TILLvisION Tutorial

TILLvisION - Image Combination

How to combine a fluorescence image with a transmission image

Sometimes it may be interesting to see fluorescence information together with the corresponding transmission image. An here you can see how powerfull TILLvisIONs image manipulation features are. This page describes a way to combine two images in color space to get this interesting result. You don't always need Photoshop to accomplish task like this.

This example also demonstrates TILLvisIONs powerfull image processing capabilities
	1. Let us acquire a transmission (TR) and a corresponding fluorescence image (FL). 2. Autoscale the transmission image and then convert it into an 8-bit image (TR8b). For the conversion of a 16-bit image to an 8-bit image use the macro conv8bit.bas.
	3. After acquisition of the fluorescence image, append a mapping and display palette to the image. For example, autoscale it and use the Rainbow2 palette. 4. Convert the image (FL) into a three channel color image (FL24b). For the conversion use the macro FColMap.bas (FullColorMapping).
5. Change the color space of the new image (FL24b) from RGB to HLS. Use the macro ColSpace.bas and select RGB to HLS conversion.
The RGB color space: R=Red, G=Green, B=Blue The HLS color space: H=Hue, L=Luminance, S=Saturation. Comment: All color information of an image in the HLS color space is stored in the first band (Hue). The second band contains the luminance of the image. By copying the transmission image into the luminance band of the fluorescence image we can "map" the brightness of the colored fluorescence with the contours of our HELA cell preparation. Had we used an "ideal" pseudocolor palette with constant luminance (like TILLvisION's Rainbow3), then the luminance band of our image would be constant too. However, as can be seen in Fig. 3, the Rainbow2 palette also produces information in the luminance band. To keep this information, we copy the FL luminance to the unused saturation band.
6. Display the new HLS image and turn off the true color mode by clicking the colored button in the "Display" toolbar. (To add a new toolbar go to View/Toolbar). 7. Switch the display of the FL24bit to the second band. Use the AnimationPlayer ("Animation" Toolbar) and set it to 1. 8. Create a new AOI that covers the whole image (second band). The corresponding "child"-image allows to apply image processing functions to one specific band of the "parent"-image (here FL24bit). 9. Switch the display to the third band (saturation). Use the AnimationPlayer and set it to 2. 10. Create another AOI (AOI1) in the third band that also covers the whole image. 11. Copy the AOI of 8.) into AOI1 of 10.) with a bit shift of 1. Use the Option dialog in the Process/Point/Copy window. A bit shift of 1 corresponds to a multiplication by 2. 12. Copy the transmission image (TR8b) into the AOI of FL24b. Use Process/Point/Copy. 13. Convert the image FL24b back from HLS to RGB. Again use the macro ColSpace.bas.

Fig.3: Transmission and Fluorescence images combined