This document discusses image scanning microscopy (ISM), a technique that improves the resolution and contrast of fluorescence microscopy images. ISM works by acquiring multiple images of the sample from different positions and combining them to generate a single image with effectively doubled resolution. The document outlines the basic principles and components of ISM systems, provides examples of applications like imaging fluorescent beads and cell structures, and discusses related techniques for further enhancing resolution and depth of imaging. It concludes by emphasizing ISM's benefits like high resolution, low photodamage, and multicolor imaging capabilities.

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
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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
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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.
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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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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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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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