At this internal conference event of the III.Institute of Physics in Quedlinburg 2011, we presented our novel "Super High resolution techniques".

1. Summerschool Quedlinburg 2011
Image Scanning Microscopy (ISM)
Superresolution Optical Fluctuation Imaging (SOFI)
Dirk Hähnel, Jörg Enderlein
III. Institute of Physics – Biophysics
Georg-August-University Göttingen
Quedlinburg 26th Sep. 2011

2. Introduction
Functional requirements:
 Usage of conventional fluorophores
 Low laser power intensity
 3D imaging
 Multi color
Objective:
 Increase the resolution without modifying size of the confocal spot
 Avoiding techniques which use blinking molecule behavior?
2 Quedlinburg 26th Sep. 2011

3. 3
Lateral Resolution Limit of Standard Light Microscopy
 Emission is considered as a planar wave from point-like-source
 Using pure diffraction optics
 Resolution limit at a phase difference of π/2
N.A. = n∙sinq
Quedlinburg 26th Sep. 2011
l
2n∙sinq
q
objective

4. Confocal Scanning Microscopy
 Confocal scanning uses diffraction limited excitation volume
 Structured excitation but gives away spatial information on the
detection side, which contains all Fourier modes from excitation
Quedlinburg 26th Sep. 2011

5. Structured Illumination Microscopy
 Scanning periodic excitation over a sample
periodic image
uniform image
Quedlinburg 26th Sep. 2011
= l = l
min 4 sin 4N.A.
y
n
Q
two-fold enhancement
in resolution

6. Image Scanning Microscopy - Setup
 Enhancement of a standard confocal setup
 Replacing point-detector by EMCCD
 Excitation intensity detection is optional
Quedlinburg 26th Sep. 2011

7. Image Scanning Microscopy - Detection
 Image of PSF on the Sensorchip
 4-dim data array contains:
 Pixel x on CCD-Chip
 Pixel y on CCD-Chip
 Position x – Confocal sample
 Position y – Confocal sample
 Each pixel acts like a small confocal aperture for a two-dimensional
image
Quedlinburg 26th Sep. 2011

8. Image Scanning Microscopy – Image
 Fluorescent bead imaging
 Increase of contrast and resolution
 Intensity profile of a single
fluorescent bead
*scalebar indicates 1 mm
Quedlinburg 26th Sep. 2011

9. Optical Saturation Microscopy
 Using saturation of the excited state
 Taking measurements on at least two different intensity-levels
 Archiving the higher harmonic in the Fourier-space
Quedlinburg 26th Sep. 2011

10. Otical Saturation Microscopy
Quedlinburg 26th Sep. 2011
 Results
Confocal image Optical saturation microscopy image
Yeast cell with GFP-labeled Ato1p membrane protein
From:
J. Humpolickova, A. Benda and J. Enderlein, Biophys. Journal, 2009 (97), 2623–2629.

11. Drawbacks and Advantages
 Drawbacks:
 Optical quality of components
 Chromatic aberration
 Data handling
 Scan speed
 “Step and settle” on each pixel required
 Limited camera speed
 Automation requires nanosecond input/output timing
 Advantages:
 Any dye works
 Excitation power can be lowered
 3D-capability
 Multicolor
Quedlinburg 26th Sep. 2011

12. Status of Implementation
 Image scanning microscopy
 Resolution increase factor of 1.75-1.80 compared to confocal
imaging with ISM-technique
 Theoretically possible is a factor of 2
 Optical saturation microscopy
 Implementation completed
 Next steps
 Combination with Dual-Excitation-Technique
 Work on possible resolution increase up to factor 3
Quedlinburg 26th Sep. 2011

13. Superresolution Optical Fluctuation Imaging (SOFI)
 The fluorescent label has to exhibit at least two different emission states. For
example, these states can be a fluorescent and a non-fluorescent one, but in
principle any two or more states which are optically distinguishable will do.
 Different emitters have to switch between states repeatedly and independently
from each other.
Quedlinburg 26th Sep. 2011

14. Image cumulants of QD625 images
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15. Resolution of image cumulants of QD625 images
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16. Resolution of image cumulants of QD625 images
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17. SOFI images of QD625 labelled 3T3 cells
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18. Fourier filtering on 2nd order QD625 SOFI image
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19. Fourier filtering on 2nd order QD625 labelled 3T3 cell SOFI image
Quedlinburg 26th Sep. 2011

20. Conclusion - ISM
 Combination of two techniques:
 Structured illumination and detection
 Optical saturation
 Multicolor
 3D Imaging
 Any probes
Easy enhance resolution of standard confocal microscopes
Get super-resolution with “conventional” fluorophores
Working on enhancement of resolution up to factor 3
Quedlinburg 26th Sep. 2011

21. Conclusion - SOFI
 Implementation of SOFI is simple
 It is inherently 3D
 It is background eliminating
 It works on all blinking probes (i.e. almost all probes)
 It works on all types of microscope
 Resolution is not limited to pixel size
 It works not only on fluorescence
Quedlinburg 26th Sep. 2011