Re: chlorophyll and associated pigment spectra

Posted by Rosemary.White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Microscopy-used-equipment-tp4594144p4596500.html

HI Christian,

Basically, you can see chlorophyll autofluorescence over much of the FP spectrum.  I either collect autofluorescence in a chlorophyll-only region, often above 650 nm (except when using Cy5 or PI then have to split between these and Chl), and add to the total image in a contrasting colour, or subtract out chlorophyll autofluorescence.  Depends on what you need to see and measure and how tricky your fluorescence quantification is if you’re going down that track.
cheers,
Rosemary


On 19/02/10 6:07 PM, "Christian" <celowsky21@...> wrote:

Rosemary,

Thank you for the rapid reply!

Often when GFP is weak (ex488/em522), such as in mitochondrial targetting, the chloroplasts often in overlay become yellow as the autofluorescence becomes very noticeable.  Furthermore, if there is GFP in the chloroplasts which is not quite bright, it can be very difficult to demonstrate its presence to reviewers.  I always set up against wild type material, and I'm always looking for ways to explain to users what is going on with their "green" chloroplasts which should be red!

Thanks.

Christian