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Identifying Unknown Fluorophores

Fluorescent tags have become a powerful tool in research and diagnostics, capable of revealing cellular mechanics in real time, identifying abnormal cells, and sounding the alarm on the presence of pathogens. Still, they are only as powerful as the system used for detection, a key part of which is intelligent selection of excitation wavelength(s). Many fluorescence experiments are performed with a commercial fluorophore like TRITC, Fura-2, or one of the many variants of green fluorescent protein (GFP), in which case determining the best excitation wavelength (or band of wavelengths) is simply a matter of looking up the excitation spectrum. Occasionally, however, it may be necessary to determine the best excitation wavelength or band for an unknown fluorophore through experimentation.

Figure 1 -- emission-excitation graph

Figure 1. Emission and excitation spectra for a fluorophore often overlap.

In this application note and the associated experiment, we’ll show you how easy it is to use Ocean Optics modular products to switch between absorbance and fluorescence to fully characterize a new or unknown fluorophore. When used in the student lab as a teaching experiment, this procedure can help students to become familiar with the principles of fluorescence as they explore the effect of center wavelength and bandwidth of the excitation source on the strength of the emitted fluorescence.

 

 

Background

 

A fluorophore’s excitation spectrum shows how efficiently fluorescence will be generated, given as a function of excitation wavelength. The excitation and emission spectra for a fluorophore often overlap (Figure 1), with emission at longer wavelengths. There are several approaches that can be taken when choosing an excitation light source for a fluorophore. If using an LED, it is best to choose one with a center wavelength close to the peak excitation spectrum wavelength. If using a laser, the excitation intensity will be so much higher that it is possible to even use a wavelength on the tail of the excitation curve. If using a broadband light source for excitation, the source can be filtered using a single bandpass filter, taking care to ensure that the excitation light minimizes overlap with the emission spectrum.

 

fluorescent bill

 

Although the excitation spectrum for a given fluorophore is not quite the same as its absorbance, the absorbance spectrum of a fluorophore can be used as a quick indicator of which wavelengths are likely to be good for excitation. (When comparing an absorbance and excitation spectrum, most of the peaks will stay the same, though their relative heights may differ.)

 

Equipment

 

The modular nature of Ocean Optics spectroscopy systems is ideal for fluorescence measurements for a number of reasons. It is very easy to switch between absorbance and fluorescence measurements, simply by the rerouting of fibers to the cuvette holder. This is facilitated by the user-configurable interchangeable slits available with spectrometers like the QE Pro and Flame series, allowing the user to quickly increase or decrease the amount of light delivered to the spectrometer by changing the spectrometer slit size.

 

 

Also, a modular system allows the type of light source used for excitation to be easily changed from laser to LED to lamp, as all are conveniently fiber-coupled. In-line filtering can be added to further limit or tune the excitation wavelengths. Our LVF filter pair is a much more dynamic and versatile option than traditional bandpass filters for excitation light filtering, as its passband can be readjusted to work for a wide range of fluorophores, or to optimize fluorescence output in a given experiment.

 

In order to determine the best excitation wavelength(s) for an unknown fluorophore, we recommend a system based on a spectrometer with interchangeable slits like the QE Pro-ABS spectrometer (a Flame series spectrometer could also be used).

 

A CUV-ALL-UV four-way cuvette holder works very well for this experiment, with the input and output fibers attached opposite one another for absorbance measurements, and at right angles for fluorescence measurements. This cuvette holder allows for the insertion of a discrete filter or linear variable filter pair to further narrow the output of the excitation source, whether it be a tungsten halogen lamp (HL-2000) or LED light source (LLS-365, LLS-470). Fibers are used to route the light between light source, cuvette holder and spectrometer.

 

Dual Absorbance/Fluorescence System

Spectrometer:

QE Pro-ABS high-sensitivity spectrometer (200-950 nm)

Light source/excitation source: HL-2000 tungsten halogen source for illumination;  LLS-365 and LLS-470 LEDs for excitation
Optical fibers/probe: QP600-2-VIS-NIR 600 µm patch cord, quantity of 2
Accessories: CUV-ALL-UV cuvette holder with 74-MSP screw plugs for increased signal (2 pcs.); INTSMA-200 200 µm replaceable slit; LVF-KIT filter kit
Software: OceanView spectroscopy software
Sample: Unknown fluorophore

 

Experiment

 

To determine the best excitation wavelengths for the unknown fluorophore, make an absorbance measurement in the cuvette holder with the QE Pro-ABS spectrometer and the tungsten halogen lamp oriented at opposite ports of the cuvette holder. The QE Pro-ABS spectrometer is configured with a 10 µm slit, which is ideal for absorbance measurements. The Absorbance Wizard in OceanView is a great tool that walks you through the steps needed to get into Absorbance mode.

 

absorbance wizard oceanview

 

Once you have collected the absorbance spectrum for your unknown fluorophore, it is easy to see which wavelengths absorb the most light, and therefore which wavelengths should generate the most fluorescence if used as excitation light. Quite often it is possible to match the main absorbance peak of the fluorophore to the center wavelength of one of the LEDs available from our LLS family of LED light sources.

 

Fluorescence QE65 Pro Setup (App Note)

 

You can now reconfigure the spectrometer system for fluorescence by replacing the broadband tungsten halogen lamp with the chosen LED, and by moving the collection fiber so that it is at 90° to the excitation light. The amount of fluorescence light to be collected by the spectrometer will be much lower than was available when making the absorbance measurements, so a more sensitive spectrometer is needed. This can easily be achieved by swapping out the standard 10 µm slit of the QE Pro-ABS with a 200 µm slit, thereby vastly increasing the amount of light coupled from the 600 µm fiber into the spectrometer.

 

Instructions for changing slits are available online, as is a video tutorial.

 

Another way to increase the amount of fluorescence generated and collected is to replace the collimating lenses on the unused ports of the cuvette holder with 74-MSP mirrored screw plugs. The mirrored screw plug opposite the LED will enable the excitation light to make a second pass through the sample, increasing the total light flux, while the mirrored screw plug opposite the collection fiber will serve to reflect more of the fluorescence to be reflected and captured by the collection fiber.

 

 

Once the system is configured for fluorescence measurements, the Fluorescence wizard in OceanView can be used to get into QuickView Fluorescence mode and measure the sample’s fluorescence. At this point, you will be able to see all the emission peaks, though the spectrum will not be corrected for the spectrometer’s response. To obtain a more accurately shaped fluorescence curve, you can employ the Relative Irradiance Wizard in OceanView, using the HL-2000 as your blackbody source.

 

Video Highlight: Basic Fluorescence Spectroscopy Setup using a Flame spectrometer.

 

If you don’t feel that the LED was the best match for your fluorophore’s excitation spectrum, you can also try using the tungsten halogen lamp as your excitation source, employing the LVF-KIT to create your own bandpass filter to precisely select the center wavelength and bandwidth of the excitation light applied to the sample.