Correct Dye Image For Background Variations

This option is designed specifically for correcting variations in the background illumination for back-lit experiments. Typically such variations reduce the amount of information directly available from the variations in intensity over an image. If, for example, dye is added to a flow illuminated from a diffuse background. The intensity of the resultant image will be some combination of the background illumination and the concentration of the dye in the flow. Typically it is the concentration of the dye which the analysis is designed to determine. DigImage is capable of dealing with dyes having either a linear or logarithmic manner.

If the dye behaves in a linear manner (doubling the dye concentration halves the amount of light transmitted) then a viewer will see an illumination proportional to F=(1-C)*B, where F is the intensity of the image containing the dye at concentration C and background illumination B. The correction imposed is thus (1-C)=F/B.

On the other hand, for a logarithmic dye we assume the transmitted intensity p through a region of dye of concentration dc varies as dF=-Bdc. Integrating this gives C proportional to log(F/B). The correction we apply is (1-C)=1+Alog(F/B), where A is specified by the user (the attenuation factor).

Strictly speaking the correction should be undertaken using absolute intensities taking into account the intensity transfer functions of the camera, VTR, video tape and frame grabber. DigImage includes a number of features aimed at relating the digitized intensities and absolute intensities. Most of these reside in the menu [;PI: Intensities] - refer to the Help System in this menu for further details. For the present analysis we shall assume the digitized intensity scale is reasonably close to an absolute intensity scale (within an aribtrary constant of proportionality). The validity of this assumption depends primarily on whether the camera being used imposes a gamma correction (see Document\Cameras.DOC for details) and whether the gain and zero offset of the frame grabber card are adjusted appropriately (refer to the frame grabber manual for details).

Buffer containing background ?

This entry specifies the buffer containing an image of the background illumination, prior to start of the experiment. This image may be contained in any buffer; it should not however be written during the correction process if more than one buffer is to be corrected as the background will be used for each buffer corrected.

Correct which foreground buffer for background ?

A list of buffers may be corrected for the same variations in the background illumination. This entry specifies one of the buffers to be corrected.

Another buffer ? If you wish to add another buffer to the list to be corrected, then a reply of yes (<Y>) will prompt for a further buffer. No (<N>) indicates that all buffers to be corrected have been specified.

Buffer to store first corrected image in (the second will be in one more than

this etc.) ?

corrected version of the first image on the list will be placed in the buffer specified by this entry, the second in the buffer one higher than this and so on. Note that the corrected image may be placed in the same buffer as the original foreground (ie. containing the dye image).

Intensity scale factor (around 1) ?

This scale factor is used to rescale the intensity range in the resultant image. If the video camera was using a manual iris and no automatic gain control, then a value of around 1 should be specified as a point on the image should have the same intensity in the background and foreground images if there has been no change in dye concentration at that point. However, if an automatic iris or automatic gain control has been employed, then a value further from unity may be required as the camera may have adjusted itself between the time of the background and foreground acquisitions.

Intensity black offset (around 0) ?

This value is used in conjuction with the intensity scale factor to specify the range of intensities produced by the correction procedure. It effectively changes the value of the correction which will be set to an intensity of zero. Increasing the offset value (up to the limit of 1.0) reduces the intensities produced.

Use Rational (F/B) or Logarithmic (log(F/B)) correction ?

The response to this question determines the type of correction made to the image. To utilise the rational (linear) correction, a response of <R> should be given, while <L> accesses the logarithmic correction which maps raw intensity to concentration.

Correct in Window or whole Screen ?

The image may be corrected in either a window (<W>) or the entire frame buffer (<S>). As the code to produce this correction is written directly in assembler, there is not too much time overhead in correcting an entire buffer.

Window Selection: Region to correct for background

If the user choses to correct for variations in background illumination in a window, then the Window Selection submenu is produced to aid the window specification process. Additional information on window specification is available through the [H Help] option within the submenu.

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DigImage User Documentation

Stuart Dalziel, last page update: 19 February 1996