Challenges of Spectrofluorometry, Part 2: Instrument-Specific Concerns

Part 1 of this three-part series provided a procedure for quickly recording acceptable fluorescence spectra with classic commercial spectrofluorometers for most samples in common 1-cm-pathlength cuvettes. Part 2 of the series describes instrument-specific concerns that require modifications to our initial procedure if we want spectra that can be accurately reproduced in other laboratories. Part 3 will discuss how the sample itself can make reproducibility between instruments challenging.

In Part I of this series, we described a simple procedure to select conditions under which fluorescence excitation and emission spectra are acquired with a commercial spectrofluorometer for the most common type of sample—a solution of emitting molecules in a common 1-cm-pathlength cuvette. This procedure is satisfactory for routine samples, particularly when the results are for relative or qualitative purposes. However, when comparisons are needed between different laboratories or different instruments, the differences between instrument responses become important to understand and rectify.

There are many reasons that spectrofluorometry of a routine sample might be inconsistent from instrument to instrument. This part of the series describes at a high level some of the reasons for these inconsistencies even when you “collect data right the first time,” and provides a procedure for how to correct for instrumental responses and artifacts.

Figure 1 in Part 1 provided a schematic diagram of a conventional spectrofluorometer with a continuous light source, and defines horizontal and vertical directions with respect to light polarization. For reproducible spectrofluorometry, we need to reproduce the instrument settings and filters at a minimum. However, even with these factors kept constant, there are reasons fluorescence data recorded on different instruments or at different times on the same instrument might be inconsistent with one another.

Beginning with the lamp and ending with the emission detector, every element of the instrument that produces, detects, or interacts with light can result in instrument-specific effects on a measured spectrum. Nearly all interactions can affect the light intensity or polarization to a greater or lesser degree.

Some of the instrumental factors that can affect the apparent spectrum and that were not discussed in Part I include the following:

• The lamp intensity and spectrum can vary from instrument to instrument and over time
• Transmission of light from the light source into the excitation monochromator can vary with wavelength and over time
• The excitation spectrometer optics can affect the overall throughput and the polarization of light reaching the sample
• The emission spectrometer optics will affect the throughput and polarization of fluorescence reaching the detector
• The reference detector and its beam splitter have overall relative efficiencies that vary with wavelength and polarization of the beam
• Any optical filters used in the system will introduce their spectral features, which may vary with the degree of collimation of the beam
• Colored glass filters used in the spectrofluorometer can fluoresce and add to the background of the measurement (1)
• Any interference-type filter used in the system will affect horizontal and vertical polarizations differently if not used at normal incidence
• The cuvette affects reflection and transmission losses as a function of its material
• The point of intersection of the excitation and detection optical paths inside the sample may vary, affecting the relative amount of inner filter effects that are observed
• The cuvette positioning affects the point of overlap of the excitation and detection volumes inside the sample
• A poorly chosen or poorly cleaned cuvette may have a measurable auto-fluorescence of its own, or may have a measurable absorption spectrum that interferes with the spectroscopy of the sample it contains (for example, optical glass in the UV)
• Some cuvettes may not be intended for precision spectroscopy because of heterogeneities in the material
• Using different sample geometries (90-degree, 180-degree, square or triangular cuvettes) will greatly affect self-absorption, polarization, and the sampling volume
• The detectors have wavelength-dependent light detection efficiencies
• Both monochromators may exhibit wavelength offset errors
• Motor drive mechanisms could be malfunctioning leading to poor repeatability of the wavelength
• Each monochromator may exhibit errors in spectral bandwidth (SBW)
• The monochromator slits may not move evenly, causing the wavelength offset errors to vary with slit width
• The detector may show nonlinearities
• Gear mechanisms for moving gratings in both monochromators may exhibit hysteresis
• Recorded spectra may have a background or intensity offset that varies with wavelength.

Reference detectors are commonly used to reduce the dependence of the spectrum on many of these factors in the excitation system. Pre-installed calibration files for the main detector may be used to largely correct for a number of these factors in the emission system. However, these compensations are not found universally, and even with them, there are factors that are not fully compensated or that change over time and require recalibration. As a result, we still observe wavelength- and polarization- dependent factors that vary between different instruments, or even between measurements made on the same instrument if parts have been replaced, if the alignment changes, if calibration files have been updated, removed, added, lost, or if components of the instrument have aged. In addition, slow phosphorescence of the sample could make a spectral profile dependent on how quickly the overall measurement is made, which could also vary between users or instruments.

Procedure: Evaluate → Collect → Correct

In Part 1, we described the method for choosing initial conditions for measurement. In Part 2, we focus on the process of modifying our initial conditions if necessary and correcting measurements for instrument response and artifacts. The procedure we are about to introduce is appropriate for situations in which you are trying to generate data for routine samples that will be reproducible on other instruments and in the future. We recommend that certain aspects (wavelength errors, SBW errors, range of linearity, polarization) of the instrument performance be determined before data is collected. By doing so ahead of data collection, final throughput correction procedures based on fluorescence standards and calibrated detectors can be applied properly.

Evaluate Your Instrument

We need to evaluate our system for wavelength errors in the excitation and emission monochromators, SBW errors, and detector linearity as a starting point to collecting data for interlaboratory comparisons. We also need to evaluate the need for polarization compensation in our instrument. These factors cannot be readily repaired after our corrected spectrum is created in the end.

Evaluation of the wavelength precision, SBW precision, and detector linearity are adapted from the literature (2), which provides much more information and is recommended to the reader.

Wavelength Precision

A gear mechanism moves optical components of most monochromators. To avoid the effects of gear backlash or hysteresis on wavelength precision, all measurements should be recorded in the same scan direction (either increasing wavelength or decreasing wavelength). To ensure the wavelength of the first points in the spectrum are correct, we recommend over-scanning by a few nanometers. For example, if we are going to make our measurements by scanning downward in wavelength starting from 600 nm, we might want to begin the scan a few nanometers higher in wavelength so that gear backlash is removed by the time the monochromator reaches 600 nm. If scanning upward, we might start the scan a few nm lower in wavelength than the planned starting point for the same reason. Commercial instruments may automatically overscan the wavelength axis to avoid this problem.

Small atomic (mercury, neon, argon, krypton, xenon) lamps that are approximately the size and shape of pencils and that provide well-known reference wavelengths are common tools in spectroscopy. There are various manufacturers of these inexpensive lamps with different brand names. If a lamp of this type is placed in the position of the sample so that the emission monochromator entrance is illuminated, and if the entrance slit is narrowed to give the highest resolution, the apparent emission wavelengths of the atomic lines from these lamps can be measured and used to correct the emission monochromator wavelength axis. Any filters placed in the spectrofluorometer need to be removed before beginning.

These lamps are difficult to use to correct the excitation wavelength. One common approach to wavelength correction of the excitation monochromator is to use the emission monochromator as a reference. It requires the emission monochromator to have its wavelength axis determined first, and is only as good as the emission monochromator wavelength correction.

The first step in this procedure is to select an emission wavelength, place a cuvette containing an aqueous dilute scattering solution of your choice into the sample compartment, and record an excitation spectrum. Large peaks in the excitation spectrum will appear at the selected wavelength of the emission monochromator. Large peaks can also appear at 1⁄2 of that wavelength because of higher orders of the emission monochromator grating if those wavelengths are produced by a lamp. A few well-chosen emission monochromator settings can potentially provide several points for correction of the excitation monochromator wavelength axis. The accuracy of the emission wavelength correction is ±0.1 nm or better (2). The excitation wavelength correction done this way is not quite as good (±0.2 nm). Because of gear backlash, wavelength measurements should be recorded while scanning in the same direction that will be used in spectrofluorometry of the target compound.

The motors that drive the monochromators and slits are typically stepper motors, which require a driver to sequence the phases of the motor. If a phase of the motor fails, or if the driver board becomes intermittent, or if a connection becomes intermittent, it is possible for the driver to step inaccurately. A symptom of this failing would be a change in the sound of the instrument as it changes wavelengths, or an inability to return to the same wavelength. Some instruments may combine motors with an indexer that reads the position of the motors independently; if so, then the indexer may be susceptible to mechanical or electrical failures, also leading to poor repeatability. Repeatability of the monochromators can be tested by repeating the calibration runs several times and overlaying the scans to look for “jitter” in the peak positions. Poor repeatability is cause for an immediate call to the manufacturer.

SBW Precision and Centering

The slit width is the main variable used to determine the SBW of a monochromator. The slits are formed as a space between two metal plates that each resemble knife-edges; the slits are each supposed to travel the same distance from the closed position so that the center wavelength is always the same regardless of SBW. The same pencil-type lamp used above can be used to determine the SBW of the emission monochromator by assuming the observed width of isolated atomic lines are solely because of the SBW of the monochromator. In this case, SBW is determined as full-width at half maximum (fwhm) of a measured peak. The excitation monochromator again requires a different procedure. By setting the SBW of the emission monochromator to its lowest setting, the SBW of the excitation monochromator can be estimated by recording an excitation spectrum of a dilute scattering solution and repeating the fwhm procedure. These procedures are thought to be subject to an uncertainty of ±0.5 nm in SBW (2). This test should be performed at a few different wavelengths to understand how any SBW error varies with wavelength. If there is any significant error in SBW, and particularly if the SBW error is a function of SBW, one possibility is that at least one side of the mechanical slit is not moving properly. If no fix or repair is possible, the wavelength error determined may be found to be a function of SBW; it is also likely the SBW will not be repeatable if the slit is not moving properly. As with wavelength unrepeatability, any indication of a SBW unrepeatability is cause for an immediate call to the manufacturer.

Linear Range of Detector

Even the best detectors become nonlinear at sufficiently high light intensities. Therefore, it is important to know the linear range over which your instrument operates. One approach is to measure a series of known concentrations of a fluorophore whose emission wavelength is similar to your target compound. To avoid nonlinearity from inner filter effects, the concentration of these standards should be kept very low (below 0.05 absorbance units). The peak excitation and emission wavelengths of the standards can be used, and the series of measured intensities can be plotted and fit to ensure they are consistent with the blank and show no systematic deviation from a straight line. If the highest detected signal is still linear, the calibration measurements can be repeated with an excitation SBW up to the limit described in Part I of this series, and an even wider SBW for the emission monochromator, to give a wide intensity range for the standard concentrations. The linearity of a detector, such as a photomultiplier tube, may change for different accelerating voltages, so care should be taken to use the detector as intended for your study. When changing the contents of a cuvette, the cuvette should be emptied, rinsed with clean solvent, rinsed with the next sample, emptied again, and then refilled before the next measurement. This procedure avoids cross-contamination and dilution of the sample.

Fluorescence Polarization

If the fluorescence of a sample in the 90-degree sample geometry is at least partially polarized, there will be effects on its measured intensity and probably at least its measured excitation spectrum. Counterintuitively, this is true even if there are no polarization artifacts in any of the optics. Fortunately, polarized fluorescence is not generally a problem for small molecules in low viscosity solvents. However, even small molecules can exhibit polarized fluorescence if their fluorescence lifetime is short enough (3), whereas large molecules and viscous solvents invite this problem. If you are concerned that you might need to eliminate fluorescence polarization as a complication, then you can add a “vertical” polarizer in the same location as the excitation filter, and insert another polarizer in the same location as the emission filter to serve as a polarization analyzer. This orientation of the emission polarizer should be set to the 54.7° magic angle to make the measurement independent of the fluorescence polarization of the sample (4).

Collect Your Data

The conditions for measurement can be determined using the methods of Part I, or they can be optimized from that starting point. The following points are specific to measurements made for publication and reproducibility:

• The conditions of measurement must be controlled and recorded. This includes making sure the instrument scan range, step size, spectral bandwidth, number of points per unit wavelength, and integration or dwell time, are appropriate for the sample and that they are all recorded and preserved. It also means that any steps taken to evaluate the wavelength accuracy, spectral bandwidth accuracy, photometric accuracy, and to prevent dependence on sample polarization, should be recorded and preserved
• The clean sample cuvette should be handled with gloves and carefully wiped on all its optical surfaces with a fresh lint-free paper or cloth to remove dust or surface contaminants before being placed into the spectrofluorometer for measurements
• If your instrument has an automated shutter to block the excitation beam, this should be engaged any time a measurement is not being made
• A background spectrum of the pure solvent should be recorded under the same conditions as the sample. Ensure that the same cuvette is used for the background and for the sample, and that it is placed into the instrument in the same orientation. This procedure helps to account for any stray light, dark signal, amplifier switching, or filter fluorescence that might take place inside the spectrofluorometer, and can be subtracted from the measured spectrum before corrections are applied.

Correct Your Data

At this point, all concerns about critical aspects of the instrument that cannot be easily corrected post-measurement have been addressed. What remains is the part of the instrument performance that affects the throughput of the excitation source and the emission of their respective monochromators. On the excitation side, the reference detector, if present, corrects for many of the wavelength-dependent behaviors of the instrumentation. Without the reference detector, fluorescence excitation spectra could be so heavily convolved with the instrument response they would be almost uninterpretable without correction. However, the response of the reference detector itself and the effects of optical components after the reference detector still require throughput corrections.

There are several alternate procedures for implementing these throughput corrections and we describe only the simplest. At present, excitation and emission spectra require different approaches for their correction. The National Institutes of Standards and Technology (NIST) and the Federal Institute for Materials Research and Testing in Germany (BAM) are both excellent sources for details on instrument concerns and corrections that we recommend to the reader (1,4).

Correcting Emission Spectra

For most users, certified reference materials or secondary emission standards are the simplest approaches to correcting emission spectra. An excellent alternative is to use a calibrated lamp, but the standards offer the opportunity to use a cuvette-type sample.

A certified reference material is either a solution or glass that can be placed into a spectrofluorometer cuvette holder, and that has known excitation or emission properties. These materials are certified by, for instance, NIST in the United States and equivalent organizations in other countries. As an example, NIST has certified a series of standard reference materials that include solid glass, cuvette-shaped standards that can be placed directly into a spectrofluorometer (SRM 2943 and SRM 2944 for the spectral ranges 350–640 nm and 530–830 nm, respectively). Certified reference materials are relatively expensive compared to available secondary emission standards (5), though both are used in the same way for the same purpose.

Whether a certified reference material or a secondary standard, what these reference materials have in common is a defined emission spectrum under defined measurement conditions. The measured emission spectrum of the reference material in your instrument is compared to its certified or reported spectrum to determine a correction factor over the range of wavelengths for which it has been evaluated. This correction factor is then used to correct the measured spectrum of your target fluorescence sample.

Correcting Excitation Spectra

There are not currently any primary fluorescence excitation spectroscopy standards like those for fluorescence emission spectroscopy. The simplest available approach to correcting fluorescence excitation spectra is to use a calibrated detector. In this case, the detector is placed in the location of the cuvette to intercept the excitation beam, and its corrected signal is recorded as a function of excitation wavelength. The reference signal of the spectrofluorometer is recorded at the same time. The ratio of these two responses then becomes a correction factor for the wavelength range over which the detector has been calibrated. If there is no reference channel, the corrected signal of the calibrated detector is used to form the correction factor by itself.

Conclusion

Although this article covers spectrofluorometry only at a very high level without much detail, it is still restricted to only one measurement case (solution cuvettes) in steady-state spectrofluorometers. The wide field of spectrofluorometry includes many more sample types and configurations, along with a wide variety of instrumentation, all suited to particular purposes. If the authors could leave the reader with two important messages, the first would be this: Read the user manual for your instrument, read literature about your measurement type, and read about your sample type to prepare yourself for making your own measurements. Having this background knowledge ahead of time will enable you to avoid as many errors as possible and enable you to compare your own measurements to those in the literature to assess whether your measurements are reasonable or not. The second message is that there are many parameters that should be recorded and transmitted about spectrofluorometry measurements in addition to the basics of the instrument settings, without which reproducing your spectra could become very difficult for others.

References

(1) W.E. Van Der Veer and D. Wolpert, Laser Focus World 44, 86–89 (2008).

(2) P.C. DeRose, “Standard Guide to Fluorescence – Instrument Calibration and Validation,” NIST Interagency/Internal Report (NISTIR) – 7458 (National Institute of Standards and Technology, Gaithersburg, MD, 2008, accessed September 27, 2021). https://doi.org/10.6028/NIST.IR.7458

(3) J. Paoletti and J-B. Le Pecq, Anal. Biochem. 31, 33–41 (1969). https://doi.org/10.1016/0003-2697(69)90238-3

(4) U.Resch-Genger, Eds., Standardization and Quality Assurance in Fluorescence Measurements I (Springer-Verlag, Berlin, 2008), vol. 5.

(5) J.A. Gardecki and M. Maroncelli, Applied Spectroscopy 52, 1179–1189 (1998). https://doi.org/10.1366/0003702981945192.

Caitlyn English, Zechariah Kitzhaber, Joshua Williams, and M.L. Myrick are with the University of South Carolina, in Columbia, South Carolina. Direct correspondence to: MYRICK@mailbox.sc.edu ●