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Image Scanning Microscopy
Dirk Hähnel - Group Seminar 30.04.2013
III. Institute of Physics – BiophysicsGeorg-August-University Göttingen
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Problem
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Methods
Schermelleh et.al, JCB
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System Overview
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Data Acquisition
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Image Information
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Each pixel acts like a small confocal aperture for a two-dimensional image
Y_Sample
X_Sample
X_CCD
Y_CCD
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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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Results
●Fluorescent bead imaging●Increase of contrast and resolution
Intensity profile of a single fluorescent bead*scalebar indicates 1 mm
Quedlinburg 26th Sep. 2011
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Using saturation of the excited state
Taking measurements on at least two different intensity-levels
Archiving the higher harmonic in the Fourier-space
Increase Resolution
Quedlinburg 26th Sep. 2011
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Optical Saturation Microscopy
Quedlinburg 26th Sep. 2011
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 oftransparent mouse brain," Nature Neuroscience, advance online publication, 30 August 2011
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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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Acknowledgements
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