RAMAN SPECTROSCOPY/CANCER DIAGNOSTICS
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High-performance near-infrared Raman for clinical application
PENG ZOU
Technology advances are relatively low cross-section with high accuracy and high sample enabling Raman spectroscopy for (<1 × 10-8 ratio of Rayleigh throughput at a much lower cost with application as a clinical tool. vs. Raman scattering). no sample preparation—capabilities High-sensitivity Raman that are either technically impossible Raman spectroscopy has seen tremen- spectrometers have been at the heart of or time- and cost-prohibitive using con- dous growth in biological and life sci- successful research and application de- ventional methods (see Table 1). With ence applications over the past two de- velopment. A Raman spectrometer com- the ability to monitor various diseas- cades. As it measures inelastic scattering prises a laser source, spectrograph, and es and key health indicators, Raman from a monochromatic light source a detector (see Fig. 1). Although free- spectroscopy can provide real-time in- incident onto a sample, Raman spec- space coupling is used in many Raman formation for surgical guidance, drug troscopy provides rich information on systems, fiber-optic probes are widely effectiveness measurement, and point- molecular structures, identities, and used in medical diagnostics because of of-care diagnostics when aided by an composition, and can be used for both their flexibility and collection efficiency appropriate novel sampling interface. qualitative and quantitative chemical enabled by fiber bundling. Progress in Designing a Raman spectrometer for analysis. This information, indicated hardware and software development— a clinical environment requires consid- by the wavelength difference (or relative including high-throughput spectro- eration of the unique optical properties Raman shift) and intensity of the scat- graphs, sensitive of biological tissues: tering, results from interac- Spectrograph • Very high water content tions between photons and Sample • Highly fluorescent in the visible molecular vibrations. (VIS) region Laser This label-free, noninva- Raman probe • Highly scattering sive technique has been wide- • Strongly absorbent in the UV- ly used in research, and has VIS region FIGURE 1. The key more recently been pursued Detector for its potential as an alterna- components of a Raman Excitation laser spectroscopy system tive or complementary clini- Autofluorescence from biological spec- are the laser, detector, cal tool for patient care and spectrograph, and probe. detectors with imens introduces severe interference disease diagnostics. This ar- high quantum to Raman spectral data and obscures ticle will discuss the unique efficiency and analysis of results. However, lon- requirements for a medical Raman in- low dark noise, novel sampling inter- ger-wavelength excitation lasers can ef- strument, recent innovations in hard- faces, and robust chemometrics tools— fectively reduce or eliminate it. As lon- ware development, and clinical cancer have made Raman spectroscopy an in- ger wavelengths penetrate much deeper diagnostic applications enabled by these creasingly practical and powerful tool than UV-VIS light, Raman spectral technologies. for complex biological cell- and tis- data collected with near-infrared (near- sue-related research. IR) lasers have more biochemical in- Raman spectroscopic systems Compared to conventional diagnos- formation needed for accurate in vivo Compared to other optical spectros- tic methods used in medicine, Raman under-skin, tissue, and tumor analy- copy methods, Raman spectroscopy is spectroscopy has potential to enable sis. Two optical windows in the near- considered a weak phenomenon with a both in vivo and ex vivo diagnostics IR spectral range are recommended
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Table 1. Comparison of Raman spectroscopy with conventional near-IR Raman spectrometer as the fun- disease diagnostic methods damental requirement for a successful clin- ical instrument. Raman spectroscopy Conventional Technology Laser-based Biopsy; endoscopy; MRI; CT; x-ray Spectrograph Throughput Fast Time-consuming Dispersive grating-based spectrographs Cost Low High have several advantages over Fourier trans- form interferogram-based systems, and Speed Real time Slow they are more broadly used as medical di- Sampling Noninvasive Invasive agnostic tools due to simple optical design, Regulatory Developing Approved lack of moving parts, and fast data col- lection with a focal-plane array detector. for Raman spectroscopy (see Fig. 2). The 785 nm Raman spectra of methanol Biological tissues are composed of main- 30000 first, and most commonly used, lasers for 25000 ly water with large molecules of proteins, Raman excitation are 785 or 830 nm la- 20000 lipids, and carbohydrates. Therefore, the TPIR 785 sers, which overlay well within the first 15000 Lens spectrometer Raman spectra from biological samples optical window. The second are 1064 nm 10000 typically have broad peaks and features lasers, which are used when autofluores- 5000 and do not need a long focal-length and 0 cence is severe with shorter wavelengths. 950 970 990 1010 1030 1050 1070 1090 high-resolution spectrograph. -1 The choice of laser also relies heavily on Wavenumber (cm ) On the other hand, a spectrograph with the appropriate detector, which will be FIGURE 3. The near-IR optimized TPIR 785 high throughput of light and low f/N is discussed in the next section. Raman spectrometer (Teledyne Princeton more desirable for high signal-collection For in vivo measurements, the selec- Instruments) achieves better light throughput efficiency. In addition, high-quality an- tion of laser power is determined by a than a commercial lens spectrometer, as tireflective coating of the optics, aberra- demonstrated in this comparison using the few factors: tion-corrected spectrograph design, and 1033 cm-1 Raman peak of methanol with a • Maximum permissible exposure 785 nm laser. high efficiency gratings are critical to • Best signal intensity within the safety achieve the highest light throughput. allowable power level measurements when high spatial resolu- As an example, Teledyne Princeton • Comfort level tion is required. Instruments’ TPIR-785 Raman spectrom- • Local heating effect While near-IR light greatly reduces flu- eter incorporates a near-IR optimized lens For ex vivo measurements, a higher laser orescence interference, signal strength spectrograph and thus easily enables a power may be used to improve the sig- decreases with longer wavelength exci- 40–60% light throughput improvement nal intensity. tation because the spontaneous Raman throughout the spectral range (see Fig. 3). Since biological tissues can have com- scattering cross-section is proportional The TPIR-785’s spectrograph uses cus- plex textures and can be highly scattering, to 1/λ4, which makes the already ‘weak’ tom designed lens optics for aberration both multi- and single-mode lasers may be Raman signal even weaker in the near-IR. correction and provides imaging qual- used for a clinical Raman system. The lat- Also, water has a small Raman cross-sec- ity far superior to that of conventional ter are preferred for Raman microscopic tion and human tissue is rich with water. Czerny-Turner spectrographs (see Fig. 4). This further reduc- Optical penetration depth (mm) es the Raman scat- 7 First Second tering from human 6 window window issue. One can po- 5 tentially increase 4 laser power to im- 3 prove Raman signal to certain level, but 2 safety concerns and 1 highly photon-sensi- 0 500 750 1000 1250 1500 1750 2000 tive tissues prohib- Wavelength (nm) it using a high-pow- FIGURE 4. Lens optics designed for aberration correction enable FIGURE 2. Optical windows of Raman er laser. All these improved imaging quality; images of a 19 × 200 µm fiber array of spectroscopy in biological tissue. (Adapted challenges point to 880 nm line (top) and an image of a 19 × 200 µm fiber array with a from A. N. Bashkatov [5]) a high-performance broadband light source (bottom) are shown.
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Direct benefits of such high-quality imaging include: QE (%) QE curves FIGURE 5. Quantum 100 BLAZE offers up efficiency (QE) curves • Multichannel or Raman imaging measurement with mini- to 7X higher QE mum crosstalk 90 of a conventional 80 • Stable high etendue and resolution across the entire spectral back-thinned deep- 70 InGaAs depletion CCD (blue); range and detector focal plane 60 super-deep-depletion • Works seamlessly with a Raman probe with a large fiber 50 40 CCD (BLAZE HR bundle and truly benefits from state-of-the-art large-format 30 Super-deep- camera from Teledyne depletion CCD detectors 20 Conventional Princeton Instruments, 10 CCD orange); and InGaAs 0 Detectors and probes 200 600 1000 1400 1800 detectors (black) Wavelength (nm) For high-performance spectrometers, charge-coupled device are shown. (CCD) detectors still provide the best quantum efficiency (QE) and lowest dark current with proper cooling in the UV-VIS re- also provides deep cooling to -95°C with air for lowest dark gion (see Fig. 5). However, the CCD QE starts to decrease at current. The high QE in near-IR region and low dark current ~800 nm and has a cutoff at ~1100 nm, which is not quite effi- makes BLAZE HR optimal for near-IR Raman. Additionally, cient for near-IR Raman measurements. A high QE detector is the camera provides ultra-high readout speed through dual desired for 785–1100 nm in order to effectively cover the fin- ports for fast spectral rate, which is critical for real-time diag- gerprint Raman spectral region (150–1500 cm-1). The TPIR-785 nostics and Raman imaging. has the option to select a recently developed super-deep-deple- Some research areas, using Raman spectroscopy, have utilized tion CCD camera (BLAZE HR) as its detector. The depletion 1064 nm lasers because they overlay with the second optical win- region of epitaxial silicon is much thicker than the typical back- dow and provide even less fluorescence background. Indium galli- thinned CCD that drives QE in the near-IR region to 2–7X high- um arsenide (InGaAs) sensors are typically paired with 1064 nm er than conventional CCD detectors. The state-of-the-art camera lasers to provide better cov- a) b) erage inRaman longer-wavelength collection bers (900–2200 nm) regions. But InGaAsRaman detectors laser ber suf- fer fromRaman greater dark noise thanshortpass silicon-based lter CCD de- tectors,Raman and with a further APPLIED SCIENTIFIC notch lter INSTRUMENTATION Raman cross-section de- DRS/IFS bers CRISP Autofocus System crease at 1064 nm, as well as eye-safety concerns due the invisibility of the laser
FIGURE 6. Raman probes for clinical diagnostics includeTwo-component lens one with a small probe tip that enablesRaman collection cone a) b) Raman laserprecise cone location Raman collection bers information for in vivo diagnostics Raman laser ber and boundary Raman detection during shortpass lter a surgery (a) and Raman a multimodality The CRISP substantially eliminates focus drift in high- notch lter power microscopy applications by sensing minute DRS/IFS probe head changes between the objective lens and the specimen’s bers that allows cover slip. It also allows a specimen to remain accurately simultaneous focused for hours at a time with an accuracy of 5% of Raman and the objective depth of focus and maintains focus while diffuse reflective scanning in XY. measurements for more accurate www.asiimaging.com Two-component lens diagnostic results [email protected] (b). (Courtesy of 541.461.8181 Raman collection cone 800.706-2284 EmVision LLC) www.asiimaging.com • [email protected] • (800) 706-2284 or (541) 461-8181 Raman laser cone
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2008LFW.indb 22 7/28/20 10:12 AM beam, 1064 nm Raman systems are not Table 2. Overview of Raman-based techniques widely used for medical diagnostics. Another critical component for clin- Advantages Disadvantage Most broadly studied; Weak signal; requires ical Raman systems is the optical fiber Spontaneous Raman simple hardware setup; high-performance probe. Raman probes not only provide spectroscopy flexible operation and easy access to dif- no sample preparation instrument ferent parts of the body for in vivo mea- Development of biocompatible SERS surements, they also can be used to im- >1010 Raman cross-section Surface-enhanced Raman substrates, which are prove Raman signal collection efficiency enhancement; greatly spectroscopy (SERS) typically made of metal and expand measurement capability. Such improves the sensitivity nanoparticles; stability novel probes enable endoscopic Raman, and repeatability Raman imaging, and multimodality mea- Coherent anti-Stokes Orders of magnitude Complex and costly surement (see Fig. 6).1,2 Raman spectroscopy larger cross-section; system setup involving (CARS) Excellent for imaging multiple lasers Cancer diagnostics and Provides depth resolution Requires high-sensitivity other applications into tissues by collecting Raman system; the unique Spatially offset Raman The noninvasive, simple, fast, and high- scattering at different SORS fiber bundle probe spectroscopy (SORS) ly reproducible characteristics of its mea- distances from the also requires large-format surements make Raman spectroscopy a laser incident point camera with tall slit strong candidate for high-profile appli- Needs to meet the cations including cancer diagnostics, mi- Resonance Raman Greatly enhanced intensity enhancement condition; crobiology identification, and noninvasive spectroscopy of the Raman scattering not for all samples blood glucose measurement.3,4 or chemicals
NEW! Open Design UUprightpright MicroscopeMicroscope • Simple coarse height adjustment • Built-in motorized focus • In vivo and In vitro eexperimentationxperimentation onon oonene ssetupetup • OCC or DIC transmitted light (LED) • Epi-fl uorescent imaging • Uses standard Olympus objectives BOB TheThe SSutterutter BOBBOB – designeddesigned toto eliminateeliminate tthehe cconventionalonventional microscopemicroscope fframerame – iiss a vversatile,ersatile, open-designopen-design uuprightpright mmicroscopeicroscope platform ideal for slice electrophysiology, widefi eld fl uorescent imaging, two-photon imaging, photostimulation and new techniques just being developed!
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A large amount of research, covering The number of publications on Raman • In vivo measurements showing poten- nearly every aspect of medical diagnos- spectroscopy for cancer diagnostics has tial for real-time diagnostics tics, has been dedicated to clinical Raman grown from less than 4000 in 2010 to • Ex vivo disease detection developed spectroscopic applications over the past more than 14,000 in 2019. Highlights in based on Raman spectroscopy from decade. Pioneering studies using Raman this field include: biopsy samples, body fluid, and blood spectroscopy for detection of cancerous • Diagnostics using Raman spectroscopic • Clinical trials tissue can be found as early as the 1990s.6,7 methods performed on different types • The development of novel chemomet- Since then, cancer diagnostics has become of cancer; some results were very prom- rics, machine learning, and artificial in- one of the most prolific research areas:4,2 ising for early detection telligence methods to build robust pre- diction models Technology development greatly improves the feasibility and viability of Raman spec- troscopy for clinical applications. Since the 1928 discovery of the Raman phe- A Metrohm Group Company nomenon, many innovations have been developed to improve certain aspects of the technique (see Table 2). Improvement of instrument performance will benefit all Raman-based methods. More is needed, however, for Raman to fulfill its promise. It is interesting to reflect that the modernization of pharmaceuti- cal manufacturing, through the adoption of optical spectroscopic process analysis tools required a joint effort involving re- search communities, equipment vendors, pharmaceutical companies, and regulato- ry bodies for decades before the technolo- gies finally became viable for the industry. Among near-term challenges that will help Raman spectroscopy gain accep- tance for clinical application are cost re- duction; robust chemometrics/artificial intelligence/machine learning tools; size, weight, and power reduction; and regu- latory acceptance.
Portable Raman Analyzer REFERENCES 1. K. Lin et al., Theranostics, 7, 14, 3517–3526 (2017). for Measurements Through 2. E. Cordero et al., J. Biomed. Opt., 23, 7, 1-23 (2018). Opaque Packaging 3. H. J. Butler et al., Nat. Protoc., 11, 664–687 (2016). 4. I. Pence et al., Chem. Soc. Rev., 45, 1958–1979 The STRam® provides easy identification (2016). 5. A. N. Bashkatov, J. Phys. D: Appl. Phys., 38, of materials through a variety of 2543 (2005). 6. M. S. Feld et al., Advances in Fluorescence packaging and barrier layers utilizing Sensing Technology II, 2388, 99–104 (1995). 7. A. Mahadevan-Jansen et al., International 785nm or 1064nm wavelengths! Conference of the IEEE Engineering in Medicine and Biology Society, 6, 2722–2728 (1997).
Learn More about the STRam Peng Zou, Ph.D., is Product Manager at Teledyne Princeton Instruments, Trenton, NJ; www.bwtek.com/STRam e-mail: [email protected]; princetonin- struments.com. +1-302-368-7824 www.bwtek.com [email protected] Laser Focus World
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