Image processing functions in Image Analyst MKII
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2D DFT Filter
2D DFT Filter Butterworth BP
2D DFT Filter Butterworth BP Tiled
2D Kernel Convolution
2D Median
2D Morphological Operator
2D Nonlinear Filter
2D Savitzky-Golay filter
Absolute Value
Affine Transformation
Align Channels
Align Series (Image Stabilizer)
Align Tiled Channels
Align Tiled Series (Image Stabilizer)
Anisotropic Diffusion Filter
Attach Intensity Gating Image
Attach Overlay Image
Automatic ROI drawing
Band Pass filter Optimization
Blind Spectral Unmix with NMF
Calculate Simple Crossbleed
Calculate Spatial Moments
Calibration Wizard Parameters
Clear Segmentation Classifiers
Close Image Window
Copy Image Window
Copy ROIs from Other Image
Correct Intensity Jump
Correct Lens Distortion
Count Division and Cell Death
Count Object Colocalization
Create ROI
Create ROIs from Segments
Crop
Crop Image in Place
Crop Image to Segments
Cross-correlation data
Cross-correlation image
Detect Nuclei Convolution
Differential Evolution Optimizer
Distance from Segments
Draw Model Mitochondrion
Draw Random Position Model Mitochondria
EndIf
Erase All ROIs
Excel Window Command
Export
Fill Mask
Fill or Mask Active ROI
FLIPR Calibration with [K+]ec steps and known [K+]ic and known kP
FLIPR Complete Calibration
FLIPR Complete Calibration with known kP
FLIPR Complete Calibration with known kP - Goldman
FLIPR Complete Iterative Calibration
FLIPR Estimate PN
FLIPR Short Calibration based on known potential during MDC and CDC
FLIPR Short Calibration between known baseline and CDC
FLIPR Short Calibration between known baseline and MDC
FLIPR Short Calibration between known baseline and separately measured fP0
FLIPR Short Calibration from Zero with fx=0
FunctionOptions
Get Image Information
Get Linked Channel
If
If Greater Than Zero
Image Arithmetic (image-image math)
Image Arithmetic In Place
Image Arithmetic Single Frame
Inpaint Mask
Input
Invert
Lens Correction Optimization
Link Image Windows
Load and Run Pipeline
Load ROIs
Mask Borders
Mask Frames by Plot Values
Mask Images
Measure Object Intensity
Measure Object Morphology
Mirror or Rotate (new image)
Mirror or Rotate in Place
Multi-Dimensional Open Information
Multi-Dimensional Open Stage Position
Multi-Dimensional Reload Channel
New Image
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Onset Image
Open File
Optical Flow
Options
Pipeline
Pipeline Optimization
Pipeline Optimization Parameter
Plot
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Plot Intensities Corresponding to Segments
Plot Morphological Parameters of Segments
Plot Ratio
Plot ROI Dimensions
Plot Tracking Parameters
Potential calibration constants
Potential calibration error propagation
Potential calibration expert overrides
Projection of Vectors from a Point
Ratio
Ratiometric ROI Classifiers
ReCount Division and Cell Death
Reevaluate Segments
Remove Blank Frames
Rename
Resample Image
ROI Classifiers
Run Membrane Potential Calibration
Save ROIs
Scalar Arithmetic (image-value math)
Scalar Arithmetic Multi
Secondary Watershed Segmentation
Select
Select by Number
Sensor Noise Characteristics
Set Reference Image
Set Scaling/LUT
Set Segmentation Classifiers
Set Segmentation Intensity Classifiers
Shift Time Scale
Simple 2D Cross-correlation
Simple Segmentation
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Spectral Unmix
Strip to Well Cell Count
Substitute Poisson Noise
Subtract Background or Normalize
T or Z-project
Template Matching
Temporal Average Filter
Temporal Block Filter
Temporal Median Filter
Temporal Rolling Projection
Temporal Savitzky-Golay Filter
Thinness Ratio Optimization
Threshold
Time Stamp and Scale Bar
TMRM Complete Calibration
TMRM Complete Calibration with known kT
TMRM Complete Calibration with known kT and K-steps
TMRM Short Calibration between known baseline and MDC or CDC
Track Objects
Truncate or Cut
Wait for All Inputs
Watershed Segmentation
Wiener filter
Window Menu Command
Write Back Scaled Values
ΔF/F0

FLIPR Complete Calibration with known kP - Goldman ( IAFLIPRCompleteKPGoldman )

Parameters:
Name Short Name Type Description
Range definitions in time courses rangedef string Defines whether the ranges given below refer to frames or seconds in the recordings
Baseline Range BaselineRange string Defines the range of the baseline (in the above defined time units). Longer baseline results more accurate calibration.
K+ Steps Ranges KStepsRanges string Enter a list of range definitions for the duration of the K+-steps (in the above defined time units), including the MDC treatment.
[K+]ec Steps (mM;mM) KStepsConcentrations string Extracellular (ec) K+ concentrations corresponding to the above defined steps, including MDC, in mM. Use the Wizard tab to calculate concentrations from added volumes.
kP kP real Previously measured value of kp
CDC Range CDCRange string The range of Complete Depolarization Cocktail addition (in the above defined time units).
Take maximum of CDC Range CDCMax boolean If set yes, the maximum FLIPR intensity within the CDC Range is taken as zero calibration value (it error is the local SD of the range). Otherwise mean is calculated.
fP0-fPX Allowed Range fP0MinusfPXRange string Trace selection criterion: the minimum and maximum allowed value of fP0-fPX
fPX r^2 Min fpXr2Min real Trace selection criterion: the minimum allowed value of r^2 for the FLIPR-regression.
Quality control by propagated error of baseline QCPMPrest boolean Removes traces where the estimated SE of baseline is greater than the threshold below. Requires error propagation.
Maximum SE of baseline (mV) QCPMPrestMaxSE real Traces where the SE of resting ΔψP parameter is larger than this number are removed.
PN PNway string Defines whether PN is calculated and used indepenedntly for each trace or a median value is used uniformly for all traces. In the latter case PN is calculated for each trace, and then the calibration is re-run using the median of the PN values for increased robustness. Note that errors in determination of PN are not propagated.
Weights for fitting K-steps PNweights string Defines weighing least squares during nonlinear fit for colaculation of fpx and PN.
Description:
This method calculates plasma membrane potential (ΔψP) time course by calibration parameters from a single time lapse with only assuming the value of kP, using a nonlinear fit for Goldman equation to calculate K+-equilibrium potentials using the FLIPR Membrane Potential Kit from Molecular Devices (aka PMPI, plasma membrane potential indicator). The model used to calculate ΔψP assumes that the plasma membrane permeability for ions other than K+ is significant. The extracellular concentration of these ions is either constant, or are replaced for K+ (e.g. Na+). Furthermore, the model is also compatible by the presence of a constant voltage offset injected by electrogenic ion pumping during K-steps.
A limitation of this approach is the larger error committed during the fit than using the linear, Nernstian approach.
The redistribution of FLIPR is relatively fast and the exact value of kP has little effect unless very fast oscillations are looked. Therefore, in many cases the kP=0.38 determined on hippocampal neurons can be used.
Required experimental protocol: any time lapse, followed by establishment of K-equilibrium potential, at least 3 additions or replacements with high K+-medium (or 2 with no error estimation) and finally complete depolarization.
The cells have to be able to maintain a constant hyperpolarized ΔψP before starting K+-steps. Do not use this method if ΔψP depolarizes substantially when adding MDC.