Dual-View™ and Quad-View™
Functionality in IW 6

The Dual-View and Quad-View models of Micro-Imager from Optical Insights, LLC, (www.optical-insights.com) are now supported in Imaging Workbench 6.

IW 6 separates the images acquired using the Dual-View or Quad-View into their respective emission wavelength channels, with one image per wavelength. Each of these images can be manipulated independently. Examples include:

• The two images can be ratio’ed and the resultant image can be displayed and analyzed. This is useful in ion imaging using indo-1 or other dual emission wavelength probe. Advantages of this method to measure free calcium include (1) the camera can be run at its full (streaming) mode, unimpeded by need to stop and switch wavelengths, for the highest rate of ratios per second; (2) acquiring both images simultaneously is more correct than acquiring them sequentially; and (3) there is less bleaching and photodamage because the total illumination time is halved.
• Each image can be separately background subtracted and shading corrected
• The individual images can be analyzed using Regions of Interest and results graphed in separate panes of a graph window.
• The two images can be superimposed with one channel colored red and the other, green, and with one of the two channels attenuated to match the intensities. The resultant image can be zoomed to the pixel level to confirm pixel-level registration of the image. A version now in development will allow live subtraction of the two images, with one channel attenuated to match intensities, a preferred way to confirm this registration quality.

The Dual-View or Quad-View is configured as an emission-side wavelength selection device, so it is quick and trivial to change between image-split mode and straight-through mode of the Dual-View.

FRET analysis procedures to allow removal of spectral bleed-through are now in development. Our version will incorporate the latest theoretical and practical considerations.