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Invitation for a walk through microscopy

Invitation for a walk through microscopy. sebastian.schuchmann@charite.de. Techniques in microscopy. Conventional (light) microscopy bright & dark field, phase & interference contrast. Fluorescence microscopy light sources, fluorescence detectors, digital image , objectives.

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Invitation for a walk through microscopy

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  1. Invitation for a walk through microscopy sebastian.schuchmann@charite.de

  2. Techniques in microscopy • Conventional (light) microscopybright & dark field, phase & interference contrast • Fluorescence microscopylight sources, fluorescence detectors, digital image, objectives • Single- & two-photon confocal microscopybasic idea & differences, advantages & disadvantages • What is the optimal technique (for my question)?

  3. Conventional (light) microscopy (http://micro.magnet.fsu.edu)

  4. Magnification Illumination Specimen Light microscope: general structure I (with modification http://micro.magnet.fsu.edu)

  5. 2f (magnifying glass) imaginary image f object F Magnification 2f f object real image Illumination F Light microscope: general structure II ocular objective specimen condensor light source

  6. ocular (magnifying glass) Intermediate (real) image (on the projector screen) objective (slide projector) Final (imaginary) image light A light microscope is a combination of a slide projector with a magnifying glass Total magnification = Mobjective x Mocular

  7. bright field with specimen specimen Bright and dark field illumination objective object plane condensor

  8. dark field without specimen specimen Bright and dark field illumination dark field with specimen objective object plane condensor - ring diaphragm (usually) - dark field condensor

  9. Dark field • part-illumination of the specimen • scattered light collected by objective • bright object on dark background Objects with high contrast Objects with a sharp rise in refraction index Bright and dark field illumination • Bright field • total illumination of the specimen • direct light collection by objective • dark/colored object on bright background (with modification http://mikroskopie.de)

  10. conjugate planes in the image path optical elements retina (eye) A. Köhler (1866-1948) ocular intermediate image plane specimen objective focused specimen diaphragm condensor aperture diaphragm field diaphragm light source Microscopy illumination after Köhler (or the mystery condensor adjustment) objective condensor

  11. 3. Choose prefered objective (at least 10x), focus specimen 4. Close down field diaphragm;focus the image of the fielddiaphragm sharply onto thealready focused specimen 5. If neccessary center the condensor;then open the field diaphragmuntil it just disappears from view 6. Take out one of the eyepieces,look down the tube andadjust the aperture diaphragm Diaphragm should be 2/3 to 3/4 open (compromise between resolution & contrast) How to do adjust the Köhler illumination 1. Light source on 2. Open up fully field diaphragmand aperture diaphragm

  12. amplitude object amplitude amplitude Reduction in amplitude is equal with a reduction in light intensity (used in bright field microscopy!) phase object phase Slow down of light wave passing the phase object Amplitude and phase objects influence light waves: Basic principle for phase & interference contrast

  13. scattered light intermediate image plane slowed down,unscattered light phase ring objective focussed specimen condensor and light source phase diaphragm Phase contrast

  14. Specimen (inhomogen phase object) Prisma (Nomarski) DIC prisma (Nomarski) Polarisator Analysator Phase difference unpolarized light linear polarized light two vertical polarized waves linear polarized light (analysator vertical vs. polarisator) Differential interference contrast (DIC)

  15. Kidney tissue(tubule with some cells> 100 µm thick section) Buccal epithelial cell (monolayer) Phase contrast DIC Phase contrast vs. DIC (with modification http://mikroskopie.de)

  16. Light microscopy: illumination & contrast techniques • Illumination Try to optimise your illumination (condensor adjustment after Köhler) Bright field illumination: standard technique for most specimen Dark field illumination: specific technique for monolayer specimen with distinct differences in the refraction index • Contrast Check and improve all contrast techniques available at your microscope Phase contrast: standard technique for low-contrast monolayer specimen DIC: standard technique for low-contrast specimen, in particularily for thick (non-monolayer) preparations

  17. Fluorescence microscopy

  18. E = hn c = ln E ~ 1 / l Basic idea of fluorescence microscopy: Stokes shift

  19. Detection system (eye, conventional camera, CCD, photo diode, PMT) emission wavelenght lem > lex (filter) light source (arc lamp, laser) excitation wavelenght lex fluorescence object/dye The use of the Stokes shift in fluorescence microscopy dichroic mirror

  20. Fluorescence microscopy requires ... • Fluorochrome (or autofluorescence) see Molecular Probes (www.probes.com) • Light source

  21. Laser types UV IR Argon 351 364 457 477 488 514 Blue diode 405 440 Helium-Cadmium 354 442 Krypton-Argon 488 569 647 Green Helium-Neon 543 Yellow Helium-Neon 594 Orange Helium-Neon 612 Red Helium-Neon 633 Red diode 635 650 Ti:Sapphire 720-980 Light source Arc lamps Xenon Mercury

  22. Fluorescence microscopy requires ... • Fluorochrome (or autofluorescence) see Molecular Probes (www.probes.com) • Light source • Fluorescence detection

  23. photo diode PMT CCD conventional photography Fluorescence detector systems ... Temporal resolution Spartial resolution

  24. Analog Image Digital Sampling Pixel Quantization Fluorescence detector systems produce digital images - observer eye - conventional photography • - CCD • PMT (in combination with scan technique) (with modification http://micro.magnet.fsu.edu)

  25. 255 255 255 0 0 0 grey level grey level grey level Fluorescence detector systems produce digital images normal contrast low contrast high contrast pixel counts (with modification http://micro.magnet.fsu.edu)

  26. Mainly fluorescence detector systems are color-blind! (Colors are based on a [pseudo-]color look-up table)

  27. Fluorescence microscopy requires ... • Fluorochrome (or autofluorescence) see Molecular Probes (www.probes.com) • Light source • Fluorescence detection • (prefers) immersion objectives

  28.  n2 n1  n1 < n2    sin  n2 = sin  n1 Immersion objectives: Remember the refraction index! total reflection

  29. water or oil immersion objective medium (water or oil) specimen Emission Excitation Light source DM Immersion objectives: Remember the refraction index! refraction index (n) air  1.00 water = 1.37 oil = 1.5 glass = 1.5 Immersion objective with specimen

  30. Conventional fluorescence microscopy • Advantage • low cost • uncomplicated handling • fast imaging technique • Disadvantage • no 3-dimentional imaging possible • low depth of light penetration • bleaching

  31. Confocal microscopy (Schmitz et al., 2001)

  32. full field detection point scan detection full field illumination point scan illumination Basic idea of confocal microscopy I Conventional fluorescence microscope Laser scanning microscope specimen Arc lamp (Hg, Xe) + excitation filter laser light source

  33. Different wavelengths require different laser, for example ... visible spectrum ultra violet infra red Argon 457 477 488 514 Green Helium-Neon 543 Red Helium-Neon 633 Laser: light source for confocal microscopy Laser (Light Amplification by Stimulated Emission of Radiation) = highly precise light source in direction, frequency, phase, polarisation - monochromatic = light has the same wavelength (continuous-wave lasers) - coherent = light is oscilating in the same phase - linear polarized = light is oscilating in the same direction - can be focussed to a very high density power (compared to arc lamps)

  34. laser y x z y x Basic idea of confocal microscopy II point scan illumination (fluorescence excitation) point scan detection (fluorescence emission)

  35. PMT excitation emission pinhole filter x/y-scanning device and dichroic mirror objective z focal plane y x Confocal microscope: general structure laser source specimen

  36. PMT PMT pinhole pinhole objective objective specimen focal plane specimen Confocal microscope: the power of the pinhole

  37. Confocal microscope: excitation profil in z-direction focal plane A(x) ~ 1/I(x)

  38. visible light 104 UV IR 103 depth of light penetration (µm) 102 101 100 wavelenght (µm) Confocal microscope: depth of light penetration

  39. Confocal fluorescence microscopy • Advantage • improved spartial resolution • 3-dimentional scanning • Disadvantage • more complicated imaging control • low depth of light penetration • bleaching

  40. Two-photon microscopy A B 100 ms 5 µm

  41. single-photon excitation two-photon excitation h* h h Absorbtion Emission Emission Absorbtion h h* E = hn E ~ 1 / l E* = 1/2 E E* ~ 1 / 2l c = ln Basic idea of two-photon microscopy Two photons at the same time and at the same place with doubled wavelenght  photons from the infra red spectrum (> 750 nm)  high photon density

  42. Titan-Sapphire spectra Excitation Emission Light source for two-photon microscopy: Ti/Sa-laser Pump laser: solid-state cw laser, 532 nm, 5 W (Millennia, Spectra Physics) • Mode-locked Titan-Sapphire laser(Tsunami, Spectra Physics) • avarage power > 0.7 W at 800 nm • pulsewidth < 100 fs • nominal repetition rate 80 MHz • turning range 720 - 850 nm

  43. photon non-excited dye molecule 2p-excited dye molecule the required photon density for two-photon excitation can be established only in the focal plan and within a laser puls Two-photon excitation (with modification Piston, 1999) laser pulse focal plane

  44. emission emission PMT PMT excitation excitation pinhole IR laser x/y-scanning device and dichroic mirror z y x Single vs. two-photon microscope: general structure

  45. full field detection point scan detection point scan illumination point scan illumination Fluorescence detection using 2-photon excitation descanned detection Non descanned detection (NDD) specimen pulsed Ti:Sa laser pulsed Ti:Sa laser

  46. Two-photon microscopy with descanned and NDD-PMT excitation beam x/y-scanning device & dichroic mirror (DM) prisma for spectral analyse descanned PMT 1 & 2 DM DM non-descanned (NDD) PMT 3 & 4 objective specimen condensor trans-non-descanned (NDD) PMT 5 DM (with modification Oertner, 2002)

  47. single-photon excitation A(x) ~ 1/I(x) Single vs. two-photon excitation: excitation profile two-photon excitation focal plane A(x) ~ 1/I2(x)

  48. 104 103 depth of ligh penetration (µm) 102 101 100 wavelenght (µm) Two-photon microscope: depth of light penetration visible light UV IR

  49. (3D-FITC-dextran gel; irradiated area ~ 10 x 20 µm) y x z x two-photon absorbtion (760 nm; Ti:Sa) focal plane single-photon absorbtion (488 nm; Ar) focal plane 20 µm 10 µm Single vs. two-photon microscopy: bleaching (with modification Kubitscheck et al., 1996)

  50. Simply doubling the excitation wavelenght? h Absorbtion h Emission 102 h 101 Calcium green (506/533) 100 Fluo-3 (505/526) Two-photon cross section 10-1 Lucifer yellow (428/533) 10-2 Cascade blue (400/420) 10-3 1000 600 700 800 900 Excitation wavelenght (nm) Two-photon microscope: excitation spectra (with modification http://micro.magnet.fsu.edu)

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