Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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University of Pécs, Medical SchoolDepartment of Biophysics
Dr. Beáta Bugyi
MEDICAL BIOTECHNOLOGYBIOPHYSICS2012. October 18.
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CELL DIVISIONCELL MIGRATION
LAMELLIPODIUM
INDIVIDUAL ACTIN FILAMENTS
INTRAVITAL MICROSCOPY IN LIVING MICEmacrophages migrating in a tumorvascular flow in the liver
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Microscopyallows us to observe living things at different levels:
from organs (cm, 10-2m)to
individual molecules (nm, 10-9m).
7 orders of magnitude!!The resolution of the human eye depends on:
wavelength: λdiameter of the pupil: d
for human eyes: the limit of resolution is ≈ 0.1 mm (1’ minute of arc)from the minimal conventional viewing distance of 25 cm: distance of clear vision
! ?
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How to observe small objects invisible for the human eye?
MICROSCOPY
MIKRÓS = small+
SKOPEIN = to see
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IMAGE FORMATION: OBJECT IMAGE
HOW?
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image formation: lightlenses: glasssignal: lighttechniques:
PHASE CONTRASTFLUORESCENCEPOLARIZATIONSTEREOULTRA (DARK FIELD)
Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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image formation: electron beamlenses: electromagnetsignal: electrons
IMAGE FORMATION: OBJECT IMAGE
HOW?
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image formation: microscopic probesignal: interaction between the object and the probetechniques:
ATOMIC FORCE (AFM)NEAR-FIELD SCANNING OPTICAL (NSOM)SCANNING TUNNELING (STM)ELECTROSTATICMAGNETIC FORCESCANNING ELECTRON (SEM)
IMAGE FORMATION: OBJECT IMAGE
HOW?
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~ 1000 Abbas Ibn Firnas (Cordóba) sand glass„reading stone” hemispherical lens
~ 1284 Salvino D'Armate (italian) first eye glasses
1590 Zaccharias brothers (dutch) first compound microscope
1665 Robert Hook (english) illumination1674 Anton van Leeuwenhoek polished lenses, 270x magnification
1830 Joseph Jackson Lister corrections for lens abberations bycombining lenses
1872 Ernst Abbe Abbe Sine Condition, diffraction limit
1893 August Köhler Köhler illumination
1903 Richard ZsigmondyNobel Prize in Chemistry 1925 ultramicroscope
1932 Frits ZernikeNobel Prize in Physics 1953 phase contrast microscope
1931 Ernst RuskaNobel Prize in Physics 1986 electron microscope (EM)
1981 Gerd Binnig, Heinrich RohrerNobel Prize in Physics 1986
scanning tunneling microsope (STM)resolution: XY: 0.1 nm Z: 0.01 nm
1994Osamu Shimomura, Martin Chalfie, RogerY. TsienNobel Prize in Chemistry 2008
GFP: green fluorescent protein
2000 Stefan Hellbreaking Abbe’s diffraction limit inlight microscopySTED
http://www.nature.com/milestones/milelight/timeline.html
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NA = 0.04 – 1.4511
Image formation is based on the use of
visible light (λ = 400 – 700 nm)glass lenses
retina
OBJECT
eye
IMAGEMAGNIFIEDvirtualerect 12
CONVERGING LENS
1 CONVEX/CONVERGNIG LENS (LOUPE)thicker in the center than the periphery
magnification through increasing the visual angle on the retina
Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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retina
IMAGE 1: objective
OBJECT
eye OBJECT
↓ objective (1st lens)
Image 1: intermediateMAGNIFIED, real, inverted
↓ ocular (2nd lens)
IMAGE 2: finalFURTHER MAGNIFIED, virtual, erect
IMAGE 2: ocular
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OCULAR - eyepiece
OBJECTIVE
2 CONVEX/CONVERGNIG LENSESaligned in series
OBJECT (Specimen)
IMAGE
OBJECTIVE
OCULAR
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IMAGE
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OBJECTIVE1
found closer to the object for magnification
OCULAR (eyepiece)2: binocular
found closer to the observer for further magnification
CONDENSERto gather or condense the illuminating light onto thespecimen into a cone of light that illuminates thespecimen with uniform intensity over the entire viewfield
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MICROSCOPE STAND
ILLUMINATIONlamplaser
CAMERA
FILTERS
MIRRORS
UPRIGHT INVERTED
LIGHT SOURCE IMAGE
LIGHT SOURCE
IMAGE
OBJECT OBJECT
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CRITICAL ILLUMINATION
set conjugate planes: what is in focus for one of the conjugate planes will also be infocus at the other conjugate planes of that set
one set of conjugate planesthe image of the light source is focused onto the specimen by the condenser
uneven illumination of the specimenthe image of the light source is obtrusively superimposed on the image of the specimen
Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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CRITICAL ILLUMINATION
one set of conjugate planesthe image of the light source is focused onto the specimen by the condenser
uneven illumination of the specimenthe image of the light source is obtrusively superimposed on the image of the specimen
set conjugate planes: what is in focus for one of the conjugate planes will also be infocus at the other conjugate planes of that set
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1893. August Köhler
COLLECTOR LENSseparate light paths: illuminating & image formingtwo sets of conjugate planes: light source & specimen
filament lampcondenser aperture diaphragmback focal plane of the objectiveeyepoint (exit pupil of the microscope)
field diaphragmfocused specimen on the microscope stagefixed diaphragm of the eyepieceretina of your eye (film plane)
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1893. August Köhler
OBJECT-POINT
IMAGE-”POINT”: PIXEL
INTENSITY MAP22
1. MAGNIFICATIONthe object is big enough to see
2. RESOLUTIONall the interesting details of the object are visible
3. CONTRASTthe interesting details of the object are distingushable from the environment
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OBJECTIVE: Nobjective = 1 – 150OCULAR: Nocular = 5 – 30
MAGNIFICATION OF THE MICROSCOPEMmicroscope = Mobjective x Mocular 24
MAGNIFICATION (M)ratio of the size of the image and the objecthow big is the image compared to the object
Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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RESOLUTION (d)the shortest distance between two points of the object that can be distinguished as separateentities on the image
OBJECT
imageINTERFERENCE
PATTERN
light source
angle of diffraction
optical gratingperiodic optical properties
objective
IMAGE-2 -1 0 +1 +2
CONSTRUCTIVEmaximum - bright
DESTRUCTIVEminimum - dark
DIFFRACTION
objectOPTICAL GRADING
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„perfect” image:all the diffration orders participate in image formationin practice it can not be fulfilled
The more higher-order rays are included in the diffraction pattern,the better the resolution of the image.
ABBE’S PRINCIPLE – DIFFRACTION LIMIT
0 order 0 - 1 order 0 - 2 order 0 - 4 order
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image of a single point = interference/diffraction pattern (concentric circles of intensity minimumand maximum)AIRY DISC: diffraction limited image of a single point
maxima - bright minima - dark
012
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OBJECT IMAGE
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George Biddel Airy
RESOLVED NOT RESOLVED
The first maximum of 1 falls in the first minimum of 2.
d
image1 image2
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1872. Ernst Abbe
αλ
sin61.0, n
d yx =
λ: wavelength (illumination)α: aperture angle (objective)n: refractive index (between the object and the objective)
XY direction – in plane Z direction – along the optical axis
2)sin(2αλ
ndz =
The better the resolution is the smaller d is.
1/d: resolving power
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RESOLUTION (d): the shortest distance between two points of the object that can be distinguished asseparate entities on the image is given as:
Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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α aperture angle: half angle of the light cone captured by the objectiveNUMERICAL APERTURE (NA)
the ability of an optical system to resolve fine details in an object being observed
α: aperture anglen: refractive index
αsinnNA =
NA = 0.04 – 1.4532
XY direction – in planed = 230 nm
Z direction – along the optical axisd = 1000 nm
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CONTRASTenhancement of the inhomogeneity of the sample
OPTICAL INHOMOGENEITY light absorptionrefractive indexshapecolour...
↓ALTERED PROPERTIES OF THE LIGHT
passing through the objectdirectionvelocityphasewavelength
NA = 0.04 – 1.4535
(a) BRIGHTFIELD
(b) DIFFERENTIAL INTERFERENCE CONTRAST (DIC)thickness, shape, refractive index
(c) PHASE CONTRASTthickness, shape, refractive index
(d) HOFFMAN MODULATION CONTRAST (HMC)thickness, shape, refractive index
(e) ULTRAMICROSCOPY –DARKFIELDsmall scattering object
(f) POLARISATIONbirefringent (doubly refracting) matter NA = 0.04 – 1.45
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FLUORESCENCE
Medical Biotechnology ‐ 14. Light microscopy 2012.10.18.
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HOMOGENEOUS
CONTRAST
human glial brain tissue in monolayer culture
Frits Zernike (1888-1966)
OPTICAL INHOMOGENEITYPHASE DIFFERENCE INTENSITY DIFFERENCE
Listeria monocytogenes in PtK2 cells
cell-free model systemmoving cells
two separate compound microscopes 14o
2 objectives + 2 oculars
OBJECT↓two 2D images (left - right)↓one 3D IMAGE
application:microsurgery
14o
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DO NOT CONFUSE WITH THE BINOCULAR!!