object

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object

2008 Small World contest -18th Prize - Dr. Tamily Weissman (Harvard University - Cambridge, Massachusetts, United States)

Specimen: “Brainbow” transgenic mouse hippocampus (40x)

Technique: Confocal


Microscope - microscopy

greek: mikron = small + szkopein = observe

How to observe small objects invisible for the human eye?

MICROSCOPE

instrument, designed to make fine details visible for

human eye

MICROSCOPY

science of investigating small objects using such an

instrument


Image formation

1. MAGNIFICATION

2. RESOLUTION

3. CONTRAST


Light (optical) microscopy

Image formation is based on

- the use of visible light to illuminate objects

- the use of glass lenses


What we need to build an optical microscope

OBJECTIMAGE1 (real, magnified, inverted)IMAGE2 (virtual, magnified, erect)

OBJECT IMAGE (virtual, magnified, inverted)


1. Magnification

OBJECTIVE: N objective = 1 – 150

OCULAR: N ocular = 5 – 30

MICROSCOPE: N microscope = N objective x N ocular


2. Resolution

the shortest distance between two points (d) on a specimen that can still be distinguished as

separate entities

d: distance between two points

λ: wavelength

NA: numerical aperture

1/d: resolving power

RAYLEIGH EQUATION

XY direction – in plane

d x y

,

0.

61


NA

Z direction – along the optical axis

d z


2

2

NA


NA = 1.4

2. Resolution - Wavelength

d x y

wavelength

Nm

,

0.

61


NA

d z

resolution - XY

nm

2

2

NA

resolution - Z

nm

360 156 367

400 174 408

450 196 459

500 217 510

550 239 561

600 261 612

650 283 663

700 305 714

↑ resolving power ↓


2. Resolution – Numerical aperture

dimensionless quantity

expresses the ability of a lens to resolve fine details in an object being observed

NA nsin


n: index of refraction of the medium found between the object and the objective

μ: half-angle of the maximum cone of light that can enter or exit the objective (angular aperture)

NA = 0.04 – 1.5


2. Resolution - Airy disC

diffraction

image: intereference pattern (concentric circles of intensity minimum and maximum )

Diffraction limited image of a single point of an object: AIRY DISC (George Biddel Airy 1801-1892)

OBJECT IMAGE

maximum minimum

3

2

1

0


3. Contrast

The properties of the light (direction, velocity, phase, …) passing through the

object are changed by the optical inhomogeneity of the sample (index of

refraction, geometry, shape,…) CONTRAST


Fluorescence microscopy

Image formation is based on

- the fluorescence emission of the sample

special requirements


INNER (INTRINSIC) FLUORESCENCE

chlorophyll

Fluorophores

OUTER (EXTRINSIC) FLUORESCENCE

fluorescent molecules

kvantum dots (d = 2-10 nm, 100-100000 atoms)

proteins (Green Fluorescent Protein) (d = 10 nm, 26 kDa)

small molecules (fluorescein) (d = 1 nm, 20 atoms)


Microscope


EYEPIECE

OBJECTIVE

Microscope

SAMPLE

FILTERS

EXCITATION

DETECTOR

MIRRORS


Excitation: Lamps, lasers

LAMPS: xenon, mercury LASERS


Filters: excitation/emission

select particular wavelengths


Dichromatic mirror

select particular wavelengths: beam splitter


emission

filter

dichromatic

mirror

Filters + Mirrors: Filter cube

EMISSION

excitation filter

EXCITATION


magnification

coverslip

glass

Objective

type

immersion immerzió típusa

NA

coverslip type

magnification color code

working

distance


PHOTOMULTIPLYER

PHOTODIODE

CCD CAMERA (charged coupled device)

Detectors

photon electronic signal


Fluorescence microscopy– Problem 1

emission

excitation

m

BACKGROUND FLUORESCENCE!!!

How to improve Z-resolution?

confocal microscopy

evanescent wave microscopy

multi photon microscopy


Confocal microscopy


detector

emission

out of focus

in focus

Confocal microscopy

aperture

pinhole

excitation

aperture

pinhole

focal planes

laser


Confocal microscopy


Confocal microscopy

resolution XY, nm Z, nm

conventional 200 500-600

confocal 200 400


TIRFM

Total Internal Reflection Microscopy


TIRFM

TOTAL INTERNAL REFLECTION



sin


sin

n

2



n


1

n

n

2

1



90

critical

o


I( z)

I0

exp( z

/ d)

TIRFM

EVANESCENT FIELD

[z], nm I(z) %

0 100

1 99

10 92

100 43

1000 0


TIRFM

high NA

immersion oil (n)

excitation laser positioned out of the

optical axis of the objective


TIRFM


TIRFM

resolution XY, nm Z, nm

conventional 200 500-600

TIRFM 200 100


Multi-photon microscopy


E

hc


Multi-photon microscopy

1

Non linear optics

2 21

t

18

10


s

Jablonski diagram

LASER!!!

impulse laser


Multi-photon microscopy


Fluorescence micropscopy – Problem 2

RAYLEIGH EQUATION wavelength

nm

d


0.

61


NA

How to improve XY-resolution?

physical

mathematical

resolution - XY

nm

360 156

400 174

450 196

500 217

550 239

600 261

650 283

700 305


STED

Stimulated Emission Depletion Microscopy


STED

Stimulated depletion of fluorescence emission

excitation

fluorescence

non-linear deexcitation

ground state- non-fluorescent

remaining fluorescence


depletion laser

STED laser

excitation laser

fázislemez

red shift

objective

sample

STED

excitation depletion

widefield STED


STED

Actin cytoskeleton

Elise Stanley, Division of Genetics & Development, Toronto Western Research Institute (TWRI), Canada


STED

resolution XY, nm Z, nm

conventional 200 500-600

STED 20-40 100


SIM

Structured Illumination Microscopy


SIM

grid

rotation + image acquisition

image analysis

important:

grid geometry

number of rotation


SIM

resolution XY, nm Z, nm

conventional 200 500-600

SIM 100 150-300


FLIM

Fluorescence Lifetime Imaging Microscopy

FADIM

Fluorescence Anisotropy Decay Imaging

Microscopy


FLIM - FADIM


FRAP

Fluorescence Recovery After Photobleacing


laser impulse


quenching

FRAP

Fluorescence intensity

KINETIC ANALYSIS

recovery of fluorescence intensity

rate

extent

kioltás

recovery

50%

time(t)

mobile

immobile


Electronmicroscopy (EM)

Ernst Ruska - 1933

Cryo-elektrontomography (cryo ET)


Electronmicroscopy (EM)

Image formation is based on

- the use of an electron beam to „illuminate” the

sample

special requirements

optical EM

wavelength 400 – 600 nm 0.004 – 0.006

resolution

~

200 nm

magnification 2000 x

0.2 nm

(50 pm)

2.000.000 x

(50.000.000 x)


SOURCE

„OPTICAL”

COMPONENTS

SAMPLE

„OPTICAL”

COMPONENTS

DETECTOR

Elektronmicroscopy (EM)

high voltage: 100-300kV

electron gun

electron beam

vacuum: 10 -4 – 10 -9 Pa

„LENSES”

electromagnetic

electrostatic

sample: negative staining (uranyl acetate),

liquid nitrogen

„LENSES”

electromagnetic

electrostatic


Cryo-elektrontomography (cryo-ET)

2D 3D

Plasmodium berghei

cell

F-actin

nuclear pore complex

HIV virus


AFM

Atomic force microscopy

Gerd Binnig - 1986


AFM

POSITION

SENSITIVE

DETECTOR

kvadrant

diode

CANTILEVER

x

z

y

SAMPLE

LASER


AFM

B16 mouse melanoma cell


További érdekességek

2010 Small World contest - 7th Prize - Mr. Yongli Shan (UTSW - Dallas, Texas, USA)

Specimen: Endothelia Cell attached to synthetic microfibers (2500x)

Technique: Epifluorescence, Confocal

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