Current status of Image Scanning Microscopy implementations and future outlook.

1. Image Scanning Microscopy
Dirk Hähnel - Group Seminar 30.04.2013
III. Institute of Physics – Biophysics
Georg-August-University Göttingen
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2. Problem
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3. 3
Diffraction
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4. Methods
Schermelleh et.al, JCB
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5. 5
System Overview
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6. 6
Data Acquisition
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7. 7
Image Information
Y_Sample
 Each pixel acts like a small confocal aperture for a two-dimensional
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image
X_Sample
X_CCD
Y_CCD

8. 8
Image Processing
 Each CCD pixel acts as nearly infitly small pinhole
 Superimpose of al pixel from all images, the resulting
image is blurred, due to parrallax effect
 Reshifting image in common reference frame results in
sharp image with ~doubled resolution
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9. 9
Results
●Fluorescent bead imaging
●Increase of contrast and resolution
Intensity profile of a single
fluorescent bead
*scalebar indicates 1 mm
Quedlinburg 26th Sep. 2011

10. 10
Increase Resolution
 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

11. 11
Optical Saturation Microscopy
Confocal Image Optical Saturation Microscopy Image
Yeast cell with GFP-labeled Ato1p membrane protein ,J. Humpolickova, A. Benda and J. Enderlein, Biophys. Journal, 2009 (97), 2623–2629.
Quedlinburg 26th Sep. 2011

12. 12
Deep Tissue with Olympus Scaleview: 2Photon
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Hiroshi Hama, Hiroshi Kurokawa, Hiroyuki Kawano, Ryoko Ando,Tomomi Shimogori, Hisayori Noda, Kiyoko Fukami,
Asako Sakaue-Sawano & Atsushi Miyawaki
"Scale: a chemical approach for fluorescence imaging and reconstruction of
transparent mouse brain," Nature Neuroscience, advance online publication, 30 August 2011

13. 13
Faster ISM CLSM & MPM
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14. 14
Software Development and Standards
• CSDISM Plugin for Fiji/Imagej
– Acquisition: Java wrapper Labview
– Post processing: Java Plugin Imagej /BioFormat et.al System Biology
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15. 15
Conclusion
• ISM – Image Scanning Microscopy
– Any Dye
– Multicolor
– Low Intensity/ Damage
– 3D
• Resolution enhancement DSOM
• FAST ISM – Super High Resolution <100k€
• 2Photon Deep Tissue
• Software Tools for System Biology Community (STANDARDS)
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16. 16
Acknowledgements
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