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Combining Imaging and Electrophysiology
Hubert Affolter
Hubert Affolter, PhD 1980: Graduated at the Institute of Technology
(ETH), Zürich, Switzerland
Ion-Sensitive Electrodes
1980-1983: University Hospital Basel, Switzerland
Postdoctoral Fellow
1984-1986: State University of North Carolina, USA Postdoctoral Fellow
1986-1988: Yale University, Medical School, USA Associate Research Scientist
1989-1991: Instrutech Corp, USA Software Engineer
since 1991: HEKA Elektronik GmbH, Lambrecht, Germany Senior Software Architect
Goals
• Preserve high time resolution and exact correlation
• Compute ROI data, on- and off-line
(ROI = mean fluorescence values of Regions Of Interest at 1 wavelength)
• Retrieve imaging frames corresponding to any time position in the
electrophysiological data stream
• Acquire imaging data simultaneously with electrophysiological
recordings
Hardware
Imaging: • Camera supporting triggered
acquisition
• Filter wheel or monochromator
Amplifier: • EPC 10, iTEV 90, … • DA/AD board
(built-in or stand-alone)
Acquisition of current and imaging with precise time synchronization
Separate ROI traces: - ROI (i.e. region of interest) - Ratio - Calcium concentration
Real-time: - acquisition of frames - current, voltage, etc. - ROI computation
Off-line: - display of current, voltage, etc. - display of corresponding frame - replay of corresponding movie - ROI re-computation
Camera trigger
Wavelength control
Cell Stimulation
Cell response
Alternating 2 wavelengths Frame 1 360nm
Frame 2 390nm
Frame 3 360nm
Frame 4 390nm
Frame 5 360nm
……
ROI trace 1
ROI trace 2
Components Interactions
PatchMaster
Imaging
Monochromator
EPC10 ADC
Current Voltage DA/AD board
Camera
DAC: Stimulus Wavelength Control Frame Trigger
Supported Cameras Camera support is via the SIDX imaging library. SIDX7 supports cameras
with BULP trigger from: - Andor Technology - AVT - Jenoptik - Photometrics - Hamamatsu
Supported Filter Wheels and Monochromators PatchMaster supports: - DG-4, DG-5, Lambda-10 from Sutter Instruments - DeltaRAM from pti - Polychrome and Oligochrome from TILL Photonics
Imaging Support in Pulse Generator Window
Wavelength control
Imaging Channel => camera control ROI trace 1
ROI trace 2
Camera and wavelength timing
Defining the stimulus in Imaging application
Priming the PGF stimulus from the Imaging application
Features Camera live mode
Camera snapshot
Auto and manual scaling (minimum / maximum)
Histogram: computed, displayed, stored
Mouse tip shows pixel position and intensity value
Frames are stored as TIFF stacks
Multiple ROIs can be defined and retrieved
ROI mask is saved with image stack
Background subtraction on ROI mean fluorescence values
Calculation of fluorescence (ROI) ratios
Calculation of free calcium concentrations
Compatibility
Display ROI data together with the electrophysiological data in the same graph, with high temporal resolution
ROI data 1
Current
ROI data 2
c
Display ROI data together with
the electrophysiological data in
the same graph, with low
temporal resolution:
Online analysis window
Wavelength 1
Series Resistance
Wavelength Ratio
Wavelength 2
Select a target (series, sweep, trace) in the replay window and the corresponding image is shown in the Imaging window
Connection between recordings and images
and this corresponding image is shown
and this corresponding image is shown
Click on this target
Click on this target
Connection between recordings and images
Click here and the and the second frame of the movie is shown
Click here and the and the first frame of the movie is shown
Example 1
First frame of recording Last frame of recording
Example 1 Online window:
ROI of the 2 wavelengths
plus their ratio
plus R-series
plus background
Example 2
First frame of recording Picture of drug application
Example 2 Oscilloscope window:
Cell current
ROI of wavelength 1
ROI of wavelength 2
ROI ratio
Example 3 Online window:
ROI of the 2 wavelengths
plus their ratio
plus R-series
plus background
Goals :
• Preserve high time resolution and exact correlation
• Compute ROI data, on- and off-line
(ROI = mean fluorescence values of Regions Of Interest at 1 wavelength)
• Retrieve imaging frames corresponding to any time position in the
electrophysiological data stream
• Acquire imaging data simultaneously with electrophysiological
recordings
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