We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.
Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.
1. Some care must be taken in preparing and placing a Raman sample within this system.
2. In order to take time-gated Raman spectra, the excitation beam must be properly prepared.
3. In order for the optical gate to operate at peak efficiency, care must be taken in the preparation of the pump beam (Kerr beam) as well.
4. With the two beams combined, the Kerr gate and collection system are set up to maximize the collected time-gated signal.
5. Collection of a single time-gated Raman spectrum requires acquisition of several spectra to correct for system artifacts.
6. Once acquired, several processing steps are helpful to improve the quality and appearance of the data.
7. Representative Results:
Figure 1. Schematic diagram of the Kerr gating system. The pump beam path is shown in red, while the SHG path is shown in blue. The path where Raman and Fluorescence are overlapped is shown in green, while the path where the Fluorescence has been temporally filtered out is shown in yellow. Abbreviations as follows: BPF, band pass filter; CCD, charge-coupled device; DCM, dichroic mirror; FI, Faraday isolator; λ/2, half wave plate; LPF, long-pass filter; NLM, nonlinear medium; P, polarizer; SHG, second harmonic generation crystal.
Figure 2. Top: Raw spectra of coumarin dissolved in immersion oil. Red curve shows the spectrum taken with the gate held open (analyzer set for maximum transmission). Black curve shows the spectrum taken with the analyzer aligned for minimum transmission and a pump beam applied (the gated spectrum). Blue curve shows the spectum taken with the analyzer aligned for minimum transmission and no pump beam applied (gate held closed). Green curve shows the spectrum taken with only the pump beam applied. Dashed magenta lines indicate spectral region shown in panel below. All spectra have been smoothed with a 11 point, 3rd order Savitzky-Golay filter. Bottom: Spectra of coumarin dissolved in immersion oil after fluorescence background subtraction. Red curve is the spectrum with the gate held open, and the blue curve is the gated spectrum. The gated spectrum clearly shows the convoluted high-wavenumber peak characteristic of oils.
The field of biomedical Raman spectroscopy has seen increasing interest over the past several years as a result of its demonstrated potential for solving several difficult challenges in biological diagnostics. For example, Raman spectra have been shown to have diagnostic value in cancer detection 3, 4, 5, 6. Raman spectroscopy has also been used in bacterial quantitation 7, 8 and bacterial drug response 9. It has also found application in a broad range of other biomedical applications ranging from bone health 10 to biofluid analysis 11, 12.
Despite such great potential, however, Raman spectroscopy has a large barrier to over-come in most biological systems: its extremely weak cross-section. Therefore, Raman signals can be easily overwhelmed by even quite modest fluorescent backgrounds. Many techniques exist for removing the fluorescence lineshape 2, 13, 14. However, none of these techniques truly address the main issue with strong fluorescent backgrounds; the shot-noise contributed to the spectrum by the presence of the fluorescent background overwhelms the Raman signal and cannot be subtracted away. Several techniques, such as coherent anti-Stokes Raman spectroscopy (CARS) and stimulated Raman scattering (or inverse Raman scattering), have been developed to attempt to amplify the strength of the Raman signal 15, 16, 17. However, all of these techniques are primarily applicable to transparent samples and have their own backgrounds and problems with chemical sensitivity 18.
Shielding the detector from the fluorescence signal is the only way to truly reject the shot noise associated with the fluorescence background in spontaneous Raman scattering. Over a decade ago, Matousek et al. demonstrated fluorescence rejection using an ultrafast Kerr shutter to temporally isolate Raman and fluorescence signals 19, 20. However, to date, this system has not found widespread use in the biological field due to the need for excessively high pulse energies. The system presented in this communication, by contrast, utilizes 1000 times weaker pulse energies than some previous reports and is compatible with biological systems.
Our system, shown in Figure 1 utilizes a collinear Kerr gate geometry to conserve power and provide as much overlap between the pump and signal beams within the nonlinear medium as possible. In addition, we take care to reduce optical losses as much as possible to ensure that we have the maximum available power to operate our Kerr shutter. Using this system we have obtained Raman spectra of highly fluorescing biological samples, namely a Jasmine plant stem, without observation of any photodamage 21. In this report we show some representative data on a model system of a plant-based fluorophore (coumarin, τF ≈ 5 ns) dissolved in microscope immersion oil. This is shown in Figure 2. In the top panel, we see the strong “ungated” spectrum (analyzer set to 0°) in red, scaled to 0.04% of its maximum value for visualization purposes. Note that there are no discernable Raman peaks visible. Below, in black, is the raw gated spectrum (i.e. the spectrum with both pump and excitation lasers on). Below that, in blue, is the spectrum with just the excitation laser on, representing residual fluorescence leaking through the crossed polarizers. Finally, in green, we see the background due to the pump laser leaking through the combination of absorption and interference filters placed in the system. In the lower panel of Figure 2 we see a subregion of the ungated and gated spectrum (region denoted by dashed magenta lines in the upper panel) after subtraction of a 5th order polynomial. The gated spectrum clearly shows the high wavenumber peak associated with lipid, while the ungated spectrum has no clear Raman features.
Although our system does not operate with great effciency at present (maxiumum transmission measured has been around 5%), absolute signal strength is typically less important than signal-to-noise. There are several broad classes of biological samples for which measuring conventional Raman spectra is either difficult or impractical due to overwhelming fluorescence backgrounds. For these samples, our system can provide a definite signal-to-noise improvement, as seen clearly in Figure 2.
The authors have nothing to disclose.
This work was funded by NSF award DBI 0852891. Part of this work was also funded by the Center for Biophotonics Science and Technology, a designated NSF Science and Technology Center managed by the University of California, Davis, under Cooperative Agreement No. PHY0120999.
Name | Company | Catalog Number | Comments |
---|---|---|---|
Lenses | ThorLabs | Various | All lenses coated to have maximum transmission losses of 1% each |
Tunable Ti:Sapph laser | Coherent | Chameleon | 30 nJ, 200 fs, 80 MHz |
40X oil immersion objective | Olympus | UApo/340 | NA = 1.35 |
Inverted microscope | Olympus | IX-71 | Modified to remove all lenses in side port |
Half wave plate | Thorlabs | AHWP05M-600 | |
Glan-Thompson polarizer | Thorlabs | GTH10M | ˜10% transmission loss |
Spectrometer | PI Acton | SP2300i | |
CCD | PI Acton | Pixis 100B | |
Mathmatical software | The MathWorks | MATLAB | version 2008a |
Faraday isolator | EOT | BB8-5I | |
Piezo-electric mirror | Newport | AG-M100 | |
BBO crystal | CASIX | custom | 1 mm thickness |
Bandpass filter 1 | Andover | 008FC14 | 808 ± 0.4 nm |
Dichroic mirror | Semrock | FF662-FDI01 | band edge at 662 nm |
Long-pass filter | Semrock | BLP01-405R | band edge at 417 nm |
Bandpass filter 2 | Semrock | FF02-447/60 | 417-447 nm |
CS2 | Sigma-Aldrich | 335266 | 99% purity |
Coumarin 30 | Sigma-Aldrich | 546127 | 99% purity |
Immersion oil | Cargille | 16242 | Type DF |