You can reduce the noise in fluorescence measurements by averaging more measurements together at each time point. With fluorescence plate readers and many spectrofluorometers, each measurement can consist of the average of multiple flashes of the lamp. Usually, you can change the number of flashes. Increasing the number reduces the noise. The disadvantage is that the number of wells that can be measured in a given amount of time (with a plate reader) is decreased since the larger number of flashes per well takes more time to complete.

Another way to reduce measurement variation is to measure a ratio of intensities at two different emission wavelengths, where one of the wavelengths is set at an isosbestic point. This may not be relevant to your resorufin system, however, and it works best on plate readers that can make two different emission measurements at the same time.

Adam,

Thanks for your suggestion.

I would look into your suggestions and comeback.

Btw, the plate reader I use can have two wavelengths being measured at the same time. I have 485:525 and 485:538. Can you please elaborate as to how can determine isosbestic point.

Regards,

Pratik

An isosbestic point is a wavelength at which the measurement (absorbance or fluorescence emission) does not change when the substance changes from one state to the other, such as going from substrate to product. You can use it as the denominator in a ratiometric measurement.

To find out if your substrate has an isosbestic point in its emission spectrum, you would have to measure the emission spectrum of the substrate and product. This measurement requires either a spectrofluorometer or a monochromator-based plate reader.

Adam,

Thanks.

I already did excitation and emission spectrum with BZres and resorufin. I would use isosbestic point to normalize the data and see what happens. I will update soon.

Regards,

Pratik