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Home > Measurement Techniques > Fluorescence

Fluorescence

Fluorescence

Custom fluorescence system using an Ocean Optics spectrometer. The customer adapted the unit for underwater use.

First observed in 1560, fluorescence has evolved into a powerful technique that enables entire fields of cutting-edge science and medicine. The only spectroscopic technique capable of resolving single molecules, fluorescence has moved from the lab to applications limited only by imagination.

 

The field of fluorescence is as diverse as it is beautiful. The sample under study may be liquid or solid, irregularly shaped, or accessible only outside the lab. A well-configured modular spectroscopy system can easily be reconfigured to study a wide range of samples in both the lab and the field.

 

Although fluorescence is sophisticated, it doesn’t need to be that complex. The beauty of modular spectroscopy is that you can invent as you go, trying different configurations until you find something that works. The tips and tricks below can help you get started. Or give us a call. It may just turn out that our team of application engineers have seen your application before. It’s not unusual, even if your application is.

 

 

Advantages

  • Sensitive: Concentrations as low as picomoles and femtomoles can routinely be detected, with even lower concentrations possible.
  • Quantitative: Fluorescence signal is generally proportional to concentration. Fluorescence intensity responds to changes in concentration within picoseconds, making it well-suited for in-situ studies and monitoring fast processes.
  • Safe: Unlike many other techniques for studying biological samples, it is non-destructive to the sample, and has no hazardous byproducts.

 

Applications

Other Common Applications

  • Nature: analysis of gemstones, minerals, chlorophyll, and crude oil residues
  • Forensics: detection of fingerprints & blood; analysis of fibers and other materials
  • Phosphor thermometry: measurement of temperature using the lifetime or intensity ratios of fluorescence peaks
  • Fundamental studies: use of laser-induced fluorescence to study the electronic structure of molecules and their interactions; concentrations in combustion, plasma, and flow phenomena
  • Biology: detection of molecules, observation of cellular processes, and sorting cells
  • Medical diagnostics: analysis of tissue for presence of cancer, glucose sensors, DNA sequencing, cytometry, and gel electrophoresis

Application Notes:

Technical Note

What Is the Difference Between Quick View Mode and Relative Irradiance Mode?

Scope 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 scope mode misleading, showing a peak in the right general location, but with a distorted shape and/or center wavelength.

Fluorescence Technical Note

This can be corrected by calibrating against a blackbody light source of known color temperature and working in relative irradiance mode. 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.

Application Note:

Featured Products for Fluorescence:

Fluorescence

USB4000-FL Spectrometer is preconfigured for fluorescence measurements from 360 – 1100 nm.
The spectrometer comes with a 200 µm slit and detector collection lens for increased light throughput.
QE Pro-FL Highly sensitive spectrometer specifically suited for low light-level applications such as fluorescence.

What light source to select?

Fluorophores can be excited using a narrowband light source like an LED or laser, or with a filtered broadband lamp. Excitation efficiency will be proportional to the integrated product of the fluorophore’s excitation spectrum and the light source emission spectrum. That means that both the spectral overlap of the excitation source with the fluorophore and the source intensity are important to maximize signal.

Narrowband excitation sources: Ocean Optics LEDs range in wavelength from 240 nm to 750 nm, with 2 μW to 1 mW of output power over a 9 to 50 nm bandwidth. Deeper UV LEDs are available and the number of visible sources continues to grow each day. We also offer a selection of lasers in the visible and near-IR.

Broadband excitation sources: Xenon arc and mercury vapor lamps are often used for their UV and short wavelength output. Mercury lamps emit only at discrete wavelengths, so they need to match the specific fluorophore to be useful. The very strong mercury line at 254 nm is often benzene derivatives and other organic compounds. Xenon lamps are more popular, since their continuous spectrum can be narrowed with a monochromator or bandpass optical filter.

Other broadband excitation sources are also available. The HPX-2000 xenon lamp is the most generally useful due to its emission range of 185-2000 nm, but the D-2000 or DH-2000 deuterium lamp with UV output starting at 215 nm might be better for some applications where high intensity below 300 nm is needed. Tungsten lamps can be used as excitation sources, but don’t have much output below about 400 nm.

For applications where photobleaching is an issue, a pulsed xenon lamp like the PX-2 works really well. It can be set up for multiple pulses in a single integration window, allowing precise control of the amount of light delivered to the sample.

Optical filtering: Bandpass optical filters are the easiest way to narrow excitation light. These colored glass or interference filters offer high transmission and narrow transmission ranges. Narrowband dichroic filters matched to almost any fluorophore are widely available.

If the right excitation wavelength isn’t known, or if using a wide range of fluorophores, try a linear variable filter pair like the LVF-HL. It gives 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. Then by translating the filter pair horizontally, the center wavelength can be varied by several hundred nanometers. The filter pair can even be taken apart and used separately as variable longpass & shortpass filters.

What sampling optics do I need?

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 the liquid, or a variable bandpass optical filter can filter the lamp to optimize excitation efficiency. Let’s take a look at the options.

Four-way cuvette holder: great for looking at solutions in transmission and at 90°. It also comes in a temperature-regulated version, capable of control to within ±0.5°C. For nanomolar-range fluorescence analysis, use FluoroVettes, UV-transparent, disposable cuvettes that hold just 50 μL of solution.

Direct-attach cuvette holder: maximizes excitation light when used with a pulsed xenon lamp. It acts like a 4-way cuvette holder, but uses free-space optics for excitation lamp routing instead of an excitation fiber.

Reflection probe: ideal for measuring dense liquids, solids, and powders. Choosing a probe with an angled tip allows 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.

Fluorescence flow cell: good for looking at samples in flowing solution to monitor a process or look at bulk samples.

How do I detect data information?

Most fluorescence instrument systems are limited to one method of detection. A fluorescence system with a scanning monochromator measures fluorescence one wavelength at a time. It doesn’t work for dynamic processes, and risks photobleaching during the scan of the sample. In contrast, filter-based systems capture a band of wavelengths and permit time-resolved studies, but at the sacrifice of the high-density information contained in the full emission spectrum.

A linear array spectrometer captures the best of both worlds, scanning and fixed. First it disperses the fluorescence spectrum using a grating, and then it detects each wavelength using a separate pixel in a linear CCD array. Though CCD arrays are not as sensitive as PMTs, we have options to handle many fluorescence applications.

USB4000: When configured with a large slit, detector collection lens, and a broad grating, it is good for fluorescence that can be detected with the human eye.

Maya2000 Pro: If higher sensitivity is needed, this spectrometer’s back-thinned 2D fast Fourier transform CCD offers higher quantum efficiency over a broader spectral range.

Torus: With an aberration-corrected concave grating for low stray light and high throughput, it is well-suited to fluorescence from solid surfaces, powders, and dense liquids.

QE Pro: For best sensitivity and low stray light. It comes with interchangeable slits, so resolution can easily be changed, or it can be reconfigured for non-fluorescence measurements like transmission and reflectance.

MonoScan 2000: This scanning monochromator can be used with a PMT, photodioide, or other single-element detector, depending on the sensitivity and readout speed needed.

How do I determine excitation wavelength?

If the excitation wavelength isn’t known, an absroption measurement can determine where to excite the sample. The maximum absorbance peak is almost always the best place to excite the sample.

If the excitation has already been optimized with a linear variable filter pair (LVF), configure the fibers at 180° to one another in the 4-way cuvette holder to see the excitation spectrum. To avoid moving fibers, use a Teflon diffuser (CVD-DIFFUSE) in the cuvette holder to scatter excitation light by 90° to the detector. This is always a good idea, to make sure that the fluorescence spectrum isn’t just scatter from the source.

If the fluorescence spectrum seems to track with movement of the LVF, something’s wrong. Most fluorophores emit with a specific spectrum, and only the fluorescence intensity should change appreciably when the excitation wavelength is varied, not the shape of the fluorescence spectrum.

Finally, remember that no excitation lamp has equal intensity at all wavelengths, and fluorescence measurements can’t usually be directly compared. Tap off a reference beam to measure the excitation intensity as a function of wavelength and then correct the signal level of the fluorescence spectrum to get accurate relative fluorescence measurements.

How do I maximize my signal?

Photons are precious, so even small changes to your system can really boost signal. Here are our favorite tricks:

  • Use mirrored screw plugs (74-MSP) opposite the excitation and collection fibers in the 4-way cuvette holder. This forces excitation light to pass through the sample multiple times, and recaptures some of the fluorescence emitted away from the collection fiber. Mirrored side cuvettes are also available for low concentration samples.
  • Optimize the focus of the lenses for the excitation and collection fibers in the center of a liquid sample using the fluorescence signal in real time.
  • Use the largest fibers possible for excitation and collection. If one of them has a larger diameter, use it on the excitation side to get the most signal. Better yet, skip the excitation fiber entirely and use the direct attach cuvette holder (CUV-FL-DA) if possible.
  • Have silver mirrors (SAG+) installed in the spectrometer instead of the standard aluminum. This can increase sensitivity by ~20% and reduce UV stray light detection significantly.
  • Use high quality quartz cuvettes. A sample holder that fluoresces will result in undesired fluorescence that convolutes the desired response.
  • Minimize ambient light by covering the measurement set up with a doubled-over piece of black laser cloth or enclose the sample.
  • Make use of the non-linearity correction and correct for electrical dark to correct for small deviations in the linearity of its response to light, and to maintain a stable baseline.

What is the best way to measure turbid or opaque samples?

These samples can be especially tricky for fluorescence measurements, as their high optical density enhances the inner filter effect. Inner filtering occurs when the concentration of fluorophore is high, and the fluorescence from molecules in the excitation beam is absorbed by near-by molecules in the ground state. This effect decreases the expected fluorescence, which, in the absence of the inner filter effect, is proportional to concentration.

Front-face illumination allows these concentrated samples to be measured. If working with a liquid in a cuvette holder, a bifurcated fiber can be used to excite and collect from the same side. A reflection probe works really well for solid samples, with the 6-fiber leg used for excitation and the central fiber for collection.

It also helps to use of an optical filter on the detection side when scatter is high, especially if using a long integration time. This same approach is also applicable to dilute samples.

What is luminescence?

Luminescence explains the broad process of emission of light from a sample that hasn’t been heated to reach an excited state. Fluorescence is just one of its forms, but it also includes chemiluminescence, bioluminescence, cathodoluminescence, phosphorescence, piezoluminescence, and sonoluminescence, to name just a few. While their excitation mechanisms differ, these techniques all typically generate broad, low intensity spectra.

A standard fluorescence spectrometer can be used for detection, but the sampling optics may need to change. A bare fiber may collect enough light, particularly if it is possible to get very close to the point of luminescence. A fiber terminated with a collimating lens may gather more light, or offer more control over the field of view. An optical post mount can be used to hold either in place.

An integrating sphere may be a tempting option since it appears to collect light from all angles, but remember that just a tiny fraction of that light actually makes it to the spectrometer through the input fiber. An integrating sphere really just samples light from all directions.

Can I use spectrometer for flow cytometry and fluorescence microscopy?

Biological techniques like flow cytometry and fluorescence microscopy often use multiple fluorophores to observe different structures inside a single cell. Overlap between the emission bands is very common, and when optical filters are used to view each fluorophore, S:N is reduced. This is usually managed using compensation algorithms; data from a set of fluorescence standards is used to calculate and subtract the crosstalk. Autofluorescence from the sample itself can also contribute interference.

Using a spectrometer for detection instead of filters preserves the full fluorescence spectrum, allowing the signals from different fluorophores to be deconvolved, not just isolated and corrected. Spectrometers are less sensitive than PMTs for detection, but don’t require multiple dichroics and bandpass or blocking filters to separate the fluorescence channels prior to measurement. Spectrometers also don’t require a new set of filters when a new dye is used.

A spectrometer can also work really well for quantitative fluorescence measurements, particularly if used with ratiometric dyes. These dyes show a shift in excitation or emission wavelength in the presence of a particular chemical or a change in pH. They allow quantitative measurements to be independent of changes in laser intensity or focus, and correct for bleaching of the fluorophore, dye concentration, and cell thickness.

Measurement of the wavelength shift has traditionally been done using two excitation lasers, or by monitoring two detection ranges with bandpass filters. Using a spectrometer, however, captures the full emission spectrum, allowing for much more detailed analysis of peak shifts.

Fluorescence Fluorescence Fluorescence

These are the basic components to perform a fluorescence measurement using  an Ocean Optics Flame spectrometer, software and sampling accessories. First observed in 1560, fluorescence has evolved into a powerful technique that enables entire fields of cutting-edge science and medicine. The only spectroscopic technique capable of resolving single molecules, fluorescence has moved from the lab to applications limited only by imagination.

OCEAN HDX Spectrometers

OCEAN HDX Spectrometers

High Definition Optics in Small Bench Design
BUNDLE-QEPRO-FL

BUNDLE-QEPRO-FL

High-sensitivity Spectrometer for Fluorescence
Education Kits for Science

Education Kits for Science

Spectroscopy Experiment Kit for Teaching STEM Labs
Flame Spectrometer

Flame Spectrometer

High Thermal Stability, Interchangeable Slits
BUNDLE-FLAME-FL

BUNDLE-FLAME-FL

Application-Ready System for Fluorescence
JAZ Spectrometer

JAZ Spectrometer

Handheld Spectrometer for UV-Vis Measurements
QE Pro (Custom)

QE Pro (Custom)

High-sensitivity Spectrometer for Low Light Level Applications
Maya2000 Pro (Custom)

Maya2000 Pro (Custom)

High Sensitivity Spectrometer
Maya2000 Pro-NIR

Maya2000 Pro-NIR

High-sensitivity Spectrometer for Raman and NIR Applications
USB4000-FL

USB4000-FL

Preconfigured Spectrometer for the Fluorescence Applications and Measurements
QE Pro-FL

QE Pro-FL

High-sensitivity Spectrometer for Fluorescence
Ventana-VIS-NIR

Ventana-VIS-NIR

High-sensitivity Spectrometer for Fluorescence Techniques
HL-2000 Family

HL-2000 Family

Tungsten Halogen Light Sources for the Vis-NIR
DH-mini Light Source

DH-mini Light Source

Compact Deuterium, Halogen Light Source for the UV-Vis-NIR
DH-2000 Family

DH-2000 Family

Deuterium-Halogen Light Sources for the UV-Vis-NIR
PX-2

PX-2

Pulsed Xenon Light Source
HPX-2000 Family

HPX-2000 Family

High-powered, Continuous Xenon Light Sources
ecoVis

ecoVis

Compact, Low-Voltage Light Source
LSM Series LED Light Sources

LSM Series LED Light Sources

High-Performance UV, Visible and Broadband LEDs
DH-2000-BAL

DH-2000-BAL

Balanced Deuterium, Halogen Light Source for the UV-Vis-NIR
Raman Sample Holders

Raman Sample Holders

Sampling of SERS, Liquids, Powders and More
74-series Collimating Lenses

74-series Collimating Lenses

Single and Achromatic Lenses
COL-UV-30 Collimating Lens

COL-UV-30 Collimating Lens

Large-Diameter Single Lens
84-UV-25 Collimating Lens

84-UV-25 Collimating Lens

Large-Diameter Single Lens
Optical Filters

Optical Filters

OF2 Filters for High-pass, Bandpass and Balancing Requirements
Linear Variable Filters

Linear Variable Filters

High-pass, Low-pass and Variable Bandpass Filtering
Linear Variable Filter Accessories

Linear Variable Filter Accessories

Adapt LVFs to In-Line and Cuvette Holder Applications