Development of a time-gated system for Raman spectroscopy of biological samples

Development of a time-gated system forRaman spectroscopy of biological

samples

Florian Knorr,1,3 Zachary J. Smith,1,3 andSebastian Wachsmann-Hogiu1,2,∗

1Center for Biophotonics Science and Technology, University of California, Davis, 2700Stockton Blvd. Suite 1400, Sacramento, CA 95817, USA

2Department of Pathology and Laboratory Medicine, University of California, Davis, 4400 VStreet, Sacramento, CA 95817, USA

3Both authors contributed equally to this work.

*[email protected]

Abstract: A time gating system has been constructed that is capableof recording high quality Raman spectra of highly fluorescing biologicalsamples while operating below the photodamage threshold. Using acollinear gating geometry and careful attention to power conservation, wehave achieved all-optical switching with a one picosecond gating time and5% peak gating efficiency. The energy per pulse in this instrument is morethan 3 orders of magnitude weaker than previous reports. Using this systemwe have performed proof-of-concept experiments on a sample composedof perylene dissolved in toluene, and the stem of a Jasminum multiflorumplant, the latter case being particularly important for the study of plants usedin production of cellulosic biofuels. In both cases, a high SNR spectrum ofthe high-wavenumber region of the spectrum was recorded in the presenceof an overwhelming fluorescence background.

© 2010 Optical Society of AmericaOCIS codes: (170.5660) Raman spectroscopy; (120.6200) Spectrometers and spectroscopicinstrumentation; (190.3270) Kerr effect.

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1. Introduction

Raman spectroscopy has been a focus of intense research as a tool for addressing biomedicalproblems for the past several years. For example, Raman spectra have been shown to pro-vide exquisite diagnostic value for discriminating between cancerous and non-cancerous cellsand tissues [1–5]. Raman spectroscopy has also been used to classify and quantify bacteria


and bacterial cultures [6–8], to assess bone health [9], and as a reagentless assay for biofluidanalysis [10]. However, despite these encouraging results, Raman spectroscopy has not foundwidespread application beyond the confines of the research lab. One barrier to more widespreadutility of Raman spectroscopy is that the weak Raman emission of most biological samples isdwarfed by a significant fluorescence background. Although several methods have been pro-posed to date to robustly eliminate the lineshape of the fluorescence background [11–14], noneof these methods address the more fundamental problem of signal to noise that arises whentrying to detect a small signal riding on top of a much larger background.

Fig. 1. (a) Schematic diagram of the Kerr gating system. The pump beam path is shown as asolid red line, while the SHG path is shown as a solid navy line. The path where Raman andFluorescence are overlapped is shown in navy with a dash-dotted line style, while the pathwhere the Fluorescence has been temporally filtered out is shown in navy with a dashed linestyle. 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. (b) Diagramof pulse propagation through the Kerr shutter. The pump beam is shown in red, while theraman and fluorescence signals are shown in green and blue, respectively. A diagram of theorientations of the polarizations of the three beams at several locations is shown above thebeam path. Note that the polarizations are linear at all locations and the elliptical shape isfor figure clarity only.


Because the signal to noise of Raman signals from tissue are often limited by the shot noisefrom the fluorescence, any scheme to eliminate the fluorescence background should necessarilyblock the fluorescence photons from ever reaching the detector. More than a decade ago, Ma-tousek et al. demonstrated such a device that utilized an ultrafast Kerr shutter to pass promptlyarriving Raman photons while blocking late-arriving fluorescence photons [15, 16]. While thisdevice has demonstrated the ability to record fluorescence free Raman and Resonance-Ramanspectra of non-living samples [17], its applicability to biological samples has been limited toanalysis of tissues in vitro [18–20]. Applicability to living samples has been hampered by theuse of high peak-power lasers operating at low repetition rates where nonthermal ablation of thetissue can result [21, 22]. Reducing the pulse power at the sample would result in prohibitivelylong acquisition times due to the low repetition rates of the reported systems.

In this paper, we present a time-gating system with a novel collinear design that utilizes lowpeak power pulses operating at high repetition rates. The resulting peak and average energiesat the sample are biologically safe, and fluorescence-free Raman spectra can be acquired. Adiscussion of the system and its construction are followed by proof-of-concept results obtainedon non-biological and biological samples.

2. Materials and Methods

2.1. Kerr-gated Raman system

The time-gating system is shown schematically in Fig. 1(a).At the heart of this instrument is a collinear Kerr gate, shown in Fig. 1(b). Transform-limited

140 fs, 30 nJ pulses, centered at 808 nm, are emitted by a mode-locked Ti:Sapphire laser sys-tem operating at 80 MHz (Chameleon, Coherent Systems, Santa Clara, CA), giving an averageintensity of 2.4 Watts. Overall power in the system is controlled by a half-wave plate placedbefore a Faraday isolator. The light is then passed through an ultra-narrowband filter (FWHM= 0.8 nm, Andover Corporation, Salem, NH), with the filtered light being frequency doubledby a β-barium borate (BBO) crystal (CASIX, Fuzhou, Fujian, P.R. China). The resulting 404nm beam has a temporal width of approximately 1.65 ps and is then coupled into an Olym-pus IX-71 inverted microscope (Olympus, Center Valley, PA) fitted with a 40x 1.35 NA oilimmersion objective. Approximately 62 pJ of energy per pulse reaches the sample (averageintensity 5 mW). Independent control over the strength of the SHG beam is obtained by a half-wave plate placed after the optical isolator. By rotating the polarization of the light enteringthe BBO crystal with respect to the crystal axis, the efficiency of the SHG process can be ad-justed. Raman scattered light is collected by the objective and separated from the excitationlight by a dichroic filter (Semrock, Rochester, NY) and polarized by a Glan-Thompson po-larizer (Thorlabs, Newton, NJ). Because of dispersion in the optical elements downstream ofthe sample, the Raman scattered light becomes slightly temporally broadened. Meanwhile, theportion of the 808 nm pulse that does not pass through the narrowband filter is sent through anadjustable delay line, a half-wave plate and Glan-Thompson polarizer (Thorlabs, Newton, NJ).The waveplate-polarizer combination is used to ensure maximum transmission through the 45◦oriented polarizer, after which the pulse has a temporal width of 500 fs.

The 808 nm pump pulse and Raman probe pulse are recombined at a dichroic filter (Semrock,Rochester, NY), and the 404 nm Raman excitation light is filtered from the beam by a long-passfilter (Semrock, Rochester, NY) that passes both the Raman scattered light as well as the 808nm pump beam. This is to prevent the residual 404 nm light from exciting any Raman scatteringwithin the nonlinear medium. The pump and Raman beams are then focused by a 35 mm focallength achromatic doublet into a 1 cm pathlength quartz cuvette (Starna Cells, Atascadero, CA)containing the nonlinear medium. In these experiments, we use CS2 as our nonlinear medium.CS2 has a high nonlinear response (n2 = 3.1×10−18 m2W), with two temporal components: a


Fig. 2. Plot of gating efficiency versus temporal delay between the pump and SHG pulses.Delays were measured moving a calibrated translation stage and converting added path-length to time delay.

fast electronic response and a slower rotational (τ ∼ 2 ps) response. As shown in Fig. 1(b), theRaman beam experiences nonlinear birefringence in the CS2 induced by the pump beam whenthe pulses are spatially and temporally overlapped. Temporal overlap is accomplished by carefultuning of the delay line by translating a stage with ≤ 25μm (85 fs) resolution. Spatial overlapis accomplished by passive piezo-electric tip/tilt controls on the pump-beam steering mirrors.Both the Raman and pump beams are focused to a spot size of approximately 5 μm in the CS2.The induced birefringence of the pump beam rotates the polarization of the Raman beam whenthe two are overlapped. A detailed description of the power requirements for optical switchingis given below in Section 2.3. Because the Raman beam experiences dispersion, not all of thespectrum can be gated at one time by our pump pulse. As a proof-of-concept, we choose to focuson the high-wavenumber region of the spectrum where the signals are strongest. Additionallythe high-wavenumber region may be important for research into plant-derived biofuels [23,24].

By contrast with earlier experiments, we operate our gate with no angle between the k-vectorsof the Raman and pump beams. Although this presents a greater challenge in adequately atten-uating the 808 nm pump before the collection system, it also allows us to take advantage of agreater interaction length between the Raman and pump light than earlier systems. Maximizingthe overlap of the two beams through the CS2 is essential since our system has at least 3 ordersof magnitude less energy per pulse available to operate the nonlinear gate compared with pre-vious reports. Therefore we utilize a combination of absorption and interference filters, with acombined OD of 10 at 808 nm, to remove the 808 nm pump beam after the pulses have beenrecollimated and analyzed by a second Glan-Thompson polarizer (Thorlabs, Newton, NJ). Thefrequency- and temporally-filtered signal is then focused into an optical fiber and sent into aspectrometer (SP2300i, Princeton Instruments, Trenton, NJ) and dispersed onto a thermoelec-trically cooled CCD (Pixis 100B, Trenton, NJ) operating at -70 degrees Celsius.

2.2. Data processing

For each spectrum shown, data were acquired over several short exposures that together com-prise the total integration time. From each exposure, a dark-noise background has been sub-tracted. Because of the relatively long integration times discussed in this paper, removal ofcosmic rays is essential. This is accomplished by comparing all exposures from a particularsample on a pixel-by-pixel basis, and replacing values that fall outside of three median devia-tions with the median value for that pixel across all exposures. Additionally, for spectra taken


when the pump beam is on, a spectrum where only the pump beam was on has been subtractedto correct for the small background arising from the presence of the pump beam. Next, all expo-sures from a particular acquisition are averaged together. Finally, the spectra are smoothed witha 7-point, 3rd-order Savitsky-Golay filter, where the filter order and window size was chosen tobe below the spectral resolution of our spectrograph to avoid broadening the Raman features.All processing was done using in-house scripts running in MATLAB (The MathWorks, Natick,MA).

Fig. 3. Top: Raw spectra of perylene dissolved in toluene. Red curve shows the spectrumtaken with the gate held open (analyzer set for maximum transmission). Black curve showsthe spectrum taken with the analyzer aligned for minimum transmission and a pump beamapplied (the gated spectrum). Green curve shows the spectrum taken with only the pumpbeam applied. Blue curve shows the spectum taken with the analyzer aligned for minimumtransmission and no pump beam applied (gate held closed). Dashed magenta lines indicatespectral region shown in panel below. Bottom: Spectra of perylene dissolved in tolueneafter fluorescence background subtraction. Red curve is the spectrum with the gate heldopen, and the blue curve is the gated spectrum. Black curve is a spectrum of pure tolueneas a reference. The gated spectrum clearly shows the high wavenumber peaks of toluene.

2.3. Power requirements for optical switching

In order to determine the feasibility of using low-peak-power laser pulses for our instrument,we calculate the phase shift Δ that can be achieved when a laser beam with average powerdensity I propagates through a nonlinear medium with a nonlinear index n2 and length L by thefollowing equation:

Δ =2πn2

λLI. (1)

In order to achieve complete switching (i.e. for the polarization of the Raman light to berotated by 90◦), a phase shift of π must be achieved. Using Eq. (1), we can calculate the energyper pulse needed to achieve the required π phase shift within the nonlinear medium [15]:

E =πd2λT8Ln2

, (2)

where d is the diameter of the laser beam within the medium and T is the duration of thepulse. From Eq. (2) it can be seen that for laser pulses with a certain pulse duration, the energy


requirement can be lowered by reducing either the diameter of the laser beam, by increasing theinteraction length, or by using a material with a very large n2. However, due to diffraction, thereare limitations to the minimum d and maximum L that can be achieved with a given lens. Sincethe nonlinear interaction is intensity dependent, we can approximate the interaction length tobe the Rayleigh length, i.e. L = zR = πd2/4λ . Thus, decreasing d increases the power densityat the beam waist (thereby increasing the nonlinear response), but at the cost of reducing theinteraction length. Therefore in Eq. (2), the quantity d2/L is approximately a constant equalling4λ/π . The required energy per pulse can then be written as follows,

E =λ 2T2n2

. (3)

For laser pulses with λ ≈ 800 nm, T ≈ 1 ps, and CS2 as the nonlinear medium (n2 = 3.1×10−18 m2W), the required energy per pulse is 100 nJ, about 10 times larger than the energy perpulse we have available in our pump beam at the nonlinear medium to drive the gate. Therefore,we expect that our overall gate efficiency will be much reduced compared to earlier reports, butstill large enough to observe a sizeable effect.

3. Results and Discussion

3.1. Efficiency and Temporal Width of the Low-Power Kerr Gate

For this experiment, the experimental setup was slightly modified. The bandpass filter before theBBO crystal was removed and the unconverted 808 nm light through the crystal was used as thepump beam. The SHG beam at 404 nm was reflected off of a mirror placed in the sample planeof the microscope and sent to the detection arm. This was done to ensure that measurementsof gating efficiency were done with the briefest probe pulse possible given our experimentalsetup. The temporal width of both the pump and SHG pulses prior to entering the Kerr gatewere approximately 500 fs. Fig. 2 shows a plot of the efficiency of the gating of the SHG signalby the pump pulse versus position of the temporal delay stage.

Efficiency in this case is defined by comparing the area under the 404 nm peak at variousdelays to the area when the gate is held open, i.e.:

η(delay) =Area404(delay)

Areaopen404

. (4)

By comparing the width of the efficiency curve, we can get an estimate of how long the gate isheld open. As can be seen in Fig. 2, the maximum efficiency of the gate is 5.5%, while the gatewidth is approximately 700 fs, indicating that we are exciting only the electronic component ofthe CS2 [25, 26].

3.2. Perylene Dissolved in Toluene

For this and following experiments, the original version of the setup, discussed in Section 2.1was used. The top panel of Fig. 3 shows the spectra taken from a dilute solution of perylene, apotent fluorophore with a lifetime of 5 ns [27], dissolved in toluene.

For these experiments, spectra were acquired with 20 minute acquisition times. The red curveshows the spectrum taken with the gate held open by rotating the analyzer to maximize through-put in the absence of a pump pulse. The black curve shows the spectrum taken through the Kerrgate (with the analyzer aligned to minimize throughput in the abscence of a pump pulse). Thegreen curve is the spectrum with only the pump beam on, showing the level of backgroundarising from the residual attenuated pump beam. Finally, the blue curve shows the spectrum


Fig. 4. Top: Raw spectra of a star jasmine stem. Red curve shows the spectrum taken withthe gate held open (analyzer set for maximum transmission). Black curve shows the spec-trum taken with the analyzer aligned for minimum transmission and a pump beam ap-plied. Green curve shows the spectrum taken with only the pump beam applied. Blue curveshows the spectum taken with the analyzer aligned for minimum transmission and no pumpbeam applied. Dashed magenta lines indicate spectral region shown in panel below. Bot-tom: Spectra of a star jasmine stem after fluorescence background subtraction. Red curve isthe spectrum with the gate held open, and the blue curve is the gated spectrum. The gatedspectrum clearly shows the characteristic high wavenumber peak of cellulose.

taken through the Kerr gate in the absence of a pump pulse. Note that the upper curve hasbeen scaled to 0.1% of its original value so that the curves can be plotted on the same y-axis.In order to compare the quality of Raman peaks obtainable from the two spectra, the ungatedand gated spectra were background corrected with a 5th order polynomial determined using themethod of Lieber and Mahadevan-Jansen [12]. Those results are shown in the bottom panel ofFig. 3, with the open-gate spectrum scaled to 0.025% of its original value. The characteristichigh-wavenumber peaks of toluene are clearly observable in the gated spectrum [28] (blue line),with the observed spectrum matching closely to a spectrum taken from a pure toluene sample(shown in black). These features are masked by noise and artifacts in the ungated spectrum,shown in red.

3.3. Stem of a Star Jasmine Plant

Study of lignin, cellulose, and related compounds in plant biology is an area of increasinginterest due to the recent emergence of cellulosic biomass as an alternative fuel source [29,30]. Cellulosic materials residing within plant cell walls can be converted to biofuels onlyafter extraction from a dense network of pectin and lignin polymers. Efforts to modify thisnetwork to permit efficient extraction of biomass is an area of highly active research [31],however efforts to study relevant plant species directly with high-resolution Raman microscopyhave been hampered by overwhelming autofluorescence, requiring either prohibitive integrationtimes or use of nonfluorescing senescent plant samples [24].

Here we present the Raman spectroscopic study of a green plant stem in the presence ofstrong autofluorescence arising from lignin, chlorophyll, and other molecules within the plant.The top panel of Fig. 4 shows spectra taken from the stem of a star jasmine (Jasminum multi-florum) plant, showing the characteristic plant autofluorescence [32].

As before, the spectra were integrated over a 20 minute acquisition time. The red curve is


the spectrum with the gate held open, the black curve is the gated spectrum, the green curve isthe background due to the pump beam, and the blue curve is the spectrum with the gate heldclosed. Once again, the curve with the gate held open has been scaled to 0.1% of its originalvalue. Comparing the gated and ungated spectra after background subtraction (shown in thebottom panel of Fig. 4), we can see the characteristic high-wavenumber peak of cellulose [33]clearly in the gated curve (blue), while it is entirely lost within the noise of the ungated spectrum(red).

Fig. 5. A curve showing the theoretical evolution of shot-noise-limited signal-to-noise ver-sus integration time for the Raman and fluorescence strengths observed in the star jas-mine stem experiment. The gated spectrum, acquired in 20 minutes, had a SNR of 118. Toachieve a comparable SNR without optical gating (and assuming shot noise is the dominantnoise source), one would have to acquire for 540 minutes.

Assuming that the peak shown in the lower panel of Fig. 4 represents purely Raman-scatteredphotons, we can compute the number of Raman photons per second emitted by the sample,compared with the number of fluorescence photons emitted (calculated from the ungated curvein the upper panel of Fig. 4). This can give us an estimate of the SNR of the cellulose peakversus integration time in an ungated experiment assuming Poisson noise, shown in Fig. 5.

Computing the shot-noise associated with our gated cellulose peak, we arrive at a SNR inthe gated experiment of 118. In order to get the same signal-to-noise in an ungated experiment,we would have to integrate for 540 minutes, 27 times longer than in the gated experiment. Itshould be noted that this analysis takes into account the reduced efficiency of Raman collectionthrough the Kerr gate, meaning that despite having only a few percent transmission through ourgate the SNR improvement due to fluorescence rejection is still significant.

4. Conclusions

We have shown that our low-power time-gated system is capable of meaningfully improving theSNR of Raman spectra taken from highly fluorescing biological samples using pulse energiesbelow the damage threshold. Our gating system operates with a timescale of approximately1 picosecond and at 5% efficiency. Using this we have extracted Raman signals of tolueneand cellulose in the presence of overwhelming fluorescence from perylene and plant autofluo-rophores, respectively, with the latter spectrum having applications in biofuel development. Ad-ditionally, we have used high-repetition laser pulse energies 3 orders of magnitude weaker thandiscussed in previous reports, avoiding both thermal and nonthermal damage thresholds [22]while maintaining reasonable integration times. Although we are at present limited to a lowgating efficiency, our system can still reduce integration times by a factor of 27 in the case


of plant autofluorescence. Additionally, novel nonlinear materials and gating designs have thepotential to improve this efficiency and these are directions our group is actively pursuing.

Acknowledgements

This work was funded by NSF award DBI 0852891. Part of this work was also funded by theCenter for Biophotonics Science and Technology, a designated NSF Science and TechnologyCenter managed by the University of California, Davis, under Cooperative Agreement No.PHY0120999.


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Development of a time-gated system for Raman spectroscopy of biological samples

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