With the ASI / TILL Photonics hardware for fluorescence microscopy, a dual-emission photometry setup can be easily assembled. It is possible to realize control and data recording from common patch-clamp software such as Axon's P-Clamp, since most common patch-clamp amplifiers have at least two A/D inputs. Therefore the dual-emission photometry setup is based on two photomultiplier's (one for each emission wavelength) whose signal outputs are connected to the A/D inputs of the amplifier or to a data digitizer.

To obtain the two separate wavelengths, we use combinations of dichroic mirrors and filters. The figure, below, shows the general optical design. The photometry setup consists of a viewfinder and a detector unit consisting of two photomultipliers, filters, and a beam splitter.

The fluorescence light originating from the specimen goes straight to the aperture stop and to the two photomultipliers. The specimen area for the analysis of the fluorescence intensity is user-defined by the aperture stop whose size and position can be adjusted with the Viewfinder.

By illumination of the aperture stop with the transmitted light of the microscope and bringing the light via beam splitter and mirror to the CCD camera, a low-intensity image of the aperture is created on the CCD sensor. This image is superimposed on the fluorescence image of the specimen giving a full control of the selection of the specimen area under investigation on a video screen.

The separation of the two emission wavelengths can be achieved either by using a 50/50 beam splitter or a dichroic mirror in beam splitter position 2.

In the case of using a dichroic beam splitter in position 2 as the wavelength separating element, a filter is necessary to block the rest of the excitation light. It can be placed in the filter cube of the microscope or in filter positions A and/or B of the detector unit.

In the second case, a 50/50 beam splitter is placed in position 2. Since beam splitter 2 then transmits/reflects the full wavelength spectrum emerging from the specimen, corresponding filters are necessary in positions A and B. The transmission characteristics of these filters are determined by the dyes and cells used in the experiment.