Digital two photon microscopy for multiple fast signals acquisition Laboratory of Advanced...
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Transcript of Digital two photon microscopy for multiple fast signals acquisition Laboratory of Advanced...
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?