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UNIVERSITY OF PÉCSMEDICAL SCHOOL

www.medschool.pte.hu

BIOPHYSICS 2.2015 18th MarchDr. Beáta BugyiDepartment of Biophysics

MICROSCOPIC TECHNIQUES 1� LIGHT MICROSCOPY� FLUORESCENCE

MICROSCOPY

courtesy: see last slide

human lung tissue (histology) microsurgery

cell migration

(phase contrast microscopy)

mitosis

actin, microtubule

(confocal microscopy)

mitosis, starfish oocyte

actin, microtubule, chromosome

(3D confocal microscopy)

blood flow in living mouse

dextran, hepatocyte

(intravital microscopy)

individual molecules - formin, actin

(TIRFM)

� IMAGING TECHNIQUES

� MICROSCOPIC TECHNIQUES

• LIGHTLIGHTLIGHTLIGHT MICROSCOPYMICROSCOPYMICROSCOPYMICROSCOPY

» principles of image formation in the light microscope

� light-matter interaction: REFRACTION, DIFFRACTIONREFRACTION, DIFFRACTIONREFRACTION, DIFFRACTIONREFRACTION, DIFFRACTION

� MAGNIFICATION, RESOLUTION, CONTRASTMAGNIFICATION, RESOLUTION, CONTRASTMAGNIFICATION, RESOLUTION, CONTRASTMAGNIFICATION, RESOLUTION, CONTRAST

– FLUORESENCE MICROSCOPYFLUORESENCE MICROSCOPYFLUORESENCE MICROSCOPYFLUORESENCE MICROSCOPY

» special components in the fluorescence microscope

Overview Microscopy, light microscopy

� MICROSCOPYMICROSCOPYMICROSCOPYMICROSCOPY = 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 10-2m) to single molecules (nm 10-9m)

� SCANNINGSCANNINGSCANNINGSCANNING PROBEPROBEPROBEPROBE MICROSCOPYMICROSCOPYMICROSCOPYMICROSCOPY

� ELECTRONELECTRONELECTRONELECTRON MICROSCOPYMICROSCOPYMICROSCOPYMICROSCOPY

� LIGHT MICROSCOPYLIGHT MICROSCOPYLIGHT MICROSCOPYLIGHT MICROSCOPY

� Image formation is based on visible light (400 – 800 nm) and the use of glass lenses.

Principles of image formation in the light microscope

� MAGNIFICATION

� RESOLUTION

� CONTRAST

� …do all of the above while introducing as little

distortion as possible

Principles of image formation in the light microscope

� MAGNIFICATION

� small objects have to be large enough to see them by eyes

� RESOLUTION

� CONTRAST

� …do all of the above while introducing as little

distortion as possible

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The simple magnifying glass, loupe1x magnification

von Leeuwenhoek

(1632-1723 Dutch

zoologist, microbiologist)

reading stone (~ 810-887

B.C. Abbas Ibn Firnas)

CONVERGINGCONVERGINGCONVERGINGCONVERGING LENSLENSLENSLENS

O I1

observer

MAGNIFIEDMAGNIFIEDMAGNIFIEDMAGNIFIED

real

inverted

Magnification in the „compound” microscope2x magnification

Hans & Zacharias Jansen

(~ 1590 Dutch spectacle-

maker)

OBJECTIVEOBJECTIVEOBJECTIVEOBJECTIVEconverging lens

close to the object

O I1I2

OCULAR, EYEPIECEOCULAR, EYEPIECEOCULAR, EYEPIECEOCULAR, EYEPIECEconverging lens

close to the observer

observer

MAGNIFIEDMAGNIFIEDMAGNIFIEDMAGNIFIED

real

inverted

MAGNIFIEDMAGNIFIEDMAGNIFIEDMAGNIFIED

virtual

inverted

Lens systems in a modern light microscope

OBJECTIVEOBJECTIVEOBJECTIVEOBJECTIVE

OCULAROCULAROCULAROCULAR

CONDENSORCONDENSORCONDENSORCONDENSORuniform illumination

Köhler

Magnification of the light microscope

OBJECTIVE lens: ���������~2.5 � 150

OCULAR lens: �������~10 � 25

�����������~20 � 1000

��������� �!�"�# $����%&�'%

!()%� &�'%��������� �!�"�# $

����%&�'%

!()%� &�'%

����������� $ ��������� ∗ ������������������ $ ��������� ∗ �������

http://www.olympusmicro.com/primer/virtual/magnifying/index.html

Lenses, lens systemsnumerical aperture

http://zeiss-campus.magnet.fsu.edu/tutorials/basics/oilimmersionrefractiveindex/indexflash.html

http://www.microscopyu.com/articles/formulas/formulasna.html

NUMERICALNUMERICALNUMERICALNUMERICAL APERTUREAPERTUREAPERTUREAPERTURE (NA(NA(NA(NA))))� light collecting ability of a lens (system)

� NA ↑ more light is captured

+, $ � ∗ &��-+, $ � ∗ &��- nnnn: refactive index of the medium between the lens

and the object

αααα: 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)

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Principles of image formation in the light microscope

� MAGNIFICATION

� RESOLUTION

� small details have to be distinguishable from each other

� CONTRAST

� …do all of the above while introducing as little

distortion as possible

Resolwing power of the light microscope

RESOLUTIONRESOLUTIONRESOLUTIONRESOLUTION LIMITLIMITLIMITLIMIT

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

SPREAD IN SPACE

lightlightlightlight sourcesourcesourcesource

OBJECTOBJECTOBJECTOBJECT

opticalopticalopticaloptical gratinggratinggratinggratingdrating constant: d

periodic optical properties

CONSTRUCTIVEmaximum - bright

DESTRUCTIVEminimum - dark

INFORMATION

http://zeiss-campus.magnet.fsu.edu/articles/basics/imageformation.html

INTERFERENCEINTERFERENCEINTERFERENCEINTERFERENCE

DIFFRACTIONDIFFRACTIONDIFFRACTIONDIFFRACTION

IMAGEIMAGEIMAGEIMAGE

intensityintensityintensityintensity distributiondistributiondistributiondistribution////diffractiondiffractiondiffractiondiffraction patternpatternpatternpattern

diffraction orders: . $ 0, 01, 02

-

1

∆& $ 1&��- $ .3

objective

back focal plane

Image as a diffraction patternJohn Herschel (1792-1871, English astronomer), George Biddell Airy (1801-1892, English astronomer)

AIRYAIRYAIRYAIRY PATTERNPATTERNPATTERNPATTERN: : : : diffraction limited image of a single point-like object (concentric dark/bright

fringes)

in 3D: PSF (POINT in 3D: PSF (POINT in 3D: PSF (POINT in 3D: PSF (POINT SPREAD SPREAD SPREAD SPREAD FUNCTION)FUNCTION)FUNCTION)FUNCTION)

Richard W Cole et al Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control Nature Protocols (2011)

XY direction – lateral

Z direction – axial

Abbe’s limit of resolutionErnst Abbe (1840-1905)

Image formation: besides the 0th order maximum at least the 1th order maximum

have to be captured.

. $ 0

. $ �1 . $ 41

Maxima are observed for angles (-):

∆& $ 1&��- $ .3. $ 0,01, 02

Participate in image formation:

for . $ 1:

1 $3

&��-5

Have to be captured:

1 $3

267869$

3

2:;

objective

-5 -5

Resolwing power of a light microscopediffraction limit

1<,= $ 0.613

+,~200��1<,= $ 0.61

3

+,~200��

1? $ 2�3

+, @~800��1? $ 2�

3

+, @~800��

λ: wavelenght of the illuminating light

NA: numerical aperture of the lens system

XY direction – lateral

Z direction – axial

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 pattern of the other point source

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Simple ways to improve the resolution

λ: decreasedecreasedecreasedecrease the wavelenght of the illuminating light

NA: increaseincreaseincreaseincrease the numerical aperture of the lens system –––– IMMERSION MEDIUMIMMERSION MEDIUMIMMERSION MEDIUMIMMERSION MEDIUM

immersion medium refractive index

air 1.000

water 1.333

glycerol 1.469

oil 1.515

wavelength (nm)

NA= 0.8dx,y (nm)

400 250

500 312.5

600 375

700 437

2014 Nobel prize in ChemistryStefan Hell, Eric Betzig and William Moerner

"for the development of super-resolved

fluorescence microscopy"

1877 Abbe’s diffraction limitErnst Abbe, Carl Zeiss

Ernst Abbe memorial, Jena

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2

014/

Principles of image formation in the light microscope

� MAGNIFICATION

� RESOLUTION

� CONTRAST

� the interesting details of the object have to be

distinguishable from the environment

� …do all of the above while introducing as little

distortion as possible

ConstrastProblem: many living unstained samples (tissues/cells…) are thin and optically transparent,

hard to see them by brightfield microscopy.

OPTICAL INHOMOGENEITYOPTICAL INHOMOGENEITYOPTICAL INHOMOGENEITYOPTICAL INHOMOGENEITY of of of of the samthe samthe samthe sample (properties that distinguish the object from its

environment)

� light absorption

� refractive index

� shape

� „colour”

results in results in results in results in ALTERED PROPERTIES OF THE LIGHT ALTERED PROPERTIES OF THE LIGHT ALTERED PROPERTIES OF THE LIGHT ALTERED PROPERTIES OF THE LIGHT passing through the object passing through the object passing through the object passing through the object

� direction

� speed

� phase

� polarity

� wavelength…

Contrast enhancing techniques:

phase-contrast-, differential interference contrast- (DIC), Hoffman-modulation contrast-,

darkfield-, polarized light-, fluorescencefluorescencefluorescencefluorescence microscopymicroscopymicroscopymicroscopy,…

Fluorescence microscopy

� FLUORESCENCE MICROSCOPYlight microscopy + fluorescence

� Image formation is based on visible light (400 – 800 nm) and

the use of glass lenses.

� The The The The objectobjectobjectobject is is is is imagedimagedimagedimaged onononon thethethethe basisbasisbasisbasis of of of of itsitsitsits fluorescencefluorescencefluorescencefluorescence emissionemissionemissionemission....

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

INNERINNERINNERINNER FLUOROPHORESFLUOROPHORESFLUOROPHORESFLUOROPHORES: autofluorescence, limited

OUTEROUTEROUTEROUTER FLUOROPHORESFLUOROPHORESFLUOROPHORESFLUOROPHORES : spectral flexibility� synthetic dye

� quantum dot

� fluorescent proteins

GFP: green fluorescent protein and its spectral variants2008 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

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How can we have a fluorescent object?photoconvertible fluorophores

http://www.olympusfluoview.com/applications/opticalhighlighters.html

STANDARDSTANDARDSTANDARDSTANDARD

PHOTOACTIVABLEPHOTOACTIVABLEPHOTOACTIVABLEPHOTOACTIVABLE

PHOTOSWITCHABLEPHOTOSWITCHABLEPHOTOSWITCHABLEPHOTOSWITCHABLE

How to image fluorescence?

http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

LIGHT SOURCEarc lamp

LASER

DETECTORCCD camera

PMT

DETECTOReye

FILTERS

MIRRORS

sample

trans

epi

How to image fluorescence?optical filters, dichroic mirrors

OPTICAL FILTERSwavelength dependent

absorption/transmission properties

� shortpass

� longpass

� bandpass

DICHROIC MIRRORSwavelength dependent reflection and

absorption/transmission properties

(see: Flow cytometry)

Optical filters, dichroic mirrors in thefluorescence microscope

emission filter

excitation

filterdichroic

mirror

VASP-GFP actin-RFP

Shows again the localization of VASP at protruding lamellipodia and filopodia tips in a fish fibroblast expressing VASP-GFP and Actin-RFP.

forrás: http://cellix.imba.oeaw.ac.at/motility/nucleationfactors

VASP-GFP actin-RFP

Shows again the localization of VASP at protruding lamellipodia and filopodia tips in a fish fibroblast expressing VASP-GFP and Actin-RFP.

forrás: http://cellix.imba.oeaw.ac.at/motility/nucleationfactors

VASP-GFP actin-RFP

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www.aok.pte.hu

Light-, fluorescence microscopekeywords

� PrinciplesPrinciplesPrinciplesPrinciples of image of image of image of image formationformationformationformation in a in a in a in a lightlightlightlight microscopemicroscopemicroscopemicroscope::::

� refractionrefractionrefractionrefraction and and and and diffractiondiffractiondiffractiondiffraction

� magnificationmagnificationmagnificationmagnification, , , , resolutionresolutionresolutionresolution, , , , contrastcontrastcontrastcontrast

� Image Image Image Image formationformationformationformation bybybyby convergingconvergingconvergingconverging lenseslenseslenseslenses

� NumericalNumericalNumericalNumerical apertureapertureapertureaperture

� AiryAiryAiryAiry patternpatternpatternpattern, , , , diffractiondiffractiondiffractiondiffraction limit, limit, limit, limit, PSFPSFPSFPSF

� SpecialSpecialSpecialSpecial elementselementselementselements in a in a in a in a fluorescencefluorescencefluorescencefluorescence microscopemicroscopemicroscopemicroscope: : : : fluorophoresfluorophoresfluorophoresfluorophores, , , , lightlightlightlight

sourcessourcessourcessources, , , , detectorsdetectorsdetectorsdetectors, , , , opticalopticalopticaloptical filtersfiltersfiltersfilters, , , , mirrorsmirrorsmirrorsmirrors

www.aok.pte.hu

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/