TWO-PHOTON MICROSCOPY Listeria monocytogenes...

Biophysics 2 Physics-Biophysics 2 2013.

1

Biophysics 2. Physics - Biophysics 2.

2013. 02. 27. & 28.

dr. Beáta Bugyi – UP MS – Dept. Biophyics

PHASE CONTRAST MICROSCOPYListeria monocytogenes in PtK2 cells

courtesy of Julie Theriot, Dan Portnoy

FLUORESCENCE MICROSCOPYB16 melanoma cell expressing EGFP-actin

courtesy of Klemens Rottner

TWO-PHOTON MICROSCOPYINTRAVITAL MICROSCOPY in living mouse

vascular flow in the liverTIRF MIKROSZKÓPIAactin filaments in vitrocourtesy of Beáta Bugyi

3D CONFOCAL MICROSCOPYstarfish oocyte undergoing meiotic division

courtesy of Péter Lénárt

FRAPlamellipodium actin dynamics in B16-F1 cells

expressing EGFP actincourtesy of Lai et al. EMBO Journal 2008

SEEING IS BELIEVING

The resolution (αααα) of the human eyes depends on:

≈ 0.1 mm (from 25 cm: distance of clear vision for human eyes)

! ?

See: I. semester 10. Vision

�~�

�~0.8’-1.68’�~

�

�~0.8’-1.68’

� wavelength: λλλλ

� pupil diameter: d

íííííííííííííííí

MICROSCOPY

optical coherence tomography (OCT)

widefield, evanescent wave

confocal

4Pi, I5M

high resolution structured illumination (hrSIM)

ground state depletion (GSD)

saturated structured illumination (sSIM)

stimulated emission depletion (STED)

single molecule localisation (PALM, STORM)

near-field optical (NSOM)

electron microscopy (EM)

PET, SPECT

MRI, CT, ultrasound

human eyeAbbe’s principle

BIOLOGICAL IMAGING TECHNIQUES

MICRO SCOPY (Greek)=

MIKRON = small + SZKOPEIN = to see

� allows to visualize objects „invisible” for the human eyes

� instrument: microscope

MICROSCOPY - MICROSCOPE

Microscopy allows us to observe living things at different levels:

from organs (cm 10-2m)

to single molecules (nm 10-9m).

7 orders of magnitude !!!!

OPTICAL – LIGHT MICROSCOPY

NA = 0.04 – 1.45

Image formation:

� visible light (λ = 400 – 700 nm)

� glass lenses


2

„SIMPLE” MICORSCOPE – LUPE (1 CONVERGING LENS)

retina

OBJECT� within focal length

szem

IMAGE� magnified

� erect

� virtual

CONVERGING LENS

See: Geometrical optics

IMAGE 1: objective� magnified

� real

� inverted

OBJECT

IMAGE 2: ocular� magnified

� erect

� virtual

„SIMPLE” COMPOUND MICROSCOPE (2 CONVERGING LENSES)

OCULAR - eyepiece

OBJECTIVE

MODERN COMPOUND MICROSCOPE

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

OBJECTIVE

OCULAR

(2)

CONDENSOR

LIGHT SOURCE

IMAGE

DETECTOR:CAMERA

IMAGE

DETECTOR:EYE

STAND

OBJECT

specimen

FILTERS

MIRRORS

10

OBJECTIVE (1)� lens system found closer to the object

� magnification

OCULAR (eyepiece, 2)� lens system found closer to the observer

� further magnification

CONDENSOR� lens system to condense and focuse the illuminating

light to the specimen

� uniform illumination

OBJECTIVE

OCULARCONDENSOR

MAIN OPTICAL COMPONENTS

TRANSMISSION

LIGHT SOURCE

IMAGE

OBJECT

ILLUMINATION - transmission

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

EPI

IMAGE

OBJECT

LIGHT SOURCE

ILLUMINATION - epi


3

MICROSCOPE STAND – upright / inverted IMAGE FORMATION: OBJECT → IMAGE

OBJECT-POINT

IMAGE-”POINT”

PIXELINTENSITY

MAP

EYE

↑

PHOTON

↓

DIGITAL SIGNAL

http://www.olympusmicro.com/primer/digitalimaging/digitalimagebasics.html

http://www.olympusmicro.com/primer/java/digitalimaging/processing/spatialresolution/index.html

IMAGE FORMATION - REQUIREMENTS

1. MAGNIFICATION� the object is big enough to see

2. RESOLUTION� all the interesting details of the object are visible

� how small things can we see?

3. CONTRAST� the interesting details of the object are distingushable from the environment

1. MAGNIFICATION

OBJECTIVE: Nobjective ≈ 2.5 – 150x

OCULAR: Nocular ≈ 10 – 25x

MICROSCOPE:

Nmicroscope ≈ 50x – 1200x

MAGNIFICATION: N � ��

��

��

��

�� !" ��#$"�%�&" ∗ ��()*�� !" ��#$"�%�&" ∗ ��()*�

v

2. RESOLUTION

RESOLUTION: d� the shortest distance between two points of the object that can be

distinguished as separate entities on the image

… not as simple as it seems �

the image of a 1D point is not a point, but a 3D pattern

RESOLUTION – DIFFRACTION

OBJECT

imageINTERFERENCE

condensor

light source

diffraction angle

optical gratingperiodic optical properties

objective

IMAGE

-2 -1 0 +1 +2

CONSTRUCTIVEmaximum - bright

DESTRUCTIVEminimum - dark

DIFFRACTION

objectOPTICAL GRATING

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

See: I. semester 18. EM waves: diffraction, interference


4

RESOLUTION – AIRY PATTERN

AIRY PATTERN: diffraction limited image of a single point� image of a single point = interference/diffraction pattern (concentric circles of intensity minima

and maxima)

max. - bright min. - dark

0 Airy disk

12

3

George Biddel Airy (1801-1892)

AIRY PATTERN

OBJECT1D

IMAGE3D

POINT SPREAD FUNCTION

resolvednot resolvedThe first maximum of 1 falls in the first minimum

of 2.

d

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

http://www.olympusmicro.com/primer/anatomy/image.htm

maximum

1. minimum

RESOLUTION – AIRY PATTERN

OBJECTIVE – NUMERICAL APERTURE

NA = 0.04 – 1.7

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

APERTURE ANGLE (α)� half-angle of the light cone captured by the objective

NUMERICAL APERTURE (NA)� the ability of an optical lens system to resolve fine details in an object being observed

� dimensionless quantity

�+ , ∗ ��,��+ , ∗ ��,�

� aperture angle: αααα

� refractive index of the medium between the objective

and the object: n

n

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

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

See: Geometrical optics

IMMERSION MEDIUM refractive index: n

air 1.0002

oil 1.5

glycerol 1.4695

water 1.3333

,-,/ sin3�,��4�,�5

sin36�76��,5,-,/

sin3�,��4�,�5

sin36�76��,5

OBJECTIVE – NUMERICAL APERTURE

RESOLUTION – ABBE’S PRINCIPLE – DIFFRACTION LIMIT

Ernst Abbe (1840-1905)

� illumination wavelength: λλλλ

� numerical aperture: NA (NA = n*sinαααα)

XY direction – in plane Z direction – along the optical axis

The better the resolution the smaller d:

48,9 1

2∗

;

�+48,9

1

2∗

;

�+4< 2 ∗

;

3�+5-4< 2 ∗

;

3�+5-

� wavelenght: λλλλ ↓� aperture angle: αααα ↑

� refractive index of the medium between the object and objective: n ↑

Resolution of the light microscope: dx,y ~ 200 nm és dz ~ 1000 nmResolution of the light microscope: dx,y ~ 200 nm és dz ~ 1000 nm

3. CONTRAST

NA = 0.04 – 1.45

CONTRAST� enhancement of the inhomogeneity of the sample (properties that distinguish the object from

its environment)

e.g.:

OPTICAL INHOMOGENEITY results in

� light absorption

� refractive index

� shape

� „colour”

ALTERED PROPERTIES OF THE LIGHT passing through the object

� direction

� speed

� phase

� polarity

� wavelenght…

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

contrast-, darkfield-, polarized light-, fluorescence microscopy

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


5

PHASE-CONTRAST MICROSCOPY

brightfield

phase-contrast

1953 Frits Zernike Nobel Prize in Physics

� refractive index difference � phase shift� intensity difference

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

, �=

�,

�=

�

n↑ c↓

human glial brain tissue in monolayer culture

actin network in biomimetic model systemcourtesy: Bugyi Beáta

PHASE-CONTRAST MICROSCOPY

Listeria monocytogenes in PtK2 cellscourtesy: Julie Theriot, Dan Portnoy

cell migrationcourtesy: Vic Small

STEREOMICROSCOPY – 3D IMAGE

two separate compound microscopes

14o

2 objective + 2 ocular

DO NOT CONFUSE WITH THE

BINOCULAR!!

OBJECT

↓

two 2D image (left - right)

↓

one 3D IMAGE

application:

microsurgery

14o

FLUORESCENCE MICROSCOPY

See: II. semester 2. Fluorescence spectroscopy

Image formation:

� fluorescence emission of the object

� glass lenses

� noninvasive

http://zeiss-campus.magnet.fsu.edu/print/basics/fluorescence-print.html

INNER (INTRINSIC) FLUOROPHORES: limited

� chlorofil

OUTER (EXTRINSIC) FLUOROPHORES : spectral flexibility

� synthetic dye

� quantum dot

� protein

� GFP (green fluorescent protein) and its variants� 2008. Nobel Prize in Chemistry: Osamu Shimomura, Martin Chalfie and Roger Tsien

� antibody

� 1942. immunofluorescence: direct, indirect

FLUORPHORES

http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/introduction.html

PHOTOCONVERTABLE FLUOROPHORES: special applications

http://zeiss-campus.magnet.fsu.edu/print/superresolution/palm/practicalaspects-print.html

STANDARD

PHOTOACTIVABLE

PHOTOSWITCHABLE

FLUORPHORES


6

MICROSCOPE STAND – EPIFLUORESCENT

OBJECT - SPECIMEN

LIGHT SOURCE

EXCITATION FILTER

EMISSION FILTER

DICHROIC MIRROR

DETECTOR

OCULAR

OBJECTIVE

http://en.wikipedia.org/wiki/Fluorescence_microscope

LIGHT SOURCE

http://zeiss-campus.magnet.fsu.edu/articles/lightsources/index.html

� xenon lamp

� mercury arc lamp

� metal halid arc lamp

� laser

� LED

WAVELENGTH RANGE ONE WAVELENGTH

See: I. semester 23. Laser

FILTERS, MIRRORS

http://zeiss-campus.magnet.fsu.edu/print/basics/fluorescence-print.html

SHORTPASS LONGPASSBANDPASS

FILTER CUBE

emission filter

excitation filter

dichroic mirror

EXCITATION EMISSION

http://www.olympusmicro.com/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

DETECTOR – PMT: photomultiplier tube

http://www.olympusmicro.com/primer/techniques/confocal/pmtintro.html

photoelectric effect

photon � electron

photon

photocatode

dynode chainanode

vacuum

See: I. semester 19. Photoelectric effect

focusing

electrode

e-

másodlagos emisszió

- +

electric signal

DETECTORS – CCD: charge-coupled device

http://learn.hamamatsu.com/articles/ccdanatomy.html

http://learn.hamamatsu.com/articles/fullframe.html

http://www.microscopyu.com/articles/digitalimaging/ccdintro.html

2009 Boyle & Smith Nobel Prize in Physics

http://learn.hamamatsu.com/articles/ccdanatomy.html

http://learn.hamamatsu.com/articles/fullframe.html

http://www.microscopyu.com/articles/digitalimaging/ccdintro.html

http://hu.wikipedia.org/wiki/F%C3%A1jl:CCD_charge_transfer_animation.gif

photon

e-

photoelectric effect

photon � electron

electric signal


7

Biophysics 2. Physics - Biophysics 2.

2013. 02. 25. & 26.

dr. Beáta Bugyi – UP MS – Dept. Biophyics

íííííííííííííííí

MICROSCOPY

optical coherence tomography (OCT)

widefield, evanescent wave

confocal

4Pi, I5M

high resolution structured illumination (hrSIM)

ground state depletion (GSD)

saturated structured illumination (sSIM)

stimulated emission depletion (STED)

single molecule localisation (PALM, STORM)

near-field optical (NSOM)

electron microscopy (EM)

PET, SPECT

MRI, CT, ultrasound

human eyeAbbe’s principle

BIOLOGICAL IMAGING TECHNIQUES

BACKGROUND FLUORESCENCE

� CONFOCAL MICROSCOPY

� MULT-PHOTON MICROSCOPY

� EVANESCENT WAVE MICROSCOPY

Light microscope: Abbe’principle, diffraction limitLight microscope: Abbe’principle, diffraction limit

HOW TO IMPROVE THE RESOLUTION

excitation

emission

µm

interestingplane

AIRY PATTERN

� STIMULATED EMISSION (STED)

� SINGLE MOLECULE LOCALISATION

(PALM, STORM)

Z Y

X

dx,y ~ 200 nm dz ~ 1000 nm

1961. Marvin Minsky

1987. first confocal microscope

CONFOCAL MICROSCOPY – PRINCIPLES

WIDEFIELD MICROSCOPY

only one plane is in focus

but all the planes contribute to

the image

sample

focus

detector

objective

in focus

out-of-focus: in front of the focal planeout-of-focus: behind the focal plane

APERTURE

APERTURE

CONFOCAL MICROSCOPY

only one plane is in focus

↓ APERTURE (pinhole)spatial filter

only one plane contributes to

the image

CONJUGATED FOCAL PLANES

pinhole size: 1 Airy unitAiry unit: diameter of the Airy disk

sample

focus

detector

objective

CONFOCAL MICROSCOPY – PRINCIPLES


8

WIDEFIELD CONFOCAL

resolution widefield confocal

XY, nm 200 180

Z, nm 1000 500 !!

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

CONFOCAL MICROSCOPY

„slicing” the sample

optical slices

OBJECT↓

many 2D images

↓

3D IMAGE

http://www.olympusmicro.com/primer/techniques/confocal/confocalintro.html

CONFOCAL MICROSCOPY – OPTICAL SLICING

pollen grain

autofluorescence

TIRFM - PRINCIPLES

See: Optics, Refractometry practical

TOTAL INTERNAL REFLECTION – EVANESCENT FIELD

>3�5 >=exp3B�

45>3�5 >=exp3B

�

45

exponential decay

CDEFGFDHI

JK

JL

JL MJK

N OPP

Q

R3Q5

RPR Q RP

S

TIRFM

conventional

,-,/ sin3�,��4�,�5

sin36�76��,5,-,/

sin3�,��4�,�5

sin36�76��,5

T~KPPJU

resolution widefield TIRFM

XY, nm 200 200

Z, nm 1000 100 !!

WIDEFIELD TIRFM

B16/F1 melanoma cell

Actin filaments

TIRFM - PRINCIPLES


9

„ONE-PHOTON” MICROSCOPY� excitation: 1 photon� absorption: t = 10-15 s� E = Eexcited – Eground

→ photon wavelenght: λλλλ

„TWO-PHOTON” MICROSCOPY� excitation: 2 photons� E = Eexcited – Eground

→ photon wavelenght : 2*λλλλ

Requirements:� „simoultaneous” absorption of two photons

(t = 10-18 s)� low probabitity

� high photon density

� focused laser beam

� mode-locked laser (*106 density)

V W�

;

MULTI-PHOTON MICROSCOPY - PRINCIPLES

See: I. semester 23. Laser, II. semester 2. Fluorescence spectroscopy

http://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html

Advantages:� improved Z resolution

� deep-tissue imaging: imaging deeper layers in the sample

� confocal: ≈ 100 µm

� two-photon ≈ 1000 µm

� less invasive (low phototoxicity)

� imaging tissues in live animals: intravital microscopy

TWO-PHOTON MICROSCOPY - PRINCIPLES

macrophages migrating in a tumor implanted in the back

of an immunocompromised mouse

nuceIi (Hoechst)

TWO-FOTON MICROCOPY – INTRAVITAL IMAGING (IVM)

blood flow (liver of a living mouse)

Dextran (TexasRed)

Hepatociták (endogenous fluorescence)

http://www.nidcr.nih.gov/Research/NIDCRLaboratories/OralPharyngeal/I

endocytosis (liver of a living mouse)

Dextran – 70 kDa (Texas Red)

Dextran - 500 kDa (FITC)

Texas Red-dextran internalization in the stromal cells of the salivary

glands

2000. Stefan Hell

excitation

�

excited state - fluorescence

stimulated emission

non-linear deexcitation

�

ground state - nonfluorescent

remainig fluorescence

STIMULATED DEPLETION OF FLUORESCENCE EMISSION

STED - PRINCIPLES

See: I. semester 23. Laser

excitation laser

STED laser

red shift

STED - PRINCIPLES

excitation depletion


10

Actin filaments

resolution widefield STED

XY, nm 200 20 !!!!

Z, nm 1000 50 !!!!

WIDEFIELD STED

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

STED

2006.

PALM - PRINCIPLES

http://zeiss-campus.magnet.fsu.edu/print/superresolution/palm/practicalaspects-print.html

http://www.nature.com/nmeth/journal/v6/n2/fig_tab/nmeth0209-124_F1.html

photoswitchable fluorophore

See: II. semester 5. Fluorescence microscopy

PALM

reolution widefield PALM

XY, nm 200 10-20 nm !!!

Z, nm 1000 10-20 nm !!!

FRAP – PHOTOBLEACHING

PHOTOBLEACHING� irreversible photochemical destruction of the fluorophore due to the excitation

Disadvantages:� anti-photobleaching medium (pl. glucose oxidase – catalase – mercaptoethanol)

� lower exposure time

� pulse excitation

� lower excitation intensity

� resistant fluorophore

Advantages:� autoquenching

� FRAP, FLIP

http://en.wikipedia.org/wiki/Fluorescence_loss_in_photobleaching

http://micro.magnet.fsu.edu/primer/java/fluorescence/photobleaching/


11

idı (t)

kioltás

visszatérés

mobilis

immobilis50%

INTENSE LASER IMPULSEfluorescence quenching

before after

fluorescent molecules non-fluorescent molecules fluorescent molecules

non-fluorescent molecules

DIFFUSION

time (t)

recovery

mobile

immobile50%

fluor

esce

nce

inte

nsity

FRAP – PRINCIPLES FRAP

idı (t)

kioltás

visszatérés

mobilis

immobilis50%

Flu

ores

zcen

cia

inte

nzitá

s

B16-F1 cellEGFP-actin

LAMELLIPODIUM DYNAMICS

Lai et al. EMBO Journal 2008

FRAP

kioltás

visszatérés

mobilis

immobilis50%

time

fluorescence quenching

fluorescence recovery

ELECTRON MICROSCOPY (EM)

OPTICAL-LIGHT EM

image formationlight

glass lenses

electron beamelectromagnet

wavelength, nm 400 – 600 0.004 – 0.006

resolution, nm 200 0.2

magnification 2000 x2.000.000 x

(50.000.000 x)

1931. Ernst Ruska

NA = 0.04 – 1.45

TEMSEM

ELECTRON MICROSCOPY (EM)

TRASMISSION EM (TEM) ≈ light microscopy

� transmitted electrons

SCANNING EM (SEM) ≈ scanning probe microscopy

� backscattered electrons� differences in atomic number, groups of heavy atoms

� secondary electrons� topography of the surface

� Auger electrons� characteristic X-ray radiation

� chemical composition of the surface


12

AFM - PRINCIPLES

CANTILEVER� sharp tip (~ nm) – probe

� Si, Si3N4

� spring

baseline

PIEZOELECTRIC STAGEX-Y: scan the sample

Z: constant force

POSITION SENSITIVE PMTLASER BEAM

OPTICAL LEVER

FEEDBACK LOOP

AFM – IMAGING MODES

probe-sample interaction

force is exerted to the cantileverrepulsive/attractive force

cantilever is deflectedup/down

position of the laser beam changes

piezoelectric stage moves the sample to maintain the initial position of the cantileverZ direction: up/down

fixed position of the cantilever

Z topography of the sample

AFM – IMAGING MODES

STATIC (contact mode)� < 0.5 nm probe-surface separation

� the cantilever’s tip is dragged across the surface of the sample

� the cantilever deflection is kept constant by the feedback loop by adjusting the average tip-to-sample distance

� surface topography

DYNAMIC (non-contact, tapping)� the cantilever’s tip is not in contact with the sample

� the cantilever is oscillated at its resonant frequency where the amplitude of oscillation is typically a few

nanometers (<10 nm)

� constant oscillation amplitude or frequency is kept constant by the feedback loop by adjusting the average tip-to-

sample distance

� surface topography

Concurrent AFM–CLSM imaging of PNT2 cells with quantum dot labelled F-

actin (QD655, red) and moesin (QD525, green). AFM-derived amplitude

images of cell structure (a, c); CLSM fluorescence images (b, d). Arrows in c

and d indicate regions where topographic structures correspond to clusters of

fluorescence.

http://www.springerimages.com/Images/RSS/1-10.1007_s00418-008-0489-5-3

SUGGESTED LINKS 1.

http://www.olympusmicro.com/index.html

http://www.microscopyu.com/

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

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

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

http://www.nature.com/milestones/milelight/index.html

SUGGESTED LINKS 2.

TWO-PHOTON MICROSCOPY Listeria monocytogenes...

Documents

Transcript of TWO-PHOTON MICROSCOPY Listeria monocytogenes...