Spectrofluorometers
Buy a Spectrofluorometer | Choose the Right System | Measurements | Spectrofluorometer Components | Resources | Technical Support
Spectrofluorometers are measurement instruments capable of taking both fluorescence, or photoluminescence, measurements and absorbance measurements, with high levels of accuracy and repeatability. Systems like the Ossila Spectrofluorometer contain a sample holder, multiple light sources, and multiple single pixel detectors. To measure both absorbance and fluorescence as accurately as possible, spectrofluorometers use wavelength selection components such as monochromators.
Separate excitation and emission monochromators are used to select wavelengths of light individually either before or after passing through a sample. This allows for you to do many different spectroscopy measurements with one instrument, achieving a high level of accuracy and resolution.
Spectrofluorometer and Spectrophotometer Equipment
Choose The Right Spectroscopy System
The terms spectrometer, a spectrophotometer, or spectrofluorometer are often used interchangeably, and every system will have different designs and specifications.
As with most measurement equipment, this comes down to your experimental needs, lab space and budget. Simple spectrometers such as the Ossila USB spectrometer can be incorporated into modular optical systems, and are a cost-effective way to take simple optical spectroscopy measurements. However, they lack the resolution and accuracy of most spectrofluorometers.
Spectrofluorometers are versatile pieces of equipment. They are more costly than modular spectrometers but provide higher resolution, accuracy and consistency in all measurements.
Reliable and Calibrated Optical MeasurementsNon-invasive Molecular ProbingAll-In-One SystemWide Range of MeasurementsNo Complex Optical AlignmentMeasure Dynamic Reactions in Real TimeSpectrofluorometers vs. Spectrophotometers
Spectrofluorometers are similar to spectrophotometers, and often the terms are used interchangeably. They use the same components, and they build a spectrum in the same way. However, there are a few key differences between spectrofluorometers and spectrophotometers.
| Spectrofluorometers | Spectrophotometers | |
|---|---|---|
| Number of Monochromators | 2 | 1 |
| Number of Detectors | 2 | 1 |
| Broadband Light Source |  |
|
| UV Light Source | |
 |
| Suitable for Absorbance Measurements? | |
|
| Suitable for Fluorescence Measurements? |
Emission and excitation spectra |
|
Spectrofluorometer Measurements
Spectrofluorometers can perform a variety of measurements:
- Fluorescence Spectroscopy
- Emission Spectra: If the excitation wavelength is fixed and the emission wavelength is varied, spectrofluorometers can measure the emission spectrum of a fluorophore. These measurements tell you about the light emitted from a fluorophore.
- Excitation Spectra: Where the measured emission wavelength remains the same (ideally where emission is strongest) but the excitation wavelength is varied. These measurements will tell you which wavelengths of light stimulate the most fluorescence.
- Absorbance Spectroscopy
- Spectrofluorometers can also measure absorbance, much like a spectrophotometer. This method takes longer than absorbance measurements taken with a USB spectrometer but with higher sensitivity and resolution as you can measure transmission intensity 1 nm at a time.
Spectrofluorometers are most commonly used in fluorescence spectroscopy. This is the study of fluorophores, either for use in fluorescent applications such as in LEDs, or as a marker to study biological processes or chemical reactions.
Fluorescence spectroscopy analyzes light emitted from a sample due to an excited electron relaxing from the first electronic state into the ground state. There are two types of fluorescence measurement: steady state fluorescence and time resolved fluorescence. Time resolved PL measurements are used to measure dynamic processes like fluorescence lifetime. Steady state fluorescence can be used to probe the molecular structure of a material and is measured using a spectrofluorometer.
Fluorescence spectroscopy is often used alongside absorbance spectroscopy, as absorbance measures the excited state of a molecule whereas fluorescence measurements probe the ground state.
The Ossila Spectrofluorometer works by combining high quality components in various configurations, with the ability to select both the excitation and detection wavelength, enabling different measurements.
For fluorescence measurements, a single wavelength light source is directed towards your sample to excite any fluorophores. This can use the UV light source or combine the broadband light source and excitation monochromator to create a variable light source. Which source is best depends on the measurement you are taking. Any emitted light from the sample enters the detection monochromator. This monochromator either selects a specific emission wavelength, or scans through various wavelengths before intensity is measured by the detector.
For absorbance measurements, the broadband light source travels through the excitation monochromator, which again selects a specific wavelength or scans through a wavelength range. This monochromatic light is directed straight through your sample, where transmission is measured by a separate detector
Which Configuration Do I Need?
To efficiently use a spectrofluorometer, ensure that you are using the correct combination of components.
| Excitation Fluorescence Measurements | Emission Fluorescence Measurements | Absorbance/Transmission Measurements | |
|---|---|---|---|
| Light Source Required | Broadband light source | Single wavelength light source | Broadband light source |
|
Number of Monochromators |
Two required:
|
One required, between the sample and detector | One required, between the light source and sample |
Spectrofluorometer Components
Spectrofluorometers contain the following components:
- Excitation sources (usually both a broadband light source and a UV only source)
- A sample holder
- A detector or multiple detectors
- Two Monochromators
The arrangement of these various components will depend on spectrofluorometer design and alignment.
Excitation Sources
Emission fluorescence spectroscopy requires an high energy excitation source with a narrow wavelength range to selectively excite fluorophores. For this, you will likely need a narrowband light emitter, such as a UV light source. However, using single wavelength sources alone limits the type of measurements you can do.
Absorbance measurements and excitation fluorescence measurements require a broadband light source. To conduct excitation fluorescence measurements, you can combine a broadband light source with a monochromator to select specific wavelengths for sample excitation. This monochromator will also increase the accuracy of absorbance measurements, by assessing transmission and absorbance of each wavelength individually. However, for fluorescence measurements, variable light sources may produce a less intense excitation signal, leading to a lower signal.
Spectrofluorometers, such as the Ossila Spectrofluorometer, usually have both an high energy UV light source and a variable light source option, allowing users to conduct a wider range of measurements with just one instrument.
Sample Holder
The sample holder is a straight-forward but vital part of the system. It ensures consistency between measurements and holds the sample steady. There are sample holders designed to measure substrates for thin film measurement. Additionally, many samples measure fluorescence within solution, usually in a quartz cuvette. Using ultra-flat quartz cuvettes or substrates helps to reduce reflection, scattering and absorbance of light. This ensures that the maximum possible light is transmitted through the holder without interaction.
Detectors
Spectrofluorometers use single pixel detectors, unlike spectrometers which use array detectors. Single pixel detectors can only measure intensity and have no way to decipher between different wavelengths of light. This makes spectrofluorometers more sensitive to smaller signals, but means that wavelength selection must take place before or after interacting with the sample.
For fluorescence measurements, detectors are combined with a monochromator. This means that the detector only measures light emitted at specific wavelengths, omitting the strong excitation wavelength. To produce a fluorescence spectra, a sample can be stimulated by a high energy light source, then scanning monochromators cycle through the different wavelengths as the detector measures the delivered intensity. This way the system builds a fluorescence spectrum piece by piece.
For absorbance measurements, the light source must be able to pass through the sample directly into the detector. Therefore, for maximum versatility, some spectrofluorometers come with multiple detectors in different locations to facilitate both absorbance and fluorescence measurements.
Often photomultipliers are used as these detectors. The type of photomultipliers used will determine the spectral range and sensitivity of the spectrofluorometer.
Resources
Ossila Spectrofluorometer Comparison
We’ve compared our specifications with one of the leading competitors to demonstrate how the Ossila Spectrofluorometers compares.
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Measuring Fluorescence with a Spectrofluorometer
Learn how to take fluorescence emission and excitation measurements with the Ossila Spectrofluorometer.
Read more... Fluorescence Spectroscopy
Fluorescence spectroscopy is used to measure fluorescence. The technique often used together with absorbance spectroscopy. Fluorescence is a type of photoluminescence where light is quickly reemitted from a material after incident photons are absorbed. This is different to phosphorescence where there is a delay between photon absorption and emission. The term fluorescence is often used interchangeably with photoluminescence.
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Choosing Spectroscopy Instruments
UV-Vis spectroscopy (or optical spectroscopy) is a simple but effective technique used to probe the molecular structure of a material. Measuring absorbance and emission of light from a sample can give you vital data about:
Read more...Technical Support