Fluorolog-3
Fluorometer
Staff Contact: Emory Chan (emchan@lbl.gov)
Instrument Location: 67-4203A
Safety Points
You must be authorized to work under two WPC activities MF-0001 & MF-0007 to operate this instrument. Those operating lasers must also be authorized under activity MF-0006.
Your staff contact must approve the materials you will be measuring.
NO sample prep is allowed in lab 4203A. All samples must be prepared in a fume hood with solvents sealed in capped cuvettes prior to use in this lab space. (See your staff contact if you need a dedicated work space.)
Laser use requires specialized training. No one is allowed to touch the laser set-up, even if just to change the configuration, without authorization.
The video provided here offers guidance on using the instrument, but you must still coordinate with the staff member listed above to schedule a formal training.
Instrument Options
UV-Vis (CCD) Range: 290nm – 850nm (exit = Axial)
UV/Vis (PMT) Range: 290nm – 850nm (exit = Lateral)
NIR Detector Range: option 1 = 850-1600nm (maroon detector)
option 2 = 900-2000nm (blue detector)
Lamp/Grating Excitation: 200nm – 640nm
Laser suite and MicroMax plate reader available
3D measurements (both emission and excitation) possible
Quantum yield using integrating sphere also possible (UV-Vis region only)
Calibration
Excitation (using lamp): 467nm
Emission (using pure water): 397nm
Standard Operating Procedure
This video offers an introduction to the operation of the Fluorolog-3 to collect fluorescence emission data in the Inorganic Facility of the Molecular Foundry. It walks you through the configuration of our instrument, discusses which detectors and sources of excitation are appropriate for the emission you are looking for, and explains the basics behind using the FluoroEssence software package version 3.5. Please note that this video only covers the basics of operating the software for excitation and emission (in both the NIR and UV-Vis regions), and does not include the install or use of specialized attachments including the laser set-up, well-plate reader, or integrating sphere.
Common Questions
Q: The calibration of the lamp was spot on, but I can't see the Raman signal of water when calibrating the emission gratings. What can I do?
A: This is the most common question we hear with this instrument. Here are the things you should try, in order:
Ensure that the beam-block is not obstructing the light path (the most common problem).
Confirm that you are using purified water and NOT deionized or tap water, which will have too much noise in the background.
Extend the data collection range and see if you can find the peak. There should a very intense peak from the lamp's excitation at 350nm and a peak from water at 397nm (meaning you'll see a high intensity peak and then a lower intensity peak that is 47nm higher... pay attention to this distance so you do not calibrate to the wrong peak).
If you still can't determine which peak is the Raman peak, contact the first staff contact on the list above for help. There may be something wrong with the gratings.
Q: Why can't I collect quantum yield data using the NIR detector?
A: The integrating sphere used for this measurement is configured in such a way that we can't install it with the sphere openings in the correct direction to allow the light path to reach the NIR side of the instrument. It's designed specifically for use with the UV-Vis emission side.
Q: I'm having trouble with signal when using the integrating sphere. Do you have some tips?
A: Before the integrating sphere is installed, be sure to check all of your calibration points. You will also want to run your sample to ensure that you know exactly which region your emission peak is in. Install the sphere and measure your sample. If the signal is over 2 million counts, check that you remembered to reduce your slit width to 0.1mm. If you are still over 2 million counts, you need to dilute the sample. The integrating sphere does a great job of boosting the signal, but too many counts will saturate your detector, which is both bad for the detector and means that you results will not be reliable.
Q: Someone else is on the instrument, but my name is on the calendar reservation tool. What should I do?
A: Everyone hears the same speech when we give our training. If your name is on the calendar then you have the right to use the tool. If you do not feel comfortable reminding the user of this then notify any staff member and we can give you an assist. We are used to politely enforcing the rules! :)
