How to pick GFP+ and GFP- colonies in the PIXL

Customers reported PIXL struggles to distinguish GFP+ from GFP- colonies, selecting only GFP+. A feature request is under review, but no timeline is set. Meanwhile, Dr. Phil Kirk's team suggests optimizing imaging conditions—lighting, filters, exposure—to detect both colony types. Testing different settings may improve fluorescence sensitivity, though low fluorescence levels remain challenging. Contact technical support for more help.

Contents

• Introduction
• Procedure

Introduction

Recent feedback from our customers has pointed out that the PIXL has trouble distinguishing between GFP+ and GFP- colonies. It will try to select the GFP+ colonies and ignore the GFP- colonies.

This feature request has been submitted to our software team for investigation but we are unable to confirm when this feature will be available. 

In the meantime, our Science & Research Team has been investigating a workaround. The following article explains the process. (written by Dr. Phil Kirk, Science & Research Team)

Procedure

It may be possible to optimise the imaging conditions to make it possible to see both types of colonies at one time. I have some images of plates with both GFP+ and GFP- (S. cerevisiae) on them and I have processed them through PIXL to see if I can select GFP- and GFP+ colonies separately. I have done this for several imaging conditions using different lighting, dichroic filters and exposures. 

These plates were constructed in a slightly unusual way which I must describe. Here, colonies were inoculated with mixtures of GFP- and GFP+ (strongly expressing) cells. Once the colonies matured, they were spread plated to determine the proportion of cells in that colony that were GFP- and GFP+. Therefore, colonies appear to have different levels of intensity. This enables PIXL sensitivity to fluorescence to be determined.

For the following figures, the imaging conditions used are written at the top. ‘Background subtraction’ with ‘B.subtilis’ was the colony detection algorithm used. Here, both GFP+ and GFP- colonies could be detected and filtering allowed these types to be distinguished. In the GFP- selection, colonies with very low levels of fluorescence (3%) could not be distinguished from WT and this is related fluorescence sensitivity of PIXL (it isn’t a microscope after all).

If the GFP- colonies are difficult to see in this lighting condition, perhaps increasing the exposure could help (Figure below). Again, lower levels of fluorescence may be difficult to distinguish from GFP- colonies.

Failing that, it may be worth testing another lighting/filter combination to see if this helps distinguish the GFP- and GFP+ colonies.

If you require more information please contact [e-mail] for assistance.