microscope, 1975 First image
Beam-scanning
Hairs on an ant’s leg Confocal microscope with computer
Cox IJ, Sheppard CJR (1983) Digital image processing of confocal images, Image & Vision Compu0ng 1, 52-­‐56 (1983)
conventional
confocal
confocal
autofocus
surface
profile
Confocal r
eflectance Stereo pair of a pollen grain
J. Microsc. 165, 103-117 (1992)
Endeavour, 10, 17-19 (and cover)(1986)
Rat brain (cerebellum)
Colour confocal reflection image of a leaf
Microtubules labeled
with 15nm gold
Inst. Phys. Conf. Ser.
No 98, 1989
Commercializa<on of the confocal microscope Oxford Optoelectronics 1982
LaserSharp SOM100, 1984
BioRad MRC500, 1987
Marvin Minsky 1957 Goldman, 1940 slit
film
cornea
lens
Confocal principle reinvented
many times in different
disciplines, e.g. slit lamp in
"Spaltlampenphotographie und -photometrie,"
ophthalmology
Ophthalmologica 98, 257-270 (1940).
Z Koana 1942 Naora, 1951 H Naora, Science 114, 279 (1951)
Microspectrophotometry and !
cytochemical analysis of nucleic acids!
Naora, 1955 Free from the
SchwartzschildVilliger effect
H Naora
Exp. Cell Res. 8, 259-278 (1955)
Zvorykin & Ramberg, 1949 http://www.davidsarnoff.org/kil-chapter09.htm
“Demonstrated to the FCC at Camden in 1940,
this early system was a two-color affair employing
a combination of photocells, color filters and
flying-spot scanners for pickup, and reproducing
its images through a pair of kinescopes
whose output was combined optically on a
single screen. …
In December 1945, an advanced sequential
system developed by Kell, Schroeder,
Gordon L. Fredendall and Richard. C. Webb
was demonstrated at Princeton. The new system
employed the Image Orthicon pickup tube for
the first time, and added a new refinement in the
form of polarizing light filters to produce a
three-dimensional effect for viewers wearing
polaroid spectacles. As demonstrations go, it was
an outstanding success.”
Zvorykin & Ramberg Resolu<on Resolution (how small a thing you can see) !
depends on the wavelength of a wave!
Different types of radia<on •Electromagnetic waves!
Light!
UV!
IR!
X-rays!
Microwaves!
• Matter waves!
Electrons!
Protons, neutrons!
Ions, atoms!
• Acoustic waves!
Transmission electron microscope (TEM) Acous<c waves Image elastic properties:!
• Stiffness!
• Viscosity!
Scanning acous<c microscope (SAM) (from Wickramasinghe)
Wavelength of Electromagne<c and Acous<c Waves Scanning X-­‐ray microscope Wavelength of Par<cles Generalized Microscope!
• A microscope forms an image of an object.!
• What does an optical microscope image?!
–  variations in reflectivity (transmissivity) of
the object. !
–  in general a microscope can image
variations in different physical properties of
the object.!
Generalized Microscope Generalized Scanning Microscope Scanning electron microscope (SEM) Spectroscopic methods • 
• 
• 
• 
• 
• 
• 
• 
Absorp<on spectroscopy Raman Resonance Raman CARS 2-­‐photon absorp<on 2-­‐photon fluorescence Photoelectron spectroscopy Photoacous<c spectroscopy Any type of spectroscopy can be performed in a spa<ally resolved fashion Scanning Second Harmonic Genera<on (SHG) Microscope Two-­‐photon imaging •  Signal propor<onal to the square of illumina<on intensity –  Op<cal sec<oning with no pinhole –  Signal increased using pulsed laser Harmonic microscopy Harmonic microscopy Second harmonic genera<on microscope Sheppard CJR, Kompfner R, Gannaway J, Walsh D (1977)
The scanning harmonic optical microscope,
IEEE/OSA Conf. Laser Engineering and Applications
Washington,
IEEE J. Quantum Elec., QE-13, 100D, post-deadline.
Op<cal sec<oning in SHG Microscopy KD*P crystal, second harmonic
images - cw NdYAG laser
1064nm
Two-­‐photon fluorescence •  Advantages – Bleaching only in plane of focus – Near IR penetrates through <ssue – Need not image (only need to collect) fluorescent light (also beUer penetra<on) •  Disadvantages – Weak signal – Slightly poorer resolu<on – Expensive laser Photo-­‐thermal microscopes (from Har<kainen) Focused modulated laser
Photothermal microscopy
Photo
acoustic
microscopy
Photodisplacement
microscopy
Photoacous<c & photothermal optical point spread function
optical absorption of
sample
acoustic psf
thermal psf
thermal and acoutic properties of sample
Conclusions •  Microscope can be of conven<onal or scanning forms •  Equivalence of conven<onal and scanning microscopes •  Generalize to different forms of radia<on •  Generalize to different contrast mechanisms •  Any physical effect can be used as the basis of a microscope contrast mechanism •  Any form of spectroscopy can be performed in a spa<ally resolved manner Quiz •  In the photoelectric effect, the photocurrent depends on the work func<on of the material. By considering the concepts of the generalized microscope and the generalized scanning microscope, devise two schemes for imaging qualita<vely varia<ons in work func<on of a sample. Compare their imaging proper<es. •  Es<mate the resolu<on aUainable in (a) a conven<onal photoelectron microscope, and (b) a scanning photoelectron microscope, if the work func<on is equivalent to a wavelength of 400nm. •  Suggest what contrast mechanisms could be important in photoelectron microscopy. Photoelectron microscope •  In the photoelectric effect, the photocurrent depends on the work func<on of the material. •  By considering the concepts of the generalized microscope and the generalized scanning microscope, devise two schemes for imaging qualita<vely varia<ons in work func<on of a sample. •  Compare their imaging proper<es.Es<mate the resolu<on aUainable in (a) a conven<onal photoelectron microscope, and (b) a scanning photoelectron microscope, if the work func<on is equivalent to a wavelength of 400nm. •  Suggest what contrast mechanisms could be important in photoelectron microscopy. Transmission electron microscope (TEM) with photon s<mula<on UV illumination
• Electrons have low energy
• So need to accelerate before focusing
with ordinary TEM column
• Resolution from electrons (~10-100nm)
• Hayes Griffith, Advances in Optical &
Electron Microscopy, Vol. 10,
R. Barer and V.E. Cosslett, eds,
Academic Press, London (1987).
Scanning photoelectron microscope UV source (preferably tunable)
• In vacuum, but
maybe not necessary
• Resolution limited to
wavelength of light
electrode
Current
(Can also change
bias voltage)
scanned
Photoelectron spectro-­‐microscope (PESM) UV source
detector
⊗
magnetic field
scanned