In recent years a lot of fluorophores were developed for in-vivo monitoring of biological processes in animals. Non-fluorescent in their native state, they generate high levels of fluorescence through enzyme-mediated release of their fluorochrome.

The task was to measure two fluorescent labels at the same time in bone cancer: ProSense680 and OsteoSense750.
ProSense and OsteoSense enable in vivo detection, measurement and monitoring of skeletal changes in a wide range of disease states, including arthritis, osteoporosis and cancer metastases. Results correlate well with traditional clinical measurements of disease.

The task has been to find the right filters since the spectral properties of these two labels.
We determined for ProSense 680 an excitation filter of 630/20 nm and a 700/20 nm filter for emission to be best choice.
For OsteoSense750 we selected a 700/20 nm filter for excitation and a 790/40 nm filter for emission, because at 790 nm emission of  ProSense680 is more or less negligible. The measurements were performed at the University of Kiel, Germany.
We filled 10 μL of each dye in a concentration of 0.2 nmol in Eppendorf tubes and placed them into the NightOWL LB 983 NC 100. First measurement was performed with 630/700 filter pair, 1 s exposure time, 15% lamp energy and 1x1 pixel binning.

Result is shown on picture 1: The left Eppendorf tube with ProSense680 emitted light, but not the right one withOsteoSense750 as expected.

When changing to the OsteoSense750 filter pair 700/790, both Eppendorf tubes emitted light. Therefore we changed to another excitation filter to avoid excitation of both dyes. We switched to an excitation filter of 740/30 nm.
With the filter pair 740/30 and 790/40 we finally managed that only OsteoSense750 emitted light (picture 2). The two spots reflect the fact that two droplets were generated after pipetting the solution into the Eppendorf tube.
With those filter pairs, identical instrument setting and threshold setting OsteoSense750 showed a 1.6 times higher signal than ProSense680.

Some techniques for generating and/or detecting light in biological subjects are patented and may require licenses from third parties. Users are advised to independently determine for themselves whether their activities infringe any valid patent. ProSense and OsteoSense are trademarks of VisEn medical.