Susan J. Kohler

November 14, 1997

NOTE: These are general instructions to get you started in using the fluorimeter. There are manuals in the lab which describe many other features of the system, and more sophisticated experiments. You are encouraged to use the manuals or see me if you need additional help.

1) First turn on the fluorimeter electronics and start the lamp. This is done as follows:

• Turn on the power strip on the table behind the fluorimeter.
• Turn on the lamp power supply (tan box on the table to the left of the fluorimeter) with its on/off switch on the front.
• Push the START button on the lamp power supply to turn on the lamp. You will hear the fan start, and after a few seconds will see light coming out of the tube on top of the lamp. DO NOT LOOK INTO THE TUBE! The lamp emits ultraviolet light harmful to your eyes!!

1. After the fluorimeter lamp is turned on it is safe to turn on the computers. There are two of them. First turn on the slave computer on the table behind the fluorimeter. The computer has a big SPECTRACQ label. The on/off button is on the right side of the front panel under the blue label. Turn it on and wait for it to finish booting. The green light under the floppy drive will blink and then stay on for a while. When it is done, the little green light by the floppy drive will blink again, then go out and stay out. Next turn on the host PC. You must turn on the monitor and the computer itself. The computer is under the table. Wait until Windows 95 starts up, and then you are ready to start.

The software is started by double clicking with the left mouse button on the Instrument Control Center icon. You will be asked if you want to initialize the instrument with the default layout. Answer YES. You will then be asked to enter the monochromator calibration values. Read the number on the front of the monochromator and enter the value in the computer window. You should read the value to the tenth of a nanometer. Note that shorter wavelengths are above the center line on the dial. You have to do this for first the excitation monochromator (on the left) and then the emission monochromator (on the right). Finally, say YES when asked if you want to bring the hardware to the last position, and make sure that the monochromator readings on the dials make sense.

There are three basic types of experiments to describe:

• Record an emission spectrum
• Record an excitation spectrum
• Measure at a constant wavelength

To start an emission spectrum or excitation spectrum, click on the spectrum icon (the leftmost icon in the Instrument Control window) or use the Applications menu at the top and choose Experiment. A new window labeled DataMax will pop up. Choose the Collect menu option at the top, and then choose Experiment. You will get a template which lets you specify the experimental details. On the right side of this template is a button labeled Exp Type which lets you choose the basic type, i.e. emission or excitation. Choose the appropriate type to get the appropriate template. Or, if you have a pre-defined set of conditions, click on the Experiment… button and load the proper file. Fill in the template as necessary specifying starting and stopping wavelengths for your experiment, and a name for the data file to store your data. When you are done, click the Run button and the experiment should start. You can print the results using the Print command under the File menu. Then choose Collect to collect another spectrum or choose Exit in the File menu to close the DataMax window.

To monitor at constant wavelength, you should first run a quick emission spectrum. For some reason, this is necessary as a first step to get the instrument started. Then close the DataMax window using Exit in the File menu on the DataMax window. Then click on the CWA icon (Constant Wavelength Analysis - farthest to the right) in the Instrument Control window or choose constant wavelength analysis from the Applications menu. You will get a template to fill in with information about the excitation and emission wavelengths. You must click the "Add" button to transfer the numbers you specify into the table at the right side of the template. Then choose S(ample) for Data Channel and S for Acquisition Mode, and click on the Add button to transfer the choice to the Current Acquisition Mode window. Click the Go! Button, then click Start Acquisition. In the next little window choose <none> for Concentration Reference. Then you will get a window labeled New Sample. Click unknown for sample type and fill in a Sample ID. Finally click Run Sample, and a measurement will be made. When you are done, click Cancel in the New Sample window, then Done in the WaveSet window. Finally choose Exit under the File menu in the Constant Wavelength Analysis window.

First close the software by choosing Exit from the main File menu. Then turn off the host computer by clicking the Start button in the lower left corner with the left mouse button and then choosing shutdown. When prompted, turn on the computer and then the monitor. Then turn off the slave computer by pushing its on/off switch. AFTER BOTH COMPUTERS ARE OFF you can turn off the lamp by pushing the STOP button on the lamp power supply. After the lamp has cooled down (about half an hour) you may then turn off the lamp power supply and the power strip for the electronics.

There are four slits which control the light path on the fluorimeter. They are under your control, not the computer’s. If the slits are closed, you will not get enough light to the photomultiplier tube detector and your spectrum will be noisy. If the slits are too far open, you will lose resolution. Make sure the slits are right for your experiment! You might start with all the slits at 0.6 mm.