Digital two photon microscopy for multiple fast signals acquisition

Laboratory of Advanced Biological Spectroscopy (L.A.B.S.)University of Milan - Bicocca

Paolo PozziNeuroscience Department

University of Pavia

Flow Cross Correlation in Zebrafish Vascular System

Neuronal Network Analysis in Cerebellar Slices

Galvanometric Mirrors

Scan head

Phototube

MicroscopeObjective

Pulsed IR Laser

Two Photon Microscope

Sample

Raster scanning

Raster scanning

I

t

Raster scanning

I

t

𝐾 +¿ ¿

𝐶𝑙−

𝑁𝑎+¿ ¿

𝐾 +¿ ¿𝐶𝑙−𝑁𝑎+¿ ¿

𝐴−

Ionic ChannelsDendrites

Axon

𝐶𝑎2+¿ ¿

𝐶𝑎2+¿ ¿

Neurons

Ca2+

Ca2+

Ca+

Ca +

Ca +Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

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

AmplitudeAmplitude Phase Phase

Galvanometric Mirrors

Scan head

Phototube

State of the art

Camera

Laser

Beam expander

SLM

MicroscopeObjective

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

SLMMicroscopeObjective

Experimental setup

Phase:

Intensity:

Phase:

Intensity:

Experimental setup

Phase:

Intensity:

Phase:

Intensity:

+

How to make an image without a galvo scanner?

12

N…

12

N…

Digital two photon imaging

It is a very long calculation, but you do it once, and forget about it!

Digital two photon imaging

12 …

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 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

So, does it work?

Averange

So, is it useful?