Before using the spinning wheel system with fluorescent dyes, and in the event of any subsequent problems, we would strongly recommend carrying out the following test and optimisation procedure. If the system is connected to a microscope then please ensure that a suitable dichroic mirror is in the light path to divert incident light throught the objective.
- With the optical adapter shutter closed, switch on the high intensity light source.
- Connect a BNC lead from SIG output of the photomultiplier to the input of the amplifier module and the output of the amplifier to an oscilloscope.
- Connect a BNC lead from the OUT(put) of the main system front panel to the external trigger input of the oscilloscope. Ensure that the oscilloscope is set to trigger to a positive going signal from this source (this OUT(put) should produce a brief 5 volt pulse once per revolution when the rotor is spinning).
- Set the amplifier gain to maximum.
- Set the oscilloscope time base to 10ms and the deflection to 1V per divison.
- Connect the HT lead (BNC lead with large stainless steel plugs) between the HV socket of the photomultiplier housing and the out(put) of the photomultiplier power supply, ensuring that the voltage control is fully anticlockwise.
- With the rotor spinning at 32Hz, open the optical adapter shutter to allow incident light into the system.
- Roughly focus the microscope onto a suitable fluorescence source such as a piece of white paper. If using uv filters (<400nm) remember to carry out all tests with a uv transmitting objective. In cuvette systems signal levels should be sufficiently high to use the appropriate fluorescent probe instead of paper.
- When bright, in focus, fluorescence light is clearly visible divert the light path to the photomultiplier through the fitted emission filter.
- Gradually increase the voltage of the PMT power supply until signals appear on the oscilloscope similar to those below. The relative sizes of the six peaks will depend on the properties of the specific filters and the type of paper. As a rule of thumb with fura 2 filters the 340nm peaks should be roughly a third as high as the 380nm ones.
- Vary the PMT supply voltage and the amplifier module gain and ensure that both of these actions have an appropriate effect on the signal levels.
- If the system has an output module fitted then connect the outputs 1 - 6 in turn to the second oscilloscope display channel. The signal level from each of the 6 ouputs should correspond to the peak height of the corresponding filter position as shown below.
- The light throughput can now be optimised by making small adjustments to the following, maximising signal levels in each case:
- The focussing of the microscope.
- The three focussing screws on the lamp house.
- The position of each end of the liquid light guide.
- The position of the end of the microscope coupling tube in direct coupled systems.
- It is worth spending some time ensuring that the signals are as strong as possible. Once this has been established the spectrophotometer should be ready for use on real cells.
Diagram shows typical curves for a system with 380nm filters in positions 1 and 2, blanks in filter positions 3 and 6, and 340nm filters in positions 4 and 5.
N.B. If the PMT voltage has to be increased beyond 650 before strong signals appear then there is probably something wrong, so please check the optical system carefully, feel free to consult with us. If you are sure that the optical set-up is fine then please check the electronics directly as descibed below.
