courtesy: seelastslide UNIVERSITY OF PÉCS MEDICAL SCHOOL www.medschool.pte.hu MICROSCOPIC TECHNIQUES 1 LIGHT MICROSCOPY FLUORESCENCE MICROSCOPY BIOPHYSICS 2. 2015 18th March Dr. Beáta Bugyi Department of Biophysics human lung tissue(histology) cellmigration (phasecontrastmicroscopy) mitosis actin, microtubule (confocalmicroscopy) microsurgery individualmolecules- formin, actin (TIRFM) mitosis, starfishoocyte actin, microtubule, chromosome (3D confocalmicroscopy) bloodflow in livingmouse dextran, hepatocyte (intravitalmicroscopy) Overview IMAGING TECHNIQUES MICROSCOPIC TECHNIQUES LIGHT MICROSCOPY» principles of image formation in the light microscope light-matter interaction: REFRACTION, DIFFRACTION MAGNIFICATION, RESOLUTION, CONTRAST FLUORESENCE MICROSCOPY» special components in the fluorescence microscope Microscopy, light microscopy MICROSCOPY = MIKROS (small) + SZKOPEIN (to see) - vizualize small objects that are invisible for the human eyes: magnifying device - observe biological objects at different levels: from organs (cm 10-2 m) to single molecules (nm 10-9 m) SCANNING PROBE MICROSCOPY ELECTRON MICROSCOPY LIGHT MICROSCOPY Image formation is based on visible light (400 800 nm) and the use of glass lenses. small objects have to be large enough to see them by eyes 1
The simple magnifying glass, loupe 1x magnification Magnification in the compound microscope 2x magnification CONVERGING LENS O I1 observer reading stone(~ 810-887 B.C. Abbas Ibn Firnas) OBJECTIVE converging lens close to the object I2 O OCULAR, EYEPIECE converging lens close to the observer I1 observer MAGNIFIED real inverted von Leeuwenhoek (1632-1723 Dutch zoologist, microbiologist) MAGNIFIED virtual inverted MAGNIFIED real inverted Hans & Zacharias Jansen (~1590 Dutch spectaclemaker) Lens systems in a modern light microscope Magnification of the light microscope OCULAR CONDENSOR uniform illumination Köhler OBJECTIVElens: ~ 2.5 150 OCULARlens: ~10 25 ~20 1000 OBJECTIVE http://www.olympusmicro.com/primer/virtual/magnifying/index.html Lenses, lens systems numerical aperture NUMERICAL APERTURE(NA (NA) light collecting ability of a lens(system) NA more light is captured n: refactiveindex of themediumbetweenthelens and the object α: aperture angle, half-angle of the light cone captured by the objective If we had a lens with infinitely high magnification could we see infinitely small things? NO! The wave nature of light has to be considered! DIFFRACTION, INTERFERENCE (previously: EM waves, X-ray diffraction) http://zeiss-campus.magnet.fsu.edu/tutorials/basics/oilimmersionrefractiveindex/indexflash.html http://www.microscopyu.com/articles/formulas/formulasna.html 2
small details have to be distinguishable from each other Resolwing power of the light microscope RESOLUTION LIMIT the shortest distance between two points of the object that can be distinguished as separate entities on the image (d) Diffraction in the light microscope Image as a diffraction pattern objective back focal plane IMAGE intensitydistribution distribution/diffraction diffractionpattern diffraction orders: 0, 1, 2 DESTRUCTIVE minimum - dark SPREAD IN SPACE John Herschel (1792-1871, English astronomer), George Biddell Airy (1801-1892, English astronomer) AIRY PATTERN: diffraction limited image of a single point-likeobject(concentric dark/bright fringes) in 3D: PSF (POINT SPREAD FUNCTION) CONSTRUCTIVE maximum - bright INTERFERENCE lightsource http://zeiss-campus.magnet.fsu.edu/articles/basics/imageformation.html DIFFRACTION OBJECT opticalgrating drating constant: d periodic optical properties INFORMATION XY direction lateral Z direction axial Richard W Coleet al Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality controlnature Protocols (2011) Abbe s limit of resolution Ernst Abbe(1840-1905) Resolwing power of a light microscope diffraction limit Image formation:besides the 0 th order maximum at least the 1 th ordermaximum have to be captured. 1 objective 0 1 Maxima areobservedforangles( ): 0, 1, 2 Participate in image formation: for 1: Have to be captured: 2 2 Rayleigh s criterion: the central maximum of the diffraction pattern of one point-source has to be centered on the first minimum of the diffraction patternof the other point source XY direction lateral, 0.61 ~200 Z direction axial 2 ~800 λ: wavelenght of the illuminating light NA: numerical aperture of the lens system 3
Simple ways to improve the resolution λ: decrease the wavelenght of the illuminating light wavelength(nm) NA= 0.8 d x,y (nm) 400 250 500 312.5 600 375 700 437 NA: increase the numerical aperture of the lens system IMMERSION MEDIUM 1877 Abbe s diffraction limit Ernst Abbe, Carl Zeiss 2014 Nobel prize in Chemistry Stefan Hell, Eric Betzig and William Moerner "for the development of super-resolved fluorescence microscopy" immersion medium refractive index air 1.000 water 1.333 glycerol 1.469 oil 1.515 Ernst Abbe memorial, Jena http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2 014/ the interesting details of the object have to be distinguishable from the environment Constrast Problem: many living unstained samples (tissues/cells ) are thin and optically transparent, hard to see them by brightfieldmicroscopy. OPTICAL INHOMOGENEITYof the sample (properties that distinguish the object from its environment) light absorption refractive index shape colour results in ALTERED PROPERTIES OF THE LIGHT passing through the object direction speed phase polarity wavelength Contrastenhancing techniques: phase-contrast-, differential interference contrast- (DIC), Hoffman-modulation contrast-, darkfield-, polarizedlight-, fluorescence microscopy, Fluorescence microscopy FLUORESCENCE MICROSCOPY light microscopy + fluorescence Image formation is based on visible light (400 800 nm) and the use of glass lenses. The object is imaged on the basis of its fluorescence emission. Advantages: spectral flexibility provided by the spectral variability of fluorophores excellent contrast less invasive special techniques (FRAP, FRET, FLIM) the resolution can be improved by special tricks built in a fluorescence microscope How can we have a fluorescent object? standard fluorophores INNER FLUOROPHORES: autofluorescence, limited OUTER FLUOROPHORES: spectral flexibility syntheticdye quantum dot fluorescentproteins GFP: green fluorescentprotein and its spectral variants 2008 Noberl prize in Chemistry: Osamu Shimomura, Martin Chalfie and Roger Y. Tsien for the discovery and development of the green fluorescent protein, GFP". antibody, immunofluorescence 4
How can we have a fluorescent object? photoconvertible fluorophores STANDARD How to image fluorescence? trans LIGHT SOURCE arc lamp LASER PHOTOACTIVABLE DETECTOR eye sample PHOTOSWITCHABLE FILTERS MIRRORS epi DETECTOR CCD camera PMT http://www.olympusfluoview.com/applications/opticalhighlighters.html http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html How to image fluorescence? optical filters, dichroic mirrors (see: Flow cytometry) Optical filters, dichroic mirrors in the fluorescence microscope OPTICAL FILTERS wavelength dependent absorption/transmission properties shortpass longpass bandpass dichroic mirror emission filter excitation filter DICHROIC MIRRORS wavelength dependent reflection and absorption/transmission properties VASP-GFP actin-rfp VASP-GFP actin-rfp VASP-GFP actin-rfp Shows again the localization of VASPat protruding lamellipodia and filopodia tips in a fish fibroblast expressing VASP-GFPand Actin-RFP. forrás: http://cellix.imba.oeaw.ac.at/motility/nucleationfactors Shows again the localization of VASPat protruding lamellipodia and filopodia tips in a fish fibroblast expressing VASP-GFPand Actin-RFP. forrás: http://cellix.imba.oeaw.ac.at/motility/nucleationfactors 5
Light-, fluorescence microscope keywords Principles of image formation in a light microscope: refraction and diffraction magnification, resolution, contrast Image formation by converging lenses Numerical aperture Airy pattern, diffraction limit, PSF Special elements in a fluorescence microscope: fluorophores, light sources, detectors, optical filters, mirrors Recomended web resources http://www.olympusmicro.com/index.html http://www.microscopyu.com / http://zeiss-campus.magnet.fsu.edu/index.html http://www.ibiology.org/ www.aok.pte.hu www.aok.pte.hu 6