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

33NA = 0.04 – 1.45

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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!!