Fluorolog-3 Spectrophotometer User Guide

Figure 1: The Fluorolog-3 is an autocalibrating spectrofluorometer. The system locates the home
position of each monochromator drive highlighted in yellow and assigns a wavelength value from a
calibration file. Scans of the xenon lamp output and the Raman scatter band of water are sufficient to
verify system calibration, repeatability, and throughput. Check the calibration prior to each day’s
session with the instrument.

1. Turn on the system

Check to make sure the electronics and computer are off.
On the back of the lamp housing turn
on the POWER and wait for the fan to
turn on.
Just above the POWER switch turn on
the MAIN LAMP. It is suggested to
let the lamp stabilize for twenty
minutes before checking and
calibrating the excitation
monochromater.
Turn on the SpectrAcq and wait for it
to boot, and then turn on the Dell
computer

Open FluorEssence software on the

desktop

  1  
FluorEssence™ Interface
There are six buttons on the main FluorEssence window for setting up, running, and analyzing
FluorEssence Experiments. From left to right, these buttons are:

Experimental Menu Button: Sets up the experimental parameters to run a particular kind
of scan. The last-saved experiment is automatically loaded. If there is no previously saved
experiment, the Fluorescent Main Experiment Menu is automatically activated.

Previous Experiment Button: Modifies slightly a previous set-up experiment and runs it.
If there is no previously saved experiment, the Fluorescent Main Experiment Menu is
automatically activated.

Auto Run Previous Experiment Button: Runs an experiment according to the

parameters set up. The last-saved experiment is automatically loaded.

Make Overlay File button: With the existing data and the last experiment run, creates a
file readable by other software. If no previous data, this button is greyed out.

3D Scan to 3D Profile button: With the existing excitation-emission matrix data displayed,
shows the excitation and emission profiles. Only operates if an excitation-emission matrix is
open.

Get Peak Information Button: Fits and provides information on a chosen peak in a
spectrum. Only operates if a spectrum with a peak is open.

2. Excitation monochromator calibration check: the Xenon Lamp Scan

NOTE: Running a scan of the lamp checks the calibration of the excitation monochromator using a
reference diode found before the sample compartment. Close the lid of the sample compartment. No
cuvette or sample is needed. This is an excitation scan of the 450 W xenon lamp’s output and
should be the first check performed every day.

  2  
In the FluorEssence Main Experiment Menu,
click the Experiment Menu Button
shown above.

The Fluorescent Main Experiment

Menu appears. Click Spectra.

  3  
The Experiment Type window
appears. Click Excitation and Next.

Check the parameters of the DfltSpectralExcitation file.

Excitation 1
Start: 200 nm
End: 600 nm
Inc: 1
Slit: 1 nm
Emission 1
Wavelength Park: 350 nm
Slit Park: 1nm

  4  
Confirm that the R1 Detector is checked for the lamp scan in “Detectors,” then click “Run”

The Intermediate Display opens, and automatically runs a xenon lamp scan, which will look similar to the
one below. The peak of interest is the calibration peak for the xenon-lamp spectrum at 467 nm. To
quantify the location of this peak, “Pause” the scan and select the “Cursor” button and drag it to the
peak of interest.

Pause the scan

Select the Cursor Button
Drag over calibration peak
Record wavelength for
Pause   calibration step

  5  
Below is a calibrated Xenon lamp scan. Note the location and appearance of the 467 nm peak in the
entirety of the spectrum for the Xenon lamp. The 467 nm calibration peak is indicated with a blue
arrow. The 467 nm peak is not the highest peak in the spectrum. There are two peaks about
the same size next to one another on the spectrum, and the 467 nm calibration line is the peak on the
left. See below.

Below, the “sample scan” reveals that the 467calibration peak revealed by the
“Cursor” is at 463, 0.0153 so the excitation peak needs to be calibrated to 467.

  6  
3. If instrument was not within 467 nm peak specification, recalibrate
the instrument
In the FluorEssence Main Experiment Menu, click the Experiment Menu Button

The Fluorescent Main Experiment

Menu appears. Click Spectra.

The Experiment Type window

appears. Click Excitation and Next.

  7  
Click on Real Time Control (RTC)

The Real Time Control Window opens.

  8  
 1. Click Monos and make sure that Excitation 1 is highlighted
2. Type in the Observed/Current Position peak. In the previous uncalibrated xenon light scan used
as an example, the 467 nm peak appeared at 463 nm, so for that example, we would type “463”
as the observed/Current Position peak in the position control window
3. Click Calibrate Excitation 1

1  

  9  
Type in the peak of interest, which for the lamp scan is always 467 nm, then click “OK” and close the
window. The calibration will be saved, and can run a scan to confirm that your calibration is accurate.

  10  
4. Emission Calibration Check NOTE: The emission calibration of the instrument is directly
affected by the calibration of the excitation monochromator and should be performed AFTER the xenon-
lamp scan. When complete, the performance of the system has been verified.
The emission calibration check is an emission scan of the Raman-scatter band of water performed in
right-angle mode. Select a cuvette from the drawer below, and fill with research quality deionized water.

Load cuvette of dionized water into sample compartment as shown above and ensure that the lid is
secure over the top.
Select the Experiment Menu Button and Spectra, exactly the same as the Excitation Calibration
Check.

  11  
Now, instead of Excitation, click Emission and Next. Notice our recently calibrated excitation spectra
of the xenon lamp just behind the Experiment Type Window. We calibrated that first.

  12  
Check the emission spectrum parameters. They should be as follows:
Excitation1
Initial wavelength 350 nm
Slit 5 nm
Emission 1
Start 365 nm
End 450 nm
Inc 1
Slit 5 nm
Confirm that the Emission Detector S1 is checked rather than R1.

  13  
Check the emission calibration peak of water, which should be 397 nm. In the example peak below,
the peak is at 391.9 so emission peak needs to be recalibrated to 397 nm.

Recalibrate the emission in Real Time Control

NOTE: Make sure that Emission 1 is highlighted. If Excitation 1 is highlighted, you will undo the work
from your xenon lamp scan. Highlight Emission I, fill in the observed/current peak in Position
control, click Calibrate Emission, type 397 nm for the water Raman peak, click OK, then close the
window. The calibration will be saved, but confirm by running a scan.

  14  
Calibrated emission scan of water Raman peak. The system is now ready to use.

System Shutdown:
Turn off the SpectraAQ
Turn off the Computer
Turn off the MAIN LAMP
Continue to let the system run for 10-15 minutes
before turning off the POWER so that the fan
cools the lamp. Neglecting to do this can shorten
the life of the lamp.

  15  
Note: The dye used at LOCI to determine system sensitivity or as standards
for lifetime measurements is coumarin 6. It is primarily used as a lifetime
reference sample, but could be used as spectral calibrant. Fluorescein is a
better known fluorescence standard (above), and might be a better choice for

  16  
this application. It has a narrow emission profile, a known intensity, and is
relatively inexpensive.

Follow the steps to the Emission Calibration Check starting on page 11, but
use the Excitation and Emission parameters specific to your sample (i.e.
Experiment Type: Emission. For example, for Fluorescein, use an initial
excitation wavelength of 470-480 nm and emission 520 nm.

  17