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Invitation for a walk through microscopy. [email protected] Techniques in microscopy. Conventional (light) microscopy bright & dark field, phase & interference contrast. Fluorescence microscopy light sources, fluorescence detectors, digital image , objectives.

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Invitation for a walk through microscopy

[email protected]


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Techniques in microscopy

  • Conventional (light) microscopybright & dark field, phase & interference contrast

  • Fluorescence microscopylight sources, fluorescence detectors, digital image, objectives

  • Single- & two-photon confocal microscopybasic idea & differences, advantages & disadvantages

  • What is the optimal technique (for my question)?


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Conventional (light) microscopy

(http://micro.magnet.fsu.edu)


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Magnification

Illumination

Specimen

Light microscope: general structure I

(with modification http://micro.magnet.fsu.edu)


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2f

(magnifying glass)

imaginary

image

f

object

F

Magnification

2f

f

object

real

image

Illumination

F

Light microscope: general structure II

ocular

objective

specimen

condensor

light source


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ocular (magnifying glass)

Intermediate (real) image

(on the projector screen)

objective (slide projector)

Final (imaginary) image

light

A light microscope is a combination

of a slide projector with a magnifying glass

Total magnification = Mobjective x Mocular


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bright field with specimen

specimen

Bright and dark field illumination

objective

object plane

condensor


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dark field without specimen

specimen

Bright and dark field illumination

dark field with specimen

objective

object plane

condensor

- ring diaphragm (usually)

- dark field condensor


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  • Dark field

  • part-illumination of the specimen

  • scattered light collected by objective

  • bright object on dark background

Objects with high contrast

Objects with a sharp

rise in refraction index

Bright and dark field illumination

  • Bright field

  • total illumination of the specimen

  • direct light collection by objective

  • dark/colored object on bright background

(with modification http://mikroskopie.de)


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conjugate planes

in the image path

optical elements

retina

(eye)

A. Köhler

(1866-1948)

ocular

intermediate

image plane

specimen

objective

focused

specimen

diaphragm

condensor

aperture

diaphragm

field

diaphragm

light source

Microscopy illumination after Köhler

(or the mystery condensor adjustment)

objective

condensor


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3. Choose prefered objective

(at least 10x), focus specimen

4. Close down field diaphragm;focus the image of the fielddiaphragm sharply onto thealready focused specimen

5. If neccessary center the condensor;then open the field diaphragmuntil it just disappears from view

6. Take out one of the eyepieces,look down the tube andadjust the aperture diaphragm

Diaphragm should be 2/3 to 3/4 open (compromise between resolution & contrast)

How to do adjust the Köhler illumination

1. Light source on

2. Open up fully field diaphragmand aperture diaphragm


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amplitude object

amplitude

amplitude

Reduction in amplitude is equal

with a reduction in light intensity

(used in bright field microscopy!)

phase object

phase

Slow down of light wave

passing the phase object

Amplitude and phase objects influence light waves:

Basic principle for phase & interference contrast


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scattered light

intermediate image plane

slowed down,unscattered light

phase ring

objective

focussed specimen

condensor

and

light source

phase diaphragm

Phase contrast


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Specimen

(inhomogen phase object)

Prisma

(Nomarski)

DIC prisma

(Nomarski)

Polarisator

Analysator

Phase

difference

unpolarized

light

linear

polarized

light

two vertical

polarized

waves

linear polarized

light

(analysator vertical

vs. polarisator)

Differential interference contrast (DIC)


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Kidney tissue(tubule with some cells> 100 µm thick section)

Buccal epithelial cell

(monolayer)

Phase contrast

DIC

Phase contrast vs. DIC

(with modification http://mikroskopie.de)


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Light microscopy: illumination & contrast techniques

  • Illumination

Try to optimise your illumination (condensor adjustment after Köhler)

Bright field illumination: standard technique for most specimen

Dark field illumination: specific technique for monolayer specimen with distinct differences in the refraction index

  • Contrast

Check and improve all contrast techniques available at your microscope

Phase contrast: standard technique for low-contrast monolayer specimen

DIC: standard technique for low-contrast specimen, in particularily for thick (non-monolayer) preparations



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E = hn

c = ln

E ~ 1 / l

Basic idea of fluorescence microscopy: Stokes shift


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Detection system

(eye, conventional camera, CCD, photo diode, PMT)

emission wavelenght lem > lex

(filter)

light source

(arc lamp, laser)

excitation wavelenght lex

fluorescence object/dye

The use of the Stokes shift in fluorescence microscopy

dichroic mirror


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Fluorescence microscopy requires ...

  • Fluorochrome (or autofluorescence)

    see Molecular Probes (www.probes.com)

  • Light source


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Laser types

UV

IR

Argon 351 364 457 477 488 514

Blue diode 405 440

Helium-Cadmium 354 442

Krypton-Argon 488 569 647

Green Helium-Neon 543

Yellow Helium-Neon 594

Orange Helium-Neon 612

Red Helium-Neon 633

Red diode 635 650

Ti:Sapphire 720-980

Light source

Arc lamps

Xenon

Mercury


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Fluorescence microscopy requires ...

  • Fluorochrome (or autofluorescence)

    see Molecular Probes (www.probes.com)

  • Light source

  • Fluorescence detection


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photo diode

PMT

CCD

conventional

photography

Fluorescence detector systems ...

Temporal

resolution

Spartial

resolution


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Analog Image

Digital Sampling

Pixel Quantization

Fluorescence detector systems produce digital images

- observer eye

- conventional

photography

  • - CCD

  • PMT (in combination with scan technique)

(with modification http://micro.magnet.fsu.edu)


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255

255

255

0

0

0

grey level

grey level

grey level

Fluorescence detector systems produce digital images

normal

contrast

low

contrast

high

contrast

pixel counts

(with modification http://micro.magnet.fsu.edu)


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Mainly fluorescence detector systems are color-blind!

(Colors are based on a [pseudo-]color look-up table)


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Fluorescence microscopy requires ...

  • Fluorochrome (or autofluorescence)

    see Molecular Probes (www.probes.com)

  • Light source

  • Fluorescence detection

  • (prefers) immersion objectives


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

n2

n1

n1 < n2

sin 

n2

=

sin 

n1

Immersion objectives: Remember the refraction index!

total reflection


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water or oil immersion objective

medium (water or oil)

specimen

Emission

Excitation

Light

source

DM

Immersion objectives: Remember the refraction index!

refraction index (n)

air  1.00

water = 1.37

oil = 1.5

glass = 1.5

Immersion objective

with specimen


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Conventional fluorescence microscopy

  • Advantage

  • low cost

  • uncomplicated handling

  • fast imaging technique

  • Disadvantage

  • no 3-dimentional imaging possible

  • low depth of light penetration

  • bleaching


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Confocal microscopy

(Schmitz et al., 2001)


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full field detection

point scan detection

full field illumination

point scan illumination

Basic idea of confocal microscopy I

Conventional fluorescence microscope

Laser scanning microscope

specimen

Arc lamp (Hg, Xe) + excitation filter

laser light source


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Different wavelengths require different laser, for example ...

visible spectrum

ultra violet

infra red

Argon 457 477 488 514

Green Helium-Neon 543

Red Helium-Neon 633

Laser: light source for confocal microscopy

Laser (Light Amplification by Stimulated Emission of Radiation)

= highly precise light source in direction, frequency, phase, polarisation

- monochromatic = light has the same wavelength (continuous-wave lasers)

- coherent = light is oscilating in the same phase

- linear polarized = light is oscilating in the same direction

- can be focussed to a very high density power (compared to arc lamps)


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laser ...

y

x

z

y

x

Basic idea of confocal microscopy II

point scan illumination

(fluorescence excitation)

point scan detection

(fluorescence emission)


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PMT ...

excitation

emission

pinhole

filter

x/y-scanning device

and dichroic mirror

objective

z

focal plane

y

x

Confocal microscope: general structure

laser source

specimen


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PMT ...

PMT

pinhole

pinhole

objective

objective

specimen

focal plane

specimen

Confocal microscope: the power of the pinhole



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visible light ...

104

UV

IR

103

depth of light

penetration (µm)

102

101

100

wavelenght (µm)

Confocal microscope: depth of light penetration


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Confocal fluorescence microscopy ...

  • Advantage

  • improved spartial resolution

  • 3-dimentional scanning

  • Disadvantage

  • more complicated imaging control

  • low depth of light penetration

  • bleaching


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Two-photon microscopy ...

A

B

100 ms

5 µm


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single-photon excitation ...

two-photon excitation

h*

h

h

Absorbtion

Emission

Emission

Absorbtion

h

h*

E = hn

E ~ 1 / l

E* = 1/2 E

E* ~ 1 / 2l

c = ln

Basic idea of two-photon microscopy

Two photons at the same time and at the same place with doubled wavelenght

 photons from the infra red spectrum (> 750 nm)

 high photon density


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Titan-Sapphire spectra ...

Excitation

Emission

Light source for two-photon microscopy: Ti/Sa-laser

Pump laser: solid-state cw laser, 532 nm, 5 W

(Millennia, Spectra Physics)

  • Mode-locked Titan-Sapphire laser(Tsunami, Spectra Physics)

  • avarage power > 0.7 W at 800 nm

  • pulsewidth < 100 fs

  • nominal repetition rate 80 MHz

  • turning range 720 - 850 nm


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photon ...

non-excited

dye molecule

2p-excited

dye molecule

the required photon density for two-photon excitation

can be established only in the focal plan and within a laser puls

Two-photon excitation

(with modification Piston, 1999)

laser pulse

focal plane


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emission ...

emission

PMT

PMT

excitation

excitation

pinhole

IR laser

x/y-scanning device

and dichroic mirror

z

y

x

Single vs. two-photon microscope: general structure


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full field detection ...

point scan detection

point scan illumination

point scan illumination

Fluorescence detection using 2-photon excitation

descanned detection

Non descanned detection (NDD)

specimen

pulsed Ti:Sa laser

pulsed Ti:Sa laser


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Two-photon microscopy with descanned and NDD-PMT ...

excitation beam

x/y-scanning device

& dichroic mirror (DM)

prisma for spectral analyse

descanned PMT 1 & 2

DM

DM

non-descanned (NDD) PMT 3 & 4

objective

specimen

condensor

trans-non-descanned (NDD) PMT 5

DM

(with modification Oertner, 2002)


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single-photon excitation ...

A(x) ~ 1/I(x)

Single vs. two-photon excitation: excitation profile

two-photon excitation

focal plane

A(x) ~ 1/I2(x)


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10 ...4

103

depth of ligh

penetration (µm)

102

101

100

wavelenght (µm)

Two-photon microscope: depth of light penetration

visible light

UV

IR


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(3D-FITC-dextran gel; irradiated area ~ 10 x 20 µm) ...

y

x

z

x

two-photon

absorbtion

(760 nm; Ti:Sa)

focal plane

single-photon

absorbtion

(488 nm; Ar)

focal plane

20 µm

10 µm

Single vs. two-photon microscopy: bleaching

(with modification Kubitscheck et al., 1996)


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Simply doubling the excitation wavelenght? ...

h

Absorbtion

h

Emission

102

h

101

Calcium green (506/533)

100

Fluo-3 (505/526)

Two-photon cross section

10-1

Lucifer yellow (428/533)

10-2

Cascade blue (400/420)

10-3

1000

600

700

800

900

Excitation wavelenght (nm)

Two-photon microscope: excitation spectra

(with modification http://micro.magnet.fsu.edu)


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Two-photon microscopy ...

  • Advantage

  • optimized z-resolution

  • reduced bleaching

  • higher efficiency (removed pinhole)

  • higher depth of light penetration

  • Disadvantage

  • complicate combination of laser and imaging control

  • cost

  • reduced temporal resolution


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Intensity and spectral resolution ...

(dynamic range, signal-to-noise-ratio)

Spatial

resolution

Temporal

resolution

Limitations of fluorescence microscopy

„Eternal triangle of compromise“ (Shotton, 1995)

light source & fluorescence dye

fluorescence detection


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Fluorescence microscopy ...

confocal

(single-photon)

conventional

two-photon

+

+ +

0

spatial resolution

+

+ +

0

depth of penetration

+

0

0

bleaching

temporal resolution

++

0

0

increasing

++

+

(+)

available dyes

cost

What is the proper technique for my question?


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conventional fluorescence imaging ...

- fast and full frame imaging

  • dual-wavelenght functional imaging (Fura-2, BCECF, etc.)

confocal (single-photon) fluorescence imaging

- thin preparation (< 100 µm): cell culture (monolayer), fixed preparations

- multilabling using different dyes (require of different wavelenght)

two-photon fluorescence imaging

- thick preparation: acute and cultured brain slice

- in vivo imaging with interest on deeper structures

What is the proper technique for my question?


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