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