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Home > Applications Blog > Light Source Characterization in Life Sciences

Applications Blog

Light Source Characterization in Life Sciences

Tags: News Applications Blog Newsletter led Technical Tips irradiance nir biomedical instrumentation laser Life Sciences

Nov 18, 2015

Photonics plays a crucial role in the life sciences, from biological research to biomedical instrumentation and agriculture. Effective UV disinfection, optimal aquarium illumination and accurate blood oximetry all depend on the performance of the LEDs and lamps used to power these applications.

 

The success of these applications and laser-based techniques used in ophthalmology, optical coherence tomography, multiphoton fluorescence excitation, DNA sequencing and cytometry all begin with a well-characterized light source. Diode-array spectrometers from Ocean Optics are uniquely positioned to fulfill this role, measuring intensity at all wavelengths simultaneously -- even for a single shot of a pulsed light source. In this technical note we discuss general best practices for light source characterization, and factors to consider in choosing the most suitable spectrometer system for each task.

 

Common Measurement Setups

The best light source measurement setup is modeled after the intended application; i.e., try to measure your light source the same way you plan to use it. To illustrate this general rule, consider microscopy, where the light is often delivered to the instrument with a fiber. For a one-time check of the light source, one can simply connect this fiber to the entrance port of the spectrometer. Measuring through the fiber being used in the system is important to account for transmission of the fiber itself. This is particularly true for ultraviolet light sources transmitted via fiber, as solarization of the fiber can reduce the amount of light delivered at wavelengths below 400 nm.

 

Blood oximetry measurements are another good example. Oximeters use the entire output of an LED to determine the blood oxygen level, so all angles of LED emission should also be captured during characterization -- best accomplished using an integrating sphere. Finally, to quantify the illumination at a specific point or distance from a source, as you would when measuring UV index to assess risk of sunburn, a cosine corrector should be used to collect light for delivery to the spectrometer via a fiber. Eliminating the cosine corrector and using just the bare end of a fiber alone can still provide relative information in this application, such as the wavelength of a peak, but the quantitative intensity information is lost.

 

Narrowband Laser Sources

The important performance parameters for a narrowband light source depend upon the application for which it is used. Wavelength and linewidth of the laser are the most frequently measured parameters. In Raman excitation, any shift in the laser wavelength leads to a corresponding shift in the resulting Raman spectral peaks, and thus unreliable or potentially “blurred” spectra. In flow cytometry, multiple single-wavelength lasers may be used to excite various fluorophores. The detection system then uses complex trains of optical filters to route specific wavelength bands to PMT detectors highly sensitive to residual laser light. In both cases high resolution and wavelength accuracy are of paramount importance for the spectrometer characterizing the laser. This can be achieved through the selection of a narrow slit and long optical bench (as in the HR-series or QE Pro spectrometer), and by using a high groove density grating (i.e., high lines/mm).

 

Figure 1 shows the spectrum of a narrowband laser recorded with an HR series (high resolution) spectrometer, equipped with a 25 µm slit and an 1800 lines/mm grating to measure the wavelength range 430-560 nm. This configuration results in a spectrometer resolution or FHWM (full width at half maximum) of 0.16 nm. As very little light is needed for this measurement, it might be tempting to simply steer a laser reflection onto the spectrometer entrance slit; however, this will lead to incorrect results. Laser beams are almost perfectly parallel, while spectrometers are designed to image a divergent point source from the entrance slit, via the grating, onto the detector. To avoid this problem, the laser emission shown here was captured with an integrating sphere and delivered to the spectrometer via a 50 µm fiber (to minimize stray light in the spectrometer).

 

 

Figure 1: Spectrum of a diode-pumped solid state laser recorded with an integrating sphere, a 50 µm fiber (QP50-2-VIS-NIR) and an HR-series spectrometer equipped with a 25 µm slit and an 1800 lines/mm holographic grating. Figure 1: Spectrum of a diode-pumped solid state laser recorded with an integrating sphere, a 50 µm fiber (QP50-2-VIS-NIR) and an HR-series spectrometer equipped with a 25 µm slit and an 1800 lines/mm holographic grating.

 

 

Short Pulse Lasers and LEDs

The enormous peak powers delivered by short pulse or “ultrafast” lasers have enabled new and exciting applications in biomedical microscopy, not the least of which is multiphoton excited fluorescence, a technique used in neuroscience, immunology and cancer research. This technique provides intrinsic three-dimensional spatial resolution (through the position of the laser focus required to generate nonlinear excitation) with deep penetration (thanks to the near-infrared wavelengths). While a spectrometer cannot measure the pulse length of a Ti:Sapphire or short pulse fiber laser directly, both the coherent bandwidth (used as a measure for the shortest possible pulses) and the center wavelength of the tunable laser are of importance for successful research or diagnostics.

 

Similarly, emission bandwidth and center wavelength are of most interest when measuring single-wavelength LEDs, typically in the visible or UV range. An LED’s center wavelength and wavelength shape can change with drive current or temperature. Figure 2 shows a fiber-coupled 455 nm LED at various drive currents, recorded with an HR-series spectrometer. The relatively broad spectrum of LEDs reduces the resolution requirement for measurement, and thus would allow use of an ultra-compact spectrometer like the STS-VIS with 1.5 nm resolution (FWHM). The slightly larger Flame-VIS spectrometer would offer the flexibility to customize the grating and wavelength range to achieve higher resolution if required.  

Figure 2: The output of a single-wavelength LED (LLS-455) at different drive currents, showing a shift of the emission wavelength due to temperature changes in the LED. Spectra were recorded with an integrating sphere, a 50 µm fiber (QP50-2-VIS-NIR) and an HR-series spectrometer equipped with a 25 µm slit and an 1800 lines/mm holographic grating. Figure 2: The output of a single-wavelength LED (LLS-455) at different drive currents, showing a shift of the emission wavelength due to temperature changes in the LED. Spectra were recorded with an integrating sphere, a 50 µm fiber (QP50-2-VIS-NIR) and an HR-series spectrometer equipped with a 25 µm slit and an 1800 lines/mm holographic grating.

 

 

Tunable Near-Infrared Lasers

The output of tunable short-pulse lasers such as Ti:sapphire lasers often spans the short-wave near infrared (SW-NIR) region up to ~1100 nm, and thus can be measured using a properly configured Flame or HR-series spectrometer. A 600 lines/mm grating, blazed at 1000 nm for highest SW-NIR efficiency, can cover the range 550-1100 nm with a resolution of ~1.3 nm (FWHM) in a Flame bench. Wavelengths above 1.1 µm -- for example, from one of the newer Yb:fiber-pumped OPOs -- demand a switch from silicon to InGaAs detectors. A NIRQuest spectrometer could be configured to cover the missing tuning range from 1100-1500 nm, with a comparable resolution of ~1.5 nm (FWHM) using a 300 lines/mm grating.

 

Filtered Broadband Lamps

Broadband emitters such as tungsten halogen or xenon lamps are still by far the most common light sources used in life sciences, with new white light LED sources advancing quickly. Optical filters are often used to select a specific wavelength range for illumination or excitation, and thus careful measurements are required to ensure that sufficient power is emitted at the desired wavelengths, while radiation in any harmful or undesirable regions is eliminated. The application defines which spectral regions constitute useful or undesirable. For example, UV disinfection requires sufficient UV output in the germicide region (UVC, below 280 nm) to insure sterility, while in contrast any UV light below 350 nm needs to be blocked for ophthalmic surgery to avoid damage to the eye.

 

Moving up the rainbow, blue light exposure in the evening, as from smartphone screens or LED lighting, has been associated with restlessness at night due to its effect on the circadian rhythm. Blue light, however, is used as an effective treatment for neonatal jaundice. On the other end of the spectrum, the majority of the output from tungsten halogen bulbs is composed of invisible near-infrared light. High intensity NIR light can cause thermal damage in the retina (the reason why one should not directly stare at the sun) and needs to be eliminated from eye surgery tools.

 

As one last example, related to indoor farming or aquariums, plants require mainly blue and red light for optimal growth, while any light in the green region is simply reflected, leading to their dominant color. Light sources for plants are therefore rated by their effectiveness for plant growth. This is often given as a measure of the photosynthetically active radiation (PAR), the emission in the range from 400-700 nm, or the photosynthetically useful radiation, which also subtracts the green region from 550-620 nm.

 

Determining Light Source Intensity

Good characterization of a light source for many of these applications also requires absolute irradiance measurements in a certain wavelength region. Spectrometers are ideally suited for this task, as they can be radiometrically calibrated over their entire wavelength range at the factory or by the user using a calibrated lamp. The exact measurement setup to use depends on the application. The emission from an LED is best measured with an integrating sphere, while the illumination at a certain distance from the light source is ideally measured with a cosine corrector at the point of use, as discussed previously. It is crucial that any radiometric calibration be made with the exact setup and accessories to be used in the light source measurement, with the calibrated lamp replacing the source to be characterized. Even seemingly insignificant changes, such as disconnecting and re-connecting the fiber will invalidate an absolute irradiance calibration.

 

After calibration the spectrometer measures the light in absolute units: the emitted energy per time, area, and wavelength slice. A simple integration over the wavelength region of interest yields the desired quantity --  the amount of useful or harmful radiation released from the source. Due to this integration, signal to noise and resolution are of minor importance for the selection of the spectrometer; rather, the analysis requires good linearity, a reproducible “dark” background and reliable wavelength calibration. While even compact spectrometers such as the STS series can fulfill these requirements and are good choices for measuring the absolute irradiance in a certain wavelength range, higher end spectrometers like the Maya2000 Pro or QE Pro feature more linear detectors and will therefore yield slightly more accurate results.

 

Measuring Residual Light

If the absence of radiation in a certain wavelength range needs to be tested for safety or performance reasons, stray light will become the most crucial spectrometer specification, which takes more effort to control. As a simple precaution, input fibers with large core diameters could overfill the first mirror in the spectrometer and should be avoided. Similarly, intense light that is not needed for the measurement is best blocked before entering the spectrometer. However, the selection of spectrometer bench and grating probably has the largest impact on stray light performance. As a rule of thumb, holographic gratings tend to cause less stray light than ruled gratings. In addition, some benches are optimized for low stray light, such as the Maya LSL or the Torus spectrometers, and would be a better choice of spectrometer in these cases.

 

Figure 3 depicts an example for the blocking of UV light from a high power xenon source, here performed by inserting safety glasses. The instrument of choice was a Deep UV Maya 2000 Pro, which uses a trap for visible light for minimum stray light in the UV. It was configured with an 1800  lines/mm grating and a  5 µm slit for measurement from  160-310 nm. The spectrometer was radiometrically calibrated and the total intensity from 250-310 nm integrated to quantify the remaining emission in the UV range. Also shown is a less careful measurement without blocking any of the visible light (> 400 nm) using a USB2000+-UV-VIS spectrometer, leading to higher stray light and an incorrect result.

Figure 3: Emission from a high-power xenon light source (red) with the UV portion blocked for eye safety (black). A measurement preformed without first removing visible light (> 400 nm) leads to significant stray light contributions and an incorrect result (blue). Figure 3: Emission from a high-power xenon light source (red) with the UV portion blocked for eye safety (black). A measurement preformed without first removing visible light (> 400 nm) leads to significant stray light contributions and an incorrect result (blue).

 

 

Conclusion

Spectrometers are an excellent tool for rapid light source characterization in the life sciences. Due to their flexibility in configuring wavelength resolution and range, modular spectrometers are especially suited to determine the center wavelength of lasers or LEDs, to measure the bandwidth of the emission, and to quantify broadband lamp irradiance in specific wavelength regions for performance or safety.