Fluorescence instrumentation
This guide will provide some high level guidelines for how to choose the right components for your fluorescence spectroscopy instrument.
There are basically two types of fluorescence spectroscopy instrument configurations – transmissive and reflective as shown on the figures below. The only real difference between the two is that in the reflective version a dichroic mirror is used to direct the excitation light to the sample and collect the emission light from the sample whereas in the transmission configuration the sample is excited directly from the light source and the emission light is collected at an angle of 90 degree measured from the excitation light.
A fluorescence instrument consists of the following basic components:
There are many choices for each type of component and these choices depend first of all on the fluorophores your instrument should be able to analyze.
Below is a graphic showing the two extremes. At one end you have a very simple instrument intended to only analyze one single fluorophore with one excitation and one emission wavelength and no time resolved experiments. In contrast, the most advanced and complicated instrument is at the other end of the scale where your instrument needs to have the flexibility to analyze any (unknown) number of fluorophores with multiple excitation and emission wavelengths and with the option to perform time resolved experiments. However, for most application the instrument need to be somewhere in between these two extremes.
In the following the components inside the fluorescence instrument is described in more details such that you can better judge what will work for your specific application.
Almost any type of light source can be used for fluorescence spectroscopy so the best choice depends on the actual requirements to spectral wavelength coverage, intensity, size, cost, efficiency and whether the light source needs to pulsed.
The table above lists the most common types of light sources and the key characteristics of these. In general, LEDs are a great choice if you want to build a low cost, compact fluorescence instrument for analyzing a limited number of known fluorophores. Pulsed table top lasers are often the preferred choice if you need very accurate time resolved measurements with nanosecond timing. And broadband sources are the best choice if you need the flexibility to analyze a large set of unknown fluorophores and thus need to select almost any excitation wavelength.
The excitation filters fall into two categories:
- Fixed low pass/band pass filters
- Variable band pass filters
Variable optical band pass filters are mostly realized as scanning grating mono-chromators and are used together with broadband light sources to select the right excitation wavelength. The main benefit is of course the flexibility to choose any wavelength but the drawback is a high cost, large size, need for electronics control and stability issues due to the moving parts inside the mono-chromator. Therefore, variable bandpass filters are mostly used in versatile large laboratory instruments. The fixed filter’s main benefits are that they are small, simple, relatively low cost and very stable. However, the price you pay is a limited flexibility in the choice of excitation wavelength. Some instruments come with the option to select between a set of filters with different center wavelength. For this reason fixed filters are mostly used in handheld/portable lower cost fluorescence instruments for dedicated applications.
The choice of sample holder really depends on the application. The main thing to consider is that in the case a transmissive cuvette or flow cell you must ensure that the sample holder material is transparent for both your excitation and emission wavelength. This is especially important for UV wavelengths where most glasses absorb light so special types of materials needs to be used.
The dichroic filter is used in a 45 degree configuration. The function of the dichroic filter is to reflect the excitation light (shorter wavelengths) and transmit the emission light (longer wavelengths). For really simple systems the dichroic filter can actually function as both the excitation and emission filter.
The emission filters falls into three categories:
- Fixed low band pass filters
- Variable band pass filters
- Full spectrum diode array spectrophotometers
Variable optical band pass filters are mostly realized as scanning grating mono-chromators and are used to select the right peak emission wavelength. The main benefit is off course the flexibility to choose any wavelength but the drawback is a high cost, large size, need for electronics control and stability issues due to the moving parts inside the mono-chromator. Therefore, variable bandpass filters are mostly used in versatile large laboratory instruments. The fixed filters main benefit is that they are small, simple, relatively low cost and very stable. However, the price you pay is a limited flexibility in the choice of emission wavelength. You may design your fluorescent instrument with multiple filters but, when you need more than 3 – 4 filter this very quickly becomes bulky and expensive. Full spectrum diode array spectrophotometers will – as the name says – collect the full spectrum. This means you have all emission peaks and details about their shape and the back-ground level. For this reason diode array spectrophotometers are a good choice when you are going to measure multiple fluorophores and/or more complex spectra.
In fluorescence spectroscopy it is common to use Photo Multiplying Tubes (PMT) as detectors due to the high sensitivity and fast response of these detectors. However, Silicon-based solid-state detectors can also be used.
The number of detectors needed depends on the system configuration as shown on the figure above. If you are using a variable band pass filter (like a mono-chromator) you only need one detector. If you are using fixed bandpass filters, you need one detector per filter. If you are going to analyze multiple emission wavelengths this however, very quickly becomes bulky and expensive and a diode array spectrometer will be the preferred option. The diode array spectrometer will typically include hundreds of detectors. Using a ultra compact diode array spectrometer like our PEBBLE VIS will record the full spectrum of all peaks sampled at several hundred wavelengths.
More diode array spectrophotometers could be our Compact FREEDOM VIS spectrometer being cost-efficient with high performance and our High Throughput ROCK VIS spectrometers for their un-compromised performance.
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