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Published byFelicity Armstrong Modified over 9 years ago
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Digital two photon microscopy for multiple fast signals acquisition Laboratory of Advanced Biological Spectroscopy (L.A.B.S.) University of Milan - Bicocca Paolo Pozzi Neuroscience Department University of Pavia
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Flow Cross Correlation in Zebrafish Vascular System Neuronal Network Analysis in Cerebellar Slices
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Galvanometric Mirrors Scan head Phototube Microscope Objective Pulsed IR Laser Two Photon Microscope Sample
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Raster scanning
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I t
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I t
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Ionic Channels Dendrites Axon Neurons
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Ca 2+ Ca + Ca 2+ Neurons
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From olympusconfocal.com Neuronal Networks
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Experiment requirements: Confocal imaging Largest possible field of view Diffraction limited resolution High sensitivity to fluorescence signals fluctuations Millisecond time scale Neuronal Networks
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Experiment requirements: Confocal imaging Largest possible field of view Diffraction limited resolution High sensitivity to fluorescence signals fluctuations Millisecond time scale Over large fields of view, two photon imaging can achieve 2-3 frames per second. We have to improve by three orders of magnitude! Neuronal Networks
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Luckily, we don’t really need an image Calcium induced calcium release Signal in the cellular body varies uniformly on the microsecond scale
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Luckily, we don’t really need an image Experiment outline: Generation of a single confocal image Selection of 1 pixel (POI) in every cell Simultaneous illumination of all POIs Simultaneous acquisition of all signals
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Spatial light modulation Back focal plane Focal Plane Fourier Transform User generate phase shift pattern Spatial light modulator
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Gerchberg-Saxton Algorithm SLM Plane Sample Plane IFFT FFT Desired pattern START END Laser intensity Amplitude Phase
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Galvanometric Mirrors Scan head Phototube State of the art Camera Laser Beam expander SLM Microscope Objective Nikolenko et Al. 2008
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Some problems: Coordinates matching!!! Zero order of diffraction Excitation power wasted Very complicated (Remember, biologists must use it!!!)
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How we do it: Camera Laser SLM Microscope Objective
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Experimental setup Phase: Intensity: Phase: Intensity:
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Experimental setup Phase: Intensity: Phase: Intensity: +
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How to make an image without a galvo scanner? 1 2 N …
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1 2 N … Digital two photon imaging It is a very long calculation, but you do it once, and forget about it!
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Digital two photon imaging 1 2 … Each bright spot is a pixel of the final image. N
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Digital two photon imaging And here comes the image (in convenient SLM input coordinates)
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Some numbers SLM refresh rate: 60 Hz Camera acquisition rate: 100 Hz @full frame, 1600 Hz in a 128x128 ROI Laser power: 4 W @ 800 nm Scan grid: -28 x 28 focuses in a 12x12 scan pattern (2.4 s) -30 x 30 focuses in a 20x20 scan pattern (6.7 s) -25 x 25 focuses in a 40x40 scan pattern (26.7 s) Time for single hologram calculation: ~30 s
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So, does it work?
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Averange So, is it useful?
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