Chapter 2. Time-Resolved Raman Spectroscopy. Technical aspects and instrumentation

Chapter 2. Time-Resolved Raman Spectroscopy Technical aspects and instrumentation 20

2.1 Photon dispersion in scattering media When measuring from a small focal volume in a transparent sample or a surface the Raman signals arrive at the detector collectively and practically instantaneously; specifically, in the time it takes photons to travel through air from the sample to the detector. These photons will arrive within a very narrow distribution in time as they will have traveled a practically identical pathlength. When studying scattering samples, the Raman signal is considerably broadened in time in its arrival at the detector however, due to the random, multiple scattering events within the translucent medium creating a greater variation in pathlength. 1,2 In the case of the scattering sample, a pulsed excitation source that is well characterized in duration and energy is required to distinguish from where within the sample the Raman signal is created. Additionally, when measuring this broader distribution of photons emanating from within scattering samples, a fast detector is also required capable of gated measurements that temporally overlap well with the broadened Raman profiles. A detector gate that is too narrow in time will be too limiting in the number of photons collected and intensity will be significantly reduced, while a gate that is too broad will reduce temporal/depth resolution within a sample. 3 2.2 Ti:sapphire pulsed laser excitation A critical aspect of the TRRS system is the use of a pulsed laser. The laser pulse length affects the temporal and spectral resolution of measurements; A laser pulse that is too long (~ns) will not provide sufficient depth discrimination in a scattering sample, while a laser pulse that is too short (~fs) will not have the well-defined photon energy needed for Raman measurements. A sufficiently high repetition rate coupled with a low peak power is also required to obtain enough measureable signal, while minimizing photodegradation. The TRRS system used in this thesis work employs a Ti:sapphire laser (Coherent, Mira 900P) pumped by a CW 532 nm Verdi laser. The Ti:sapphire produces 3 ps laser pulses at a repetition rate of 76 MHz, or a pulse every 13.2 ns. The high laser pulse repetition rate allows for sufficient signal accumulated over a second, despite the low peak power of each individual pulse. The spectral broadening associated with excitation via such short laser pulses can be approximated by the Heisenberg uncertainty principle, which describes the relationship between the uncertainty in time and energy. If the 3 ps laser pulse length is considered as full width at half maximum (FWHM) to be 2σ, and where h is Planck s constant: 21

σ (t) σ (E)= h/4π; FWHM(t)/2 FWHM(E)/2 = h/4π; (3 10-12 s)/2 FWHM(E)/2=(6.62 10-34 m 2 kg/s) / 4(3.14); FWHM(E)/2= 3.5 10-23 J, or 1.76 cm -1 ; FWHM(E) = 3.5 cm -1 This expected value corresponds well with practical measurements on this system by Efremov et al., 2007, 4 in which the measured width of an Argon calibration lamp line (2.4 cm -1 ) was compared to the laser line width (5.1 cm -1 ) at 405 nm observed with the same spectrograph settings. The extra contribution to the bandwidth from the laser can be approximated as a propagation of errors; (5.1) 2 (2.4) 2 + (FWHM laser) 2 ; FWHM laser 4.5 cm -1 An additional advantage of this laser system is that its fundamental wavelength is tunable over a broad range of red to NIR wavelengths: ca. 690 nm 980 nm. Through the use of a frequency doubler/tripler (TP-2000B, u-oplaz) the fundamental wavelength can be doubled or tripled in frequency to create additional blue and UV ranges of excitation, specifically including 350-480 nm and 240-320 nm. This flexibility in excitation wavelength is advantageous for studying a wide variety of samples under resonant or near resonant conditions. For measurements described in this and subsequent thesis chapters, various excitation wavelengths were employed including fundamental 720 nm, and frequency doubled 412 nm and 460 nm. Different filters and other optics also are needed for different wavelength ranges. We employed long-pass filters from Semrock: type 735 AELP, 420 AELP and 470 AELP, respectively. The exact cut-off wavelength can be optimized by tilting the edge filter. The Raman signal is backscattered to a triple-grating, 50-cm spectrograph (Acton) which has two CCD cameras mounted to different exit ports; a CCD camera (Andor, DV420-OE) for CW Raman measurements, and an ICCD (La Vision, Picostar) for gated detection. The detector spectral sensitivity plays an important role in determining which excitation wavelength to implement for a particular measurement, as the quantum efficiency of the ICCD camera is greatest for UV and visible wavelengths, and drops off dramatically at wavelengths beyond ~800 nm. The detector performance also plays a crucial role in TRRS through the creation of a detector gate, and will therefore be discussed in detail in the next section. 22

2.3 Gated detection via an intensified charge-coupled device (ICCD) camera For TRRS measurements via an intensified charge coupled device (ICCD) camera, the detector gate size is regulated by voltage parameters within the intensifier of the ICCD. Specifically, two potentials, average voltage (V av ) and clamping voltage (V cl ), are applied between the photocathode screen and the multichannel plate (MCP), as shown in Figure 2.1. A positive average voltage keeps the camera closed in between gate pulses, while the clamping voltage relative to this determines the gate size. 4,5 Optimum voltage settings of the intensifier must be chosen to create a gate that provides high signal intensity, while maintaining good temporal resolution. The gate should also achieve fluorescence rejection by being relatively narrow in time and having sharp or steep opening and closing slopes. 4,5 The narrow gate width is important for rejecting fluorescence of longer lifetimes, while the steep closing slope is most critical for rejecting fluorescence of shorter lifetimes. 3,4 Efremov et al., 2007, 4 provides a detailed overview of the quantification of fluorescence rejection, as well as the spectral resolutions possible with the TRRS system used in this work, under varying parameters. The specific optimal voltage values of the intensifier vary with excitation wavelength, and a characterization of the gate size provided by various settings of the intensifier, performed at 412 nm excitation, can be found in Figure 2.2. At this wavelength, a combination of V av and V cl of 19.6 and 10.3, respectively, provides a gate of approximately 250 ps, and is the best compromise between intensity and resolution. Table 2.1 summarizes the gate widths created by this and various other voltage combinations for which the delay time versus intensity profiles are shown in Figure 2.2. These profiles of Raman signal intensity versus detector delay time correlate with detector gate size, and they are a convolution of laser pulse width and temporal broadening from the sample. The FWHM (Full Width at Half Maximum) of these profiles is approximately equal to the detector gate width when measuring non-retarded Raman photons from a small detection volume of a transparent sample. In that case the contributions of the laser pulse width and pathlength differences are negligible. A thin (1 mm) quartz cuvette of cyclohexane was used to measure the profiles in Figure 2.2. Since the optimal intensifier settings depend on the kinetic energy of the electrons released from the photocathode, different settings were needed for measurements in the NIR. For λ ex = 720 nm we used V av = 21.0 V and V cl = 9.1 V. 23

OFF ON Figure 2.1. Intensifier of the ICCD camera. Photons are converted to electrons by the photocathode. The intensifier is OFF when the average and clamping voltage settings do not facilitate the flow of electrons through the multichannel plate (MCP). The intensifier is ON when the average and clamping voltage settings allow electrons to reach the multichannel plate (MCP) where they are multiplied. These electrons subsequently knock photons off of a phosphor screen to be detected by the CCD camera. Resulting Gate Width (ps) V Av (Volts) V Cl (Volts) (FWHM) 19.6 10.7 150 19.6 10.6 175 19.2 10.3 200 19.6 10.3 250 19.6 6.2 500 Table 2.1. The approximate gate widths as FWHM provided by various combinations of V Av and V Cl settings of the ICCD camera for an excitation wavelength of 412 nm. I. E. Iping Petterson, unpublished work. 24

Net Peak Intensity 5000 4000 3000 2000 1000 250 ps 500 ps 175 ps 150 ps 200 ps 0 0 100 200 300 400 500 600 Net Delay Time (ps) Figure 2.2. ICCD gate profiles; net peak intensity of a representative Raman peak of cyclohexane plotted versus detector delay time for various settings of the average voltage and clamping voltage (see Table 2.1), respectively, of the ICCD camera. The FWHM of these profiles is practically identical with the detector gate width. Excitation 412 nm. I. E. Iping Petterson, unpublished work. 2.4 Depth profiling measurements The short detector gate of 250 ps can be delayed in time after the laser pulse, as shown in Figure 2.3A. When a very short delay time is used, the detector will collect Raman photons that are backscattered primarily from the surface of the sample. However, when a longer delay time is used, a greater percentage of photons emanating from deeper within a scattering sample will be detected. These photons have traveled deeper into the sample, and thereby have a much longer total pathlength. Their travel time within the sample and to scatter back towards the detector will also be longer and with a broader temporal distribution than those photons from the surface, as shown in Figure 2.3B. By first using a short detector delay to obtain a spectrum from primarily the sample surface, and then obtaining subsequent spectra while increasing the delay in small increments, a depth profile 25

of a sample can be formed. This principle is put into practice and discussed further in Chapter 3 of this thesis. A 1 st layer 2 nd layer B Figure. 2.3. (A) Raman photons created on the surface and within the second layer of a scattering medium have different pathlengths and arrive at the detector at different times. (B) Diagram of the profiles in time of Raman photons from first and second layers of a 2-layer sample when measured with a pulsed laser system and ICCD camera with a 250 ps gate. Raman photons from the second layer have a less intense, broadened time profile. Implementing a relatively short delay time for the detector gate also allows for the avoidance of considerable amount of fluorescence background. 26

2.5 Effect of focusing In conventional Raman measurements of the surface of a sample, the focal distance of the excitation and collection optics plays a critical role in the efficiency of Raman signal detection. In performing TRRS measurements of a two-layer sample, this is true for the intensity of the signal from the surface as well. However, a different relationship is found between Raman signal intensity from the second layer and accurate focus on the sample surface. Figure 2.4 shows Raman intensity versus detector delay time profiles, recorded at different sample distances from the objective (0 mm- 4 mm) for a 2-layer sample, of which the first layer was a 3 mm thick layer of Teflon, with a second layer of a 2 mm thick block of Arnite. While the distance from the sample surface does have a significant effect on the intensity of signal from the first layer material, the intensity of Raman signal from the second layer, Arnite, appears to remain more or less constant for all distances from the objective we measured. This indicates that for depth measurements, there is an advantage towards improved ratio of signal from deeper layers to surface signal by not focusing on the surface layer. Teflon 1st Layer 3.5 mm distance Intensity Arnite 2nd Layer 0 250 500 750 1000 Delay time (ps) Figure 2.4. Effect of focusing on Raman intensity in depth profile measurements with TRRS. The first layer intensity changes significantly with sample distance, having a maximum corresponding with the collection lens focus, in this case 3.5 mm. The sample distance is not critical for the intensity of second layer Raman signal (for distances 0-4 mm). I. E. Iping Petterson, unpublished results. 27

2.6 Advanced Raman techniques combined with TRRS Spatially Offset Raman Spectroscopy and TR-SORS As Time-Resolved Raman Spectroscopy is a depth Raman technique that makes the distinction between surface and deeper Raman photons emanating from a sample on the basis of their temporal difference by detecting with a delayed time gate, Spatially-Offset Raman Spectroscopy (SORS) 6 makes this discrimination spatially. The SORS approach was first reported by Matousek, et al., 2005. In SORS, a lateral offset is created between the laser excitation point and the Raman photon collection point; effectively exciting the sample at a different location from where the Raman signal is collected. Due to the random-walk propagation of photons in a scattering material, the further from the excitation spot, the higher the probability that collected photons have emanated from deeper within the sample. This spatial offset is typically most effective on the scale of a few mm, as the total number of collected photons significantly decreases with an offset, thus with too large a spatial offset, too few photons will make it back to the detector. The optimal offset distance is dependent on the thickness and scattering properties of the first-layer material, however even a small offset will significantly reduce the contribution of Raman signal from the surface, as can be seen in Figure 2.5. This will provide an advantage for signal from deeper layers in a 2 layer sample, similar to the effect noted for such a sample when adjusting the sample distance. Although SORS is usually carried out with CW lasers and detectors, with our TRRS setup, combining Spatially-Offset excitation geometry with Time-Resolved detection is also possible, and this approach may be advantageous in particular sample scenarios. 7 Figure 2.5 shows a comparison of signal intensity from first and second layers in a 2-layer scattering sample when measured with conventional Raman spectroscopy and SORS (2.5A), and TRRS and TR-SORS (2.5B). This provides an overview of the effect of these different techniques on the signal intensity from first and second layers. Chapters 3 and 6 of this thesis discuss these effects and the combination of Time-Resolution with Spatially-Offset Raman spectroscopy in greater detail. 28

Intensity 60000 50000 40000 30000 20000 A 1st Layer 2nd Layer Intensity 250 200 150 100 B 1st Layer 2nd Layer 10000 50 0 RS SORS 0 TRRS TR-SORS Figure 2.5. Intensity differences in measurements of a 2-layer sample of Teflon; 2 mm (first layer), and Arnite; 2 mm (second layer), when measured by (A) Continuous wave Raman spectroscopy (RS) versus SORS with a 5 mm offset, and by (B) TRRS with a 300 ps detector delay time versus TR-SORS with a 300 ps delay time plus 5 mm spatial offset. It should be noted that while intensity can be compared among bars within Figure A or B, intensity differences between frames A and B are arbitrary. I.E. Iping Petterson, unpublished results based on the spectra of Chapter 3. 29

Surface-Enhanced (Resonance) Raman Spectroscopy and TR-SERS Raman signals can be selectively enhanced by using a technique called Surface-Enhanced Raman Spectroscopy (SERS). This selective enhancement is provided primarily by roughened metal surfaces or nanoparticles, most frequently Au or Ag. 8-10 In general, SERS is understood to provide Raman scattering enhancement by various mechanisms: physical, chemical and resonance effects. These may include electronic associations between the plasmon of the charged metal nanoparticle and particular molecular vibrations of the analyte in resonance with the excitation wavelength. The electronic associations between plasmons and the analyte have been observed even if the molecule is only in the vicinity of the metal nanoparticle, occurring by plasmon charge transfer, however the strongest enhancement is generally provided for adsorbed molecules. 9,10 In this latter scenario SERS provides an additional advantage in that binding to metal nanoparticles by the analyte of interest, or by an impurity, may also quench their fluorescence. Additionally, when a molecule is excited with an incident excitation beam of an energy that coincides with or approaches that of a molecular transition to an excited electronic state (for example S 0 to S 1 ), the vibrations (= geometry changes) that are associated with this electronic transition will be in resonance with the excitation energy and can be selectively enhanced. This phenomenon is also possible without the presence of SERS nanoparticles, in which case it is called Resonance Raman Scattering (RRS), a mode of Raman spectroscopy introduced in Section 1.2. Improvements in enhancement can be obtained by creating a resonance between the absorption or molecular transition of the molecules and the resonance of the metal plasmon, through exciting the sample with approximately this wavelength. The strongest enhancement is generally obtained when the plasmon resonance of the metal and molecular absorption both overlap with the excitation wavelength. In this scenario absorption by the molecule and nanoparticle is also then at its maximum, which can have a detrimental effect on the Raman scattering intensity when concentrations of molecules and analytes are too high. Often in these resonant scenarios not all molecular vibrations are equally enhanced, however this loss of information may be advantageous for simplicity in studying samples in which a particular structure of interest is found within a complex matrix. 30

The type of nanoparticle used for SERS enhancement of an analyte is also an important consideration as it must be compatible chemically, as well as in its spectroscopic properties. Ag and Au nanoparticles are commonly used, and their specific properties are dependent on the particle sizes and shapes that have been synthesized. Adjusting these physical parameters of the nanoparticles allows them to be tuned to provide greatest enhancement at certain wavelengths. Also generally speaking Au can be more chemically inert that Ag, which may also be advantageous for certain applications. SERS is a technique that can be combined with numerous geometries and detection techniques, including TR, SORS, and TR-SORS. Chapter 5 of this work will discuss these combinations with SERS in greater detail. 31

2.7 References 1 Matousek, P., Towrie, M., Stanley, A., and Parker, A.W. Efficient rejection of fluorescence from Raman spectra using picosecond Kerr gating. Appl. Spectrosc., 1999, 53(12): 1485 1489. 2 Everall, N., Hahn, T., Matousek, P., Parker, A.W., and Towrie, M. Photon migration in Raman spectroscopy. Appl. Spectrosc., 2004, 58(5): 591 597. 3 Efremov, E.V., Ariese, F., and Gooijer, C. Achievements in resonance Raman spectroscopyreview of a technique with a distinct analytical chemistry potential. Analytica Chimica Acta, 2008, 606(2): 119 134. 4 Efremov, E.V., Buijs, J.B., Gooijer, C., and Ariese, F. Fluorescence rejection in resonance Raman spectroscopy using a picosecond-gated intensified CCD camera. Appl. Spectrosc., 61, 2007, 571. 5 Kentech, Didcot UK. High Rate Imager tutorial. http:// www.kentech.co.uk/ tutorials. 23/11/2011 updated. 6 Matousek, P., Clark, I. P., Draper, E.R.C., Morris, M.D., Goodship, A. E., Everall, N., Towrie, M., Finney, W.F., and Parker, A.W. Subsurface Probing in Diffusely Scattering Media Using Spatially Offset Raman Spectroscopy. Appl. Spectrosc., 2005, 59(4):393 400. 7 Iping Petterson, I. E., Dvorak, P., Buijs J.B., Gooijer, C., and Ariese, F. Time-resolved spatially offset Raman spectroscopy for depth analysis of diffusely scattering layers, Analyst, 2010, 135: 3255 3259. 8 Lee, P. and Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem., 1982, 86: 3391 3395. 9 Le Ru, E.C. and Etchgoin, P.G. A quick overview of surface-enhanced Raman spectroscopy. In: Principles of Surface-Enhanced Raman Spectroscopy and related plasmonic effects. Amsterdam, the Netherlands: Elsevier, 2009. pgs. 1 25. 10 McNay, G., Eustace, D., Smith, W. E., Faulds, K., and Graham, D. Surface-Enhanced Raman Scattering (SERS) and Surface Enhanced Resonance Raman Scattering (SERRS): A Review of Applications. Appl. Spectrosc., 2011,65(8): 825 837. 32