Ratiometric images

Ratiometry refers to the experimental techniques in which two different signals are recorded to calculate their ratio, in order to find out if there is any relation between them.

In a Fluorescence Microscope, for example, you can image the spatial intensity distribution of two different dyes in a two Channel image.

If you want to compare signals of two separate images (one fluorophore), you should deconvolve them similarly. For more details see this page about signal quantification.

Ratiometric comparisons can be made in various ways in Huygens. See for example in the Huygens Professional the Operations Window -> Analysis -> Ratio Image.

This can be also used at some extent as a CoLocalization analysis tool but, unlike with most of the colocalization coefficients, ratiometry is very much affected by a different scaling of the involved signals.

Keeping the dynamic range

Image Restoration by Doing Deconvolution always implies a gain in Dynamic Range. This means that in a restored image you can distinguish more different levels of intensity that in the original one.

In many cases preserving this gain requires a rescaling. A 8 bit (Byte Type) original in which only 256 gray levels can be distinguished must be "upgraded" to a higher data type to keep the gained information in intensity resolution, and a scaling factor applied to make use of the new dynamic range.

The Huygens Compute Engine will generate 16 bit (Integer Type) restored images in low memory conditions (generally, in all 32 bit machines), but it will go for 32 bit (Float Type) output images in 64 bit machines. Independently of the format of the final result, for the internal calculations 32 bit is always used.

If your originals are in 8 or 12 bit type, a scaling factor may be applied to the data before deconvolution that will be kept in the final 16 or 32 bit results. This factor, which is a benefit from many other points of view, will affect your ratiometric results, and should be taken into account in your ulterior analysis.

How to know these factors

The Huygens Professional and the newest versions of the Huygens Essential will return these factors in their Task Report windows, and in the task log files of the Batch Processor.

The restoration commands in Tcl Huygens will return the scaling factors per channel when the -tclReturn option is used. (See the Scripting Manual).

File exporting

Beware that, apart from this restoration scaling, another factor will be applied if you save your results in any of the available TIFF File Formats!!! Read more in Tiff Scaling.