Confocal & Two-photon Microscopy

Contents

1. Two-Photon Microscopy : Basic principles and Architectures

2. Resolution and Contrast in Confocal and Two-Photon microscopy

3. Example of two-photon images

4. Extensive application: Fluorescence Correlation Microscopy,

Life-time imaging

Figure 2, Relevant time scale.

1. Two-Photon Microscopy (vs One-photon)

Figure 1. Jablonski diagram

• Nonlinear optical excitation , I2p P2

1. Two-Photon Microscopy (vs One-photon)

Figure 3. Quadratic dependence.

3D localized uncaging and photobleaching

in subfemto-liter volume

1. Two-Photon Microscopy (vs One-photon)

Figure 4. Excitation region in one & two-photon microscopy

Z-axis Z-axis

1. Two-Photon Microscopy (vs One-photon)

Figure 3. Photobleaching in one & two-photon microscopy

10 15 20 25 30

0

50

100

150

200 *100, NA:1.3

030530.orjFluorescence distribution of octupole film

Flu

ore

sce

nce

In

ten

sity (

arb

.un

it)

Z-position (m)

1. Two-Photon Microscopy (vs One-photon)

One-photon

Two-photon

Figure 5. Z-direction scanning spectra in One & Two-photon microscopy

• Quantum Theory of Two-photon excitation

P ~ <f Er •

r m><m Er

•ri>

r - mi

2

m

* Multi-Photon transition probability (P)

1. Two-Photon Microscopy (vs One-photon)

• Two-Photon transition Probability

2222

22

2

)()(

)(

c

NAtPP

tIP

P

P

* Time-averaged two-photon fluorescence intensity per molecule

TT PP dttPTc

NAdttP

TP

0

2

2

0

222 )(

1

2

)()(

1

1. Two-Photon Microscopy (vs One-photon)

a. Continuous Wave Laser22

20

2

2

)(

c

NAPP P

cw where

0)( PtP

b. Pulsed Laser222

0

0

22

22

202

2

)(1

2

)(

c

NA

f

Pdt

Tc

NA

f

PP

pp

Pp

where0)(

)( 0

tP

f

Ptp

p 0 < t < for

1 < t < fp

for

1. Two-Photon Microscopy (vs One-photon)

• Architecture of Two-Photon microscopy

1. Two-Photon Microscopy (vs One-photon)

Safety Box

BeamroutingSupplement Mira

Mira-900

DMIR

Verdi

1. Two-Photon Microscopy (vs One-photon)

Leica confocal systems : TCS SP2

2. Two-photon confocal microscopy combined with femtosecond laser Thickness, depth and more precise images measurement by 3D sectioning

1. The spectral detector for brilliant confocal 2D, 3D images by emitted fluorescence

: Femtosecond Ti-Sapphire system with 80MHz, 100fs delivering peak powers of over 100kW !! wide tuning range , 700~1000nm !! At the entrance of scanning head, ~20mW Before the objective lens, 9~13mW At the sample, 3~5mW

Picosecond, CW (required higher average power !!)

• Light source

1. Two-Photon Microscopy (vs One-photon)

Mira 900 : 76MHz, 180fs, 400~500mW,

1. Two-Photon Microscopy (vs One-photon)

• Advantages of Two-photon Deep-specimen imaging

a. Lower absorption & scattering coefficient due to IR : Deeper penetration effect !

b. Excitation only in a subfemtoliter-sized focal volume : It reduce photodamage !

The resolution, defined as the minimum separation of twoPoint objects that provides a certain contrast between them,

depends on

The wavelength of the light !Numerical Aperture of the optical arrangement !

Specimen !

2. Resolution and Contrast (confocal vs two-photon)

•Three-dimensional distribution of light near the focus of lens Point Spread Function

1

0

2/0

sin/2

22

)(sin2

),(

devJenA

ivuh iuiu

sin2 nr

v

2sin2 nzu Where,

The intensity PSF ( related to its FWHM) ,

),(),(),( *2vuhvuhvuh

2. Resolution and Contrast (confocal vs two-photon)

* Confocal system

Pointwise-illumination

Pointwise-detection

22det

2),,(),,(),,( zyxhzyxhzyxh illconfocal

222det

2

2 ]),,([),,(),,( zyxhzyxhzyxh illconfocalp

222]),,([),,( zyxhzyxh illphotontwo Cf)

Cf) Uniform detector

2. Resolution and Contrast (confocal vs two-photon)

Table1, FWHM

2. Resolution and Contrast (confocal vs two-photon)

Figure 3. Calculated Point Spread Function

FWHM extent (m)

Illumination (ill)

Detection (det)

Lateral AxialPSF

Confocal=illdet

Two-photon=(ill)2

2p-confocal=(ill)2xdet

0.20 0.84

0.19 0.78

0.14 0.57

0.23

0.16

0.93

0.63

* Lateral Resolution

NAr

NAr

NAr

emphotontwoxy

emconfocalxy

emxy

7.0

4.0

6.0

,

,

2. Resolution and Contrast (confocal vs two-photon)

2,

2,

2

3.2

4.1

2

NA

nr

NA

nr

NA

nr

emphotontwoz

emconfocalz

emz

* Axial Resolution

vdvvuIuI

vdvvuIuI

Pz

Pz

)2/,2/()(

),()(

22

1

Depth discrimination !

2. Resolution and Contrast (confocal vs two-photon)

3. Examples

1 43 52

OP

Top Bottom

TP

Z-scanning range : ~28m (5 sections, 7.0417 m step)

3. Examples : Neuron cell imaging

Lysosome(DND-189)

Nucleus(Propidium

iodide)

3. Examples : Neuron cell imaging

3. Examples : Neuron cell imaging

Side-view 3D-reconstruction

50m

50m

4. Application : Fluorescence Correlation

Microscopy

2)(

)()()(

tF

tFtFG

Where F(t)=F(t)-<F(t)>

])1)(1([

1)(

5.02DD

NG

D ~ r0

2 / 4D

4. Application : Life-time two-photon imaging

4. Application : Life-time two-photon imaging

Steady state intensity image

Time resolved intensity image

Autofluorescence of human skin

: 2-photon image

Ca image of Cortex neutron

: 2-photon image