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Home > News & Events > Fluorescence Spectroscopy

Fluorescence Spectroscopy

Modular Tools for Fluorescence Measurements

Fluorescence is the absorption and emission of light of two different frequencies, or wavelengths. This is typically seen in experimental setups when a lower wavelength of incident light is absorbed from one direction, and a higher wavelength of light is emitted in all directions. This is most striking when a sample absorbs UV light and emits visible light.

A sample molecule may be excited electronically and vibrationally by an incoming photon, relax to a lower vibration state by heating the sample around it, and return to the electronic ground state by emitting a lower energy (higher wavelength) photon than the absorbed one.

Fluorescence is used to investigate a number of samples, as fluorescent molecules will absorb a certain wavelength and emit another. With a known incident light wavelength, a sample may be identified by its fluorescent emission spectrum. As fluorescence occurs on a molecular scale, it is the only spectroscopic technique capable of identifying single molecules.

Making Fluorescence Measurements

A fluorophore’s excitation spectrum shows how efficiently fluorescence will be generated as a function of excitation wavelength. The excitation and emission spectra for a fluorophore often overlap, with emission at longer wavelengths (Figure 1).

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

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

Modular spectrometers, excitation sources and accessories allow users to optimize systems for a variety of parameters, and to easily switch between fluorescence and absorbance measurements. In addition, spectrometers such as the QE Pro and Flame have interchangeable slits to accommodate different measurement needs.

Spectrometers

Figure 2: Spectrometers that use a high sensitivity, back-thinned CCD detector are excellent options for fluorescence measurements.

Figure 2: Spectrometers that use a high sensitivity, back-thinned CCD detector are excellent options for fluorescence measurements.

There is a robust marketplace for fluorescence spectrometers, with pricing and flexibility depending on instrument sensitivity and configuration. Ocean Optics offers various spectrometers suitable for fluorescence, with back-thinned CCD array detector spectrometers like the Maya2000 Pro and QE Pro-FL (Figure 2) at the top of the list.

Excitation Sources

There are several possible approaches when choosing an excitation light source for a fluorophore. If you’re using an LED, it is best to choose one with a center wavelength close to the peak excitation spectrum wavelength. If you’re 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 you’re using a broadband light source for excitation, the light can be filtered using a single bandpass filter, taking care to ensure that the excitation light minimizes overlap with the emission spectrum.

Although the excitation spectrum for a given fluorophore is not exactly 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.

Filtering Options

Bandpass optical filters are the simplest way to narrow the excitation light. These colored glass or interference filters offer both high and narrow transmission ranges. Narrowband dichroic filters matched to almost any fluorophore are also available.

Linear Variable Filters Collection

Figure 3. Linear variable filters have an adjustable center wavelength and bandpass.

If the optimum excitation wavelength is not known, or if you’re using a wide range of fluorophores, we recommend a linear variable filter pair like our LVF-HL. Linear variable filters give users the flexibility to adjust both the center wavelength and the bandwidth of excitation. The bandwidth can be set as narrow as 20 nm or as wide as 100 nm. With a pair of these filters, you can position the filters horizontally to vary from center wavelength by several hundred nanometers. Also, you can use the filters as separate variable longpass and shortpass filters (Figure 3).

 

Sampling Accessories

A standard fluorescence system collects fluorescence at 90° to the incident light beam to minimize interference from transmitted and scattered light. This improves signal to noise, and lowers the detection limit by up to a million times as compared to a straight-through, 180° transmission geometry.

The great thing about a modular fluorescence system is that it’s quick and easy to change how samples are viewed with a single excitation source and detector. An optically dense solution can be measured at 0°, 90° and 180° within minutes just by changing optical fiber routings. Alternatively, a probe can be used for immersion into a liquid, or a variable bandpass optical filter can filter the lamp to optimize excitation efficiency. Let’s take a look at the options.

CUV-ALL-UV with fibers

Figure 4. A popular choice for fluorescence measurement of solutions is this cuvette holder with collimating lenses for straight-through or 90° degree measurements.

The CUV-ALL-UV cuvette holder is great for looking at solutions in transmission and at 90° (Figure 4). Add 74-MSP screw plugs to enhance sensitivity in the sample compartment. For nanomolar-range fluorescence analysis use FluoroVettes, which are UV-transparent, disposable cuvettes that hold just 50 μL of solution.

The CUV-FL-DA attaches to Ocean Optics light sources and maximizes excitation light. It acts like a 4-way cuvette holder, but uses free-space optics for excitation lamp routing instead of an excitation fiber.

A reflection probe is a good option for measuring dense liquids, solids and powders. Choosing a probe with an angled tip allows the fluorescence measurement to happen right at the interface, and reduces backscatter. Just remember to filter the excitation wavelengths out of the signal, since scatter will still be high.

Use a fluorescence flow cell to measure fluorescence in flowing solutions, typically to monitor a process or look at bulk samples.

Software Considerations

OceanView is our flagship desktop spectroscopy application. When using OceanView for fluorescence measurements, an important consideration is the distinction between QuickView Mode and Relative Irradiance Mode.

QuickView mode data shows the raw number of counts for each pixel in the array without any processing or correction for spectrometer sensitivity. This is important to remember, because each spectrometer has a different response function that comes from a combination of its individual elements and alignment. That can make QuickView mode misleading, showing a peak in the correct location, but with a distorted shape and/or center wavelength.

QuickView is great for fast measurements, but can give fluorescence spectra with emission maxima and peak ratios that do not match those reported in the literature. Data that will be published or shared in other formats should be acquired in a processed mode like Relative or Absolute Irradiance to ensure accurate peak shapes.

In Relative Irradiance mode, instrument response can be corrected in QuickView mode by calibrating against a blackbody light source of known color temperature. A tungsten halogen lamp is a convenient standard, and works well for visible and NIR wavelengths. A relative irradiance measurement generates a corrected spectrum of relative intensity as a function of wavelength, scaled from 0 to 1 in arbitrary units.

Is relative irradiance mode always necessary for fluorescence measurements? No. Measurements taken with a single spectrometer are accurate relative to one another, even if the spectral shape is uncorrected. That means you can take the ratio of one fluorescence measurement to another and get an accurate change in the percent signal as a function of wavelength.

Relative irradiance is important when comparing measurements taken by different spectrometers, when determining the spectral shape, or when looking for peak location and shifts.

Summary

Traditional fluorescence systems were often inflexible, unwieldy and limited to lab settings. Today, modular spectroscopy makes possible dozens of different fluorescence applications by giving users the flexibility to combine optical bench components and accessories in various configurations.

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