Traces can be displayed as intensity change (I) versus time or as fractional intensity change (I/I) versus time. Segments of the traces may be expanded graphically to focus on critical features.

The Trace Display can display:
1. Traces from individual pixels
2. The spatial averages of selected pixels
3. The FFT of selected traces,
4. The correlation between traces, and
5. Separated or superimposed traces. Superimposed traces can be scaled to equal height for comparing time courses.

Page Display (Spatial Array):
The Page Display can display an array of intensity-versus-time traces for all pixels. The Page Display can also display gray scale or pseudocolor images of the Resting Light Intensity or intensity differences between user selected frames. Multiple groups of pixels may be identified and color-coded on the Page Display; the corresponding individual or average traces appear in the Trace Display. An omit array may be created to exclude selected pixels from the display and analysis. An option is available to replace bad pixels with the average of the four adjacent pixels. The Page Display can display:
1. A map of Traces
2. Differences between user selected frames
3. RMS Noise Levels
4. Light Intensities relative to a Dark Frame (Resting Light Intensity)

Filtering:
The following kinds of temporal high- and/or low- pass filtering are available:
· Gaussian,
· Butterworth,
· Bessel
· median,
· binomial, and
· tau

In addition, spatial filtering options include up to 10 iterations of a 3x3 Median, 3x3 Mean, or 5x5 Gaussian low-pass filter and a variable-width Gaussian high-pass filter.

Other Options:
Both the Page Display and the Trace Display can be exported as TIFF, PostScript, or ASCII files.



Cardio/Movie

The analytical process continues with a Movie of the surface membrane potential, progressing for up to 10240 frames. For immediate review, a grey-scale image of unfiltered data may be scanned by scrolling manually using the Scan feature.

The second analysis interface within CardioPlex is the Cardio/Movie interface. It provides pseudo-color displays of the data (shown at right). Pseudo-color images may be displayed as single frames, multiple frame layouts, and as an animated movie. The color-coded pixel intensities may be displayed directly, as contour plots, or as surfaces in 3D. A variety of color schemes, scaling, and filtering options are available. A trace inset is available to link the current movie frame to a time series for an individual pixel or group of pixels.

Trace Inset:
The Trace Inset supplies an intensity-versus-time plot for an individual or group of pixels selected from the pseudo-color movie frame on display. A red bead on the trace identifies the current frame while the movie is running. A frame may be selected by clicking on the trace inset, as well.

Multiple Frame Layouts:
Primary movie frames may be selected in regular sequences automatically or manually edited to select irregular sequences for simultaneous display in the movie window. Special CardioAnalysis frames may be associated by frame number or frame type in various patterns and accumulated in an editable list for simultaneous display.

Spatial Filtering:
Spatial smoothing and filtering options for the pseudo-color images include up to 10 iterations of a 3x3 Median, 3x3 Mean, or 5x5 Gaussian low-pass filter and a variable-width Gaussian high-pass filter.

Overlay of grey-scale Image:
For anatomical reference, a grey-scale image of the preparation, imported as a TIFF or BMP file, can be overlaid on the pseudo-color images.

Pseudo-Color Scaling:
The pseudo-color scaling of the frames can be manipulated by using different color tables and four different scaling schemes:

The scaling schemes are:
1. Variable (with each detector scaled individually to a standard range);
2. Fixed, using the overall minimum and maximum among all traces;
3. Fixed, using the range of a user-chosen detector for all traces; and
4. Manual.
(When you've tried all these options, it will be clear why it's called pseudo-color - as in "lies, damn lies, and pseudo-color").

Other Options:
The movie can be saved as a movie file and the file can then be read and replayed by CardioPlex. The display on the movie window can be exported (in color or grey-scale) as a TIFF file and a sequences of frames from the movie may be exported as a TIFF-Stack or MPEG file.



CardioAnalysis

A third analysis interface within CardioPlex provides a CardioAnalysis panel for setting up and executing the CardioAnalyis, which generates a CardioReport panel (shown below) for navigating the extended movie and multiple frame displays and associated traces and tables. Types of analysis for paced data include activation time maps and conduction vector plots. Analysis for fibrillation and spontaneous activity include phase angle and phase singularity indicator movies.

For Paced Data

Action Potential Duration
For each pixel, the times of depolarization (i.e., activation) and subsequent repolarization (i.e., recovery) are computed. Depolarization and repolarization times are computed using either threshold values or temporal derivatives of the signal. Action potential duration for each pixel are determined from the difference between the repolarization time and depolarization time. Spatial maps of each of these parameters can be displayed as images, where the values are color-coded.

IsoChrone Maps
An isochrone map displays the spatial variation in a quantity at or in relation to a specific point in time. (Isochrone means "same time"; thus, isochrone maps indicate events that happen at the same time.) Isochrone maps of depolarization or repolarization times are generated using contour plots of the depolarization or repolarization time maps described above. For example, an isochrone map generated from a depolarization time map indicates the position of the wave front as a line or curve. Therefore, in one picture, an isochrone displays the sequence of depolarizations or repolarizations.

Conduction Vectors
For each pixel, the times of depolarization (i.e., activation) and subsequent repolarization (i.e., recovery) are computed. Depolarization and repolarization times are computed using either threshold values or temporal derivatives of the signal. Action potential duration for each pixel are determined from the difference between the repolarization time and depolarization time. Spatial maps of each of these parameters can be displayed as images, where the values are color-coded.

For Fibrillation Data

Phase Angle
For each pixel and a selected time delay (under user control), the relationship between the pixel value at a given time and its value at the specified delay is summarized by its phase (expressed as an angle associated with the ratio of the coordinates as a tangent). This phase angle captures useful information on the dynamics of the surface membrane potential at each location; in particular, the existence of an axis of rotation or phase singularity in the neighborhood of a pixel. For the signal at each pixel, F(t), a cardiac phase variable, (t), can be computed as:



Where indicates a specified time delay and F* indicates the threshold for depolarization. This phase variable provides a unique value (from - to ) indicating the time course of each action potential. The construction of this variable can be explained easily graphically. The process involves plotting F(t+) versus F(t) and making a transformation into a polar coordinate system where F* is the origin and (t) indicates the angular position. CardioPlex generates a spatial map of phase angles, which are displayed as a color-coded image, representing phase cyclically on the standard color wheel (with purple as phase ± ).

The CardioReport panel includes a Phase Trajectory plot of F(t+) versus F(t) for a selected pixel or group of pixels.

Phase Movie
The phase movie displays a moving spatial image of instantaneous phase as a function of time. The instantaneous spatial display is a color-coded image, representing phase cyclically on the standard color wheel (with purple as phase ± ).

Phase Singularity Movie
From each phase angle map, the local contour integral algorithm is applied to score each pixel as a potential phase singularity. The phase singularity movie displays a moving spatial image of instantaneous phase singularity time. The instantaneous spatial display is a color-coded image, representing 'direction of rotation' (red for counterclockwise and blue for clockwise) on a yellow background indicating absence of phase singularity.