“Breaking the diffraction barrier in fluorescence microscopy at low

light intensities by using reversibly photoswitchable

proteins”M. Hoffmann, C. Eggeling,S. Jakobs, S.W. Hell

JOURNAL CLUB PRESENTATION2/13/2006

Mehmet Dogan

OUTLINE

• Background:– Resolution,– STED, – RESOLFT

• Photoswitching • Characterization of switch kinetics of protein: asFP595• Demonstration of RESOLFT idea : ~100nm resolution• Conclusions

Resolution Limit

Abbe’s Diffraction Limit:

sin2n

x

Abbe’s Equation Modified for Fluorescence:

1sin2nx

0 Abbe Limit

0x

Saturation FactorsatIxI /)(

Requirements for Subdiffraction Resolution

• Large saturation factor– Either large I(x)

– Or small saturation intensity Isat

• Spatial intensity zero

satIxI /)(

Isat

I(x)

x

saturated saturated

Reversible Saturable Optical Fluorescent Transition (RESOLFT)

A B)(xIk ABAB

BAk BAAB

BAA kk

kN

At Equilibrium:

BAAB

BAA kxI

kN

)(

sat

A

IxI

N)(

1

1

AB

BAsat

kI

dt

dNNkNk

dt

dN BBBAAAB

A

Rate Equations:

tkk

k

k

kk

ktN BAAB

AB

BA

BAAB

ABA exp)(

Normalized Populations:

Spatial Intensity Zero for Increased Resolution

A Subset : STEDStimulated Emission Depletion

• State A: Fluorescent State• State B: Non-fluorescent ground state

IkStE

Stimulated Emission vs. Spontaneous Emission

spk

I2/100 cmMW

kI spsat

Too high saturation intensity Photo induced damage

Alternative Approach: Reduced Isat

)/(1sin2 satIInx

Systems with weak spontaneous interstate conversions

Remember:

spsat kI

Photoswitchable Fluorophores:ssFP595 : Photochromic Fluorescent Protein

ON State (A) : fluorescence-activated

OFF State (B) : fluorescence-inhibited450 nm 560 nm

Photoswitching

Photoswitching of protein in E-coli with wide field epifluorescence microscope

Photoswitching of thin protein layer on a 0.3 µm focal spot

Iy= 2 W/cm2

Ib=0.1 W/cm2

Iy= 4.4 W/cm2

Ib=3.6 W/cm2

Py=3.3 nW

Pb=2.2 nW

8 orders of magnitude less than STED

Drawbacks

1) Low quantum yield: <1%

2) Incomplete OFF (15% fluorescence)

3) Photobleaching with cycling

4) Intensity to be adjusted for fluorescence settling

Effects of Iy and Ib on Inhibition

Isat~ 1 W/cm2

Effect of Iy

Larger Iy gives larger Residual Fluorescence

Strong inhibition and small fluorescence settling time

Subdiffraction focal spots

Solid lines: calculated

Dashed lines: measured

Focal spot with two offset peaks using phase plate

y

x

Effective PSFCalculated effective PSF using experimental values

Calculated Effective PSF using theoretical values

Incomplete inhibition of fluorescence at the periphery:

RESOLFTdiff PSFPSFPSFE )1(

0.3

Imaging Test Samples Grooves on test slides with focused ion beam milling

10µm long

100nm wide

0.5-1µm deep

Separation: 500nm

Immersion into buffer with asFP595:Grooves filled by adsorption

scan

a-c

a-f

d-e

20nm steps

50ms dwell time

Iy= 600W/cm2

Ib=30 W/cm2

Conclusion• Demonstration of resolution increase with

photoswithing at low power• New proteins should be engineered

Challenges• Low quantum yield (1%)• Slow switching requires ms integration• Action cross-talk