Your lens` transmittance at 340 nm may be too low (

Hi Silke,

From your description I find it unlikely that the problem is in your optical system, since the raw values from your LED are many many times higher than that of your arc lamp (unless there was a change in the camera exposure, but I will assume there wasn't).

One issue of using LEDs for ratiometric assays is that there is no guarantee that your different LEDs will have the same output power. There is a chance that your 380nm LED is simply a lot stronger than your 340nm, since it is a significantly longer wavelength (so that you will have a lot more photons for the same output power with your 380nm). It might be interesting to check the output power and calculate the luminace of each LED to see if your raw values make sense.

Alternatively, you might consider switching over to a non-ratiometric method. I don't really know for synthetic dyes, but for genetically encoded calcium indicators, such as GCaMP, non-ratiometric measurement is pretty much the standard. You would need to change your LEDs to match another wavelength, but it might be worth a shot.

Hope this helps and good luck!

Hi Janos and Ronan,

thank you both for your answers!!

I use an opimised 340nm LED(tested by the manufacturer), so the transmittance should be good.

I will check the output power. But how can I calculate the luminace?

I don´t have any experience in that...

Thanks a lot!

Check your neutral density filters that will attenuate the light passing through

I think the simplest way to measure your output would be to use your camera without a filter cube, if you are able to poke around with your microscope. This way you can use the setup that is as close as possible to your actual experiment.

Remove the cube, put a random sample under the objective, take pics using the reflected light and see which LED gives the brightest picture by quantifying the raw signals.

Hope this is actually feasible!