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

Flow Cross Correlation in Zebrafish Vascular System Neuronal Network Analysis in Cerebellar Slices

Galvanometric Mirrors Scan head Phototube Microscope Objective Pulsed IR Laser Two Photon Microscope Sample

Raster scanning

I t

I t

Ionic Channels Dendrites Axon Neurons

Ca 2+ Ca + Ca 2+ Neurons

From olympusconfocal.com Neuronal Networks

Experiment requirements: Confocal imaging Largest possible field of view Diffraction limited resolution High sensitivity to fluorescence signals fluctuations Millisecond time scale Neuronal Networks

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

Luckily, we don’t really need an image Calcium induced calcium release Signal in the cellular body varies uniformly on the microsecond scale

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

Spatial light modulation Back focal plane Focal Plane Fourier Transform User generate phase shift pattern Spatial light modulator

Gerchberg-Saxton Algorithm SLM Plane Sample Plane IFFT FFT Desired pattern START END Laser intensity Amplitude Phase

Galvanometric Mirrors Scan head Phototube State of the art Camera Laser Beam expander SLM Microscope Objective Nikolenko et Al. 2008

Some problems: Coordinates matching!!! Zero order of diffraction Excitation power wasted Very complicated (Remember, biologists must use it!!!)

How we do it: Camera Laser SLM Microscope Objective

Experimental setup Phase: Intensity: Phase: Intensity:

Experimental setup Phase: Intensity: Phase: Intensity: +

How to make an image without a galvo scanner? 1 2 N …

1 2 N … Digital two photon imaging It is a very long calculation, but you do it once, and forget about it!

Digital two photon imaging 1 2 … Each bright spot is a pixel of the final image. N

Digital two photon imaging And here comes the image (in convenient SLM input coordinates)

Some numbers SLM refresh rate: 60 Hz Camera acquisition rate: 100 frame, 1600 Hz in a 128x128 ROI Laser power: 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

So, does it work?

Averange So, is it useful?