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| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 2, Iss. 10 — Oct. 31, 2007
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Development of a handheld Raman microspectrometer for clinical dermatologic applications

Chad A. Lieber and Anita Mahadevan-Jansen  »View Author Affiliations


Optics Express, Vol. 15, Issue 19, pp. 11874-11882 (2007)
http://dx.doi.org/10.1364/OE.15.011874


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Abstract

Although skin is easily accessible to optical methodologies, a portable measurement head is necessary to allow ready spectroscopic interrogation of all anatomic locations. However, most conventional Raman microspectrometers and even dermatologic-specific Raman systems are fixed systems ill-suited to anatomic accessibility. To this end, we have developed a portable Raman microspectrometer system for future dermatologic studies. An in-house-built bench-top system was used to qualify the optical components and design. Based on this system’s layout, a handheld microspectrometer was developed for future clinical application. This system produces similar operating characteristics to the bench-top prototype, and is shown to provide clear Raman spectra from skin tissue measured in vivo in clinically-feasible integration times.

© 2007 Optical Society of America

1. Introduction

Raman spectroscopy is used in several fields for a myriad of analytical purposes. The requirements of the Raman system vary with each application, from temperature and pressure resistant sampling probes for geological study and process monitoring [1

1. Y. E. Gorbaty and G. V. Bondarenko, “High-pressure high-temperature Raman cell for corrosive liquids,” Rev. Sci. Instrum. 66, 4347–4349 (1995). [CrossRef]

,2

2. D. Schiferl, S. K. Sharma, T. F. Cooney, S. Y. Wang, and K. Mohanan, “Multichannel Raman spectrometry system for weakly scattering materials at simultaneous high pressures and high temperatures,” Rev. Sci. Instrum. 64, 2821–2827 (1993). [CrossRef]

], to miniaturized systems for space applications [3

3. D. L. Dickensheets, D. D. Wynn-Williams, H. G. M. Edwards, C. Schoen, C. Crowder, and E. M. Newton, “A novel miniature confocal microscope/Raman spectrometer system for biomolecular analysis on future Mars missions after Antarctic trials,” J. Raman Spectrosc. 31, 633–635 (2000). [CrossRef]

5

5. A. Wang, L. A. Haskin, and E. Cortez, “Prototype Raman spectroscopic sensor for in situ mineral characterization on planetary surfaces,” Appl. Spectrosc. 52, 477–487 (1998). [CrossRef]

]. One area of increasing interest is the use of Raman spectroscopy for biological tissue analysis and diagnosis [6

6. E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, “Prospects for in vivo Raman spectroscopy,” Phys. Med. Biol. 45, R1–59 (2000). [CrossRef] [PubMed]

8

8. N. Stone, C. Kendall, N. Shepherd, P. Crow, and H. Barr, “Near-infrared Raman spectroscopy for the classification of epithelial pre-cancers and cancers,” J. Raman Spectrosc. 33, 564–573 (2002). [CrossRef]

]. Perhaps the most important design considerations for such a system are high signal-to-noise, portability and simplicity. Medical devices must be easily transportable within the clinical environment, and their operation must be simplified for use by clinicians and support personnel with little or no spectroscopic experience. More critically, clinical application requires short acquisition times despite inherently weak biological Raman activity. Furthermore, many cancers originate in specific locations within tissue, e.g. many skin cancers are known to originate in the stratum basale, the single layer of cells that separates the epidermis from the dermis. Measurement of Raman signal from this layer, without interference from surrounding tissue, could increase the effectiveness of Raman spectroscopy for early diagnoses.

Confocal Raman microspectroscopy is a technique that allows for small, fixed measurement volumes, and can thus be used to obtain Raman signal from specific depths and regions, without the need for physical sectioning. This “optical sectioning” capability has been recently utilized to measure hydration gradients across the skin [9

9. P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol. 116, 434–442 (2001). [CrossRef] [PubMed]

,10

10. L. Chrit, P. Bastien, G. D. Sockalingum, D. Batisse, F. Leroy, M. Manfait, and C. Hadjur, “An in vivo randomized study of human skin moisturization by a new confocal Raman fiber-optic microprobe: assessment of a glycerol-based hydration cream,” Skin pharmacology and physiology 19, 207–215 (2006). [CrossRef] [PubMed]

], determine molecular composition of arterial walls [11

11. H. P. Buschman, G. Deinum, J. T. Motz, M. Fitzmaurice, J. R. Kramer, A. van der Laarse, A. V. Bruschke, and M. S. Feld, “Raman microspectroscopy of human coronary atherosclerosis: Biochemical assessment of cellular and extracellular morphologic structures in situ,” Cardiovasc. Pathol. 10, 69–82 (2001). [CrossRef] [PubMed]

,12

12. H. P. Buschman, E. T. Marple, M. L. Wach, B. Bennett, T. C. Schut, H. A. Bruining, A. V. Bruschke, A. van der Laarse, and G. J. Puppels, “In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy,” Anal. Chem. 72, 3771–3775 (2000). [CrossRef] [PubMed]

], and characterize biochemistry of ocular tissue [13

13. R. Erckens, M. Motamedi, and W. March, “Raman spectroscopy for non-invasive characterization of ocular tissue: potential for detection of biological molecules,” J. Raman Spectrosc. 28, 293–299 (1997). [CrossRef]

]. Unfortunately, most published reports of confocal Raman microspectrometer (CRM) design and application involve large, fixed systems which do not lend themselves to easy transportability within a clinical environment [14

14. P. J. Caspers, G. W. Lucassen, H. A. Bruining, and G. J. Puppels, “Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin,” J. Raman Spectrosc. 31, 813–818 (2000). [CrossRef]

16

16. A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol. 119, 64–69 (2002). [CrossRef] [PubMed]

]. Additionally, most commercial CRM systems are based on epi-illumination microscope frames, which require sample placement on an upright or inverted stage. While these are acceptable for in vitro studies or in vivo limb measurements, such designs are ill-suited for routine clinical application.

Recent advances in commercial designs have made Raman systems with portable probes more readily available for applications such as process monitoring and inline inspection, but are not especially suited for clinical use. Raman fiber probe designs such as the InPhotonics RamanProbe (Norwood, MA), the Visionex Enviva (formerly of Atlanta, GA), and Raman immersion probes provide no appreciable depth resolution, typically on the order of several hundred microns or more. Microscope objective-based commercial Raman probes such as the Kaiser Optical Systems MkII (Ann Arbor, MI) and Renishaw RP10 (Gloucestershire, United Kingdom) do not provide a translatable depth of focus or any means for stabilization of the probe/target interface. The River Diagnostics model 3510 (Rotterdam, the Netherlands) is a confocal Raman microspectrometer with stabilizing platform and translating objective, but in a fixed bench-top design. Thus, these commercially available probes fail to incorporate all the features necessary for portable clinical application.

Several research groups have reported in-house-built confocal microspectrometers and microscopes to support or replace commercial systems for in vivo use. Caspers et al. [9

9. P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol. 116, 434–442 (2001). [CrossRef] [PubMed]

,14

14. P. J. Caspers, G. W. Lucassen, H. A. Bruining, and G. J. Puppels, “Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin,” J. Raman Spectrosc. 31, 813–818 (2000). [CrossRef]

,17

17. P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J. 85, 572–580 (2003). [CrossRef] [PubMed]

] have developed several CRM systems for skin study, including an inverted design for in vivo application (now sold by River Diagnostics), similar to the design described by Chrit et al. [10

10. L. Chrit, P. Bastien, G. D. Sockalingum, D. Batisse, F. Leroy, M. Manfait, and C. Hadjur, “An in vivo randomized study of human skin moisturization by a new confocal Raman fiber-optic microprobe: assessment of a glycerol-based hydration cream,” Skin pharmacology and physiology 19, 207–215 (2006). [CrossRef] [PubMed]

,15

15. L. Chrit, C. Hadjur, S. Morel, G. Sockalingum, G. Lebourdon, F. Leroy, and M. Manfait, “In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe,” J. Biomed. Opt. 10, 44007 (2005). [CrossRef] [PubMed]

]. However, all of the reported systems were designed for bench-top use. Rajadhyaksha et al. [18

18. M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: Advances in instrumentation and comparison with histology,” J. Invest. Dermatol. 113, 293–303 (1999). [CrossRef] [PubMed]

,19

19. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995). [CrossRef] [PubMed]

] developed a confocal microscope head mounted to a mechanical arm for clinical study of skin morphology, and were able to obtain confocal images in vivo with axial resolution less than 6 µm. Though this system obtained confocal images at speeds up to 30 frames per second, it collected no spectroscopic data and thus lacks the quantitative and biochemical information provided by the Raman phenomenon. Swindle et al. [20

20. L. D. Swindle, S. G. Thomas, M. Freeman, and P. M. Delaney, “View of normal human skin in vivo as observed using fluorescent fiber-optic confocal microscopic imaging,” J. Invest. Dermatol. 121, 706–712 (2003). [CrossRef] [PubMed]

] describe a modified confocal fluorescence microscope fitted with a scanning fiber imager, and while high resolution fluorescence images are produced, these images required extrinsic fluorescent dye. Kelly et al. [21

21. J. P. Kelly, A. H. Weiss, Q. Zhou, S. Schmode, and A. W. Dreher, “Imaging a child’s fundus without dilation using a handheld confocal scanning laser ophthalmoscope,” Arch. Ophthalmol.-Chic. 121, 391–396 (2003).

] similarly show the resultant images from-, but few of the details of a handheld version of a laser scanning opthalmoscope. Scopes such as these rely on Rayleigh scatter and selective absorption of vessels; a much more intense effect than the weak Raman cross-section. Thus, while several clinical imaging systems hit on various elements of the combination, none bring the ingredients of Raman sensitivity, high spatial resolution, and true clinical portability together.

Alternately, a suitable archetype for a portable CRM for clinical use may be found in space-based applications, which require compact instrumentation to minimize payload weight. Specifically, a portable, pseudo-confocal Raman system for planetary mineral analysis developed by Wang et al. [5

5. A. Wang, L. A. Haskin, and E. Cortez, “Prototype Raman spectroscopic sensor for in situ mineral characterization on planetary surfaces,” Appl. Spectrosc. 52, 477–487 (1998). [CrossRef]

] was reported to be limited primarily by the focusing error induced by the robotic arm which attached the system to a carrier vehicle. This system consisted of a compact probe head, with internal diode laser source, connected to a remote detection system and power supply via a flexible cable. With some modification to the low-power source, this system serves as an ideal model for a clinical CRM.

Several groups have thus developed CRM systems but are limited due to portability, flexibility, or ability to detect the faint biological Raman signal of living tissue. Therefore we have designed a portable Raman microspectroscopy system for optical diagnosis of biological tissues in a clinical setting. First, a benchtop prototype system was constructed using readily available optical mounts and a microscope frame. This system was used as a lab-based system for in vitro testing and served as proof-of-concept for the design and component testing of a truly portable system. The clinical system was built with a combination of commercial mounts and custom-machined parts to allow volume-limited Raman spectral measurements in the clinical environment via a cart-mounted detection system and handheld measurement head. Here, we present a description of the design of the clinical system, along with operating characteristics and sample spectra.

2. Benchtop Raman microscope

An in-house built Raman microscope was used for comparative validation of the handheld system and its optical components, and is shown in figure 1. The laser source, microscope objective, spectrograph, and CCD detector chip from this system were also used in the handheld CRM. The laser source is an external-cavity diode laser (ECDL, Littrow configuration, built in-house) with an 825 nm, 150 mW laser diode (DL8032, Sanyo Electronics, Japan), and 1800 line/mm gold-coated, high modulation diffraction grating (ThermoRGL, Rochester, NY). A near-infrared-optimized, infinity-corrected microscope objective (10 mm f o, 0.35 NA, Nachet, France) focuses the excitation light onto the sample and collects the Raman scatter (an accessory lens corrects for the 200 mm rear-conjugate of the objective, but is not shown in figure). A 200 mm focal length (f i) achromatic doublet launches the Raman collection into a 100 µm-core optical fiber (slightly enlarged over diffraction limit to permit higher collection efficiency and larger collection volume). This fiber is connected to an imaging spectrograph (HoloSpec f/1.8i, Kaiser Optical Systems, Ann Arbor, MI), where the light is dispersed and detected by a liquid nitrogen-cooled, back-illuminated, deep-depletion CCD array (EEV1024EB, Roper Scientific, Trenton, NJ). Spectral resolution of this system is <7 cm-1. Spatial resolution is 2.4 µm by 6.6 µm laterally and 22 µm axially, based on the full-width-at-half-maximum (FWHM) of Raman intensity profiles of a spatially-scanned 1 µm polystyrene bead. The discrepancy between orthogonal lateral resolutions and the enlarged axial resolution was ascribed to residual astigmatism (and, hence, longer beam waist) in the laser beam. Magnification between the tissue and the collection fiber is 20× (=fi/fo).

Fig. 1. Schematic of prototype bench-top confocal Raman microscope (CRM). ECDL=laser, AP=anamorphic prism pair, C+=positive cylindrical lens, BP=bandpass filter, DM=dichroic mirror, M=mirror, BS=50/50 beamsplitter, SP=shortpass filter, LP=longpass filter, Obj=microscope objective.

As this system was designed as a validation prototype for the handheld system, Raman spectra were measured in vivo from the dorsal region of a volunteer’s hand. The hand was placed on the microscope stage while Raman depth spectra were obtained at the surface (as indicated by the video image) and at 10 µm depth intervals. Despite constraining the hand and arm of the subject to restrict movement, muscular and pulsatile micromotions severely reduced the collected Raman intensity, as shown in figure 2 (60 sec. integration). By attaching a thin fused-silica window (1mm thickness) to the microscope stage, and pressing (from beneath the window) the dorsal region of the hand against this window, these micromotions were reduced or eliminated. In this configuration, it was possible to obtain clear Raman spectra from the surface of the skin (as determined by video image) with much higher intensity and signal-to-noise ratio in the same integration times. Similar observations have been made by other groups applying confocal imaging techniques to skin in vivo [9

9. P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol. 116, 434–442 (2001). [CrossRef] [PubMed]

,15

15. L. Chrit, C. Hadjur, S. Morel, G. Sockalingum, G. Lebourdon, F. Leroy, and M. Manfait, “In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe,” J. Biomed. Opt. 10, 44007 (2005). [CrossRef] [PubMed]

,18

18. M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: Advances in instrumentation and comparison with histology,” J. Invest. Dermatol. 113, 293–303 (1999). [CrossRef] [PubMed]

], indicating that a clinical Raman instrument must contain a stabilizing device in order to preserve the spatial resolution of the spectral measurements.

Fig. 2. Noticeable effect of stabilizing window for in vivo Raman measurement of stratum corneum; 60 sec. integration.

3. Clinical Raman microspectrometer

The goal of this portable design was a handheld measurement head and a cart-mounted illumination and detection system suitable for clinical use. The handheld probe utilizes the same detection system and ECDL laser source, slightly modified to allow fiber-coupling. The fiber-coupled ECDL utilizes an 825 nm, 150 mW laser diode with fused cylindrical microlens (VPSL-0830-150, Blue Sky Research, Milpitas, CA) and a high-modulation, 1800 line/mm gold-coated diffraction grating (ThermoRGL, Rochester, NY). A steering mirror allows positioning of the ECDL output for coupling into a 5.5 µm core single-mode (SM) fiber, which is mounted in a stabilized fiber positioner with translatable coupling lens (PAF-X-5-825, Optics for Research, Caldwell, NJ). Output power at the distal end of fiber was 75 mW.

The optical layout of the clinical microspectrometer is similar to that of the bench-top system, but built on a miniaturized scale to enable handheld operation. The chassis for the measurement head was milled from a single block of aluminum, and drilled to accept a combination of ½” diameter commercial optical mounts (9762-K, New Focus, San Jose, CA) and custom holders. As seen in figure 3, light from the ECDL fiber is collimated (C), and the beam is steered using two gold-coated mirrors (M). A narrow bandpass filter (BP) (λcenter=825 nm, bandwidth=5 nm, Chroma Technology, Rockingham, VT) rejects Raman scatter generated in the laser delivery fiber. A shortpass dichroic mirror (DM) (λtransition=835 nm, Chroma Technology, Rockingham, VT) separates the elastic scatter from the Raman signal. A near-infrared-optimized, infinity-corrected objective (same as bench-top system) serves both to focus the laser on the sample and to collect the Raman scatter. The objective is mounted to a translation stage with a motorized linear actuator (T-LA28-S, Zaber Technologies, Richmond, BC) with <0.1 µm resolution to enable automated positioning of the objective. This translation allows control of focal depth within the sample, which is placed against a fused silica window (2 mm thickness) mounted to the chassis. The collected Raman scatter is reflected by the dichroic mirror, and residual elastic scatter is removed via a longpass edge filter (LP) (λtransition=835 nm, Chroma Technology, Rockingham, VT). A concave silver mirror (CM, f i=100 mm) focuses the collected light into a 100 µm core diameter optical fiber, which serves as the confocal aperture. Magnification between the tissue and the collection fiber is 10×. This collection fiber is connected to the detection system, which is housed on a wheeled cart for portability within the clinic. The detection system consists of the same holographic imaging spectrograph and TE-cooled version of the CCD detector chip (256BR, Roper Scientific, Trenton, NJ) used in the bench-top system.

Fig. 3. Schematic of handheld CRM with components and approximate dimensions. C=collimator, M=mirror, BP=bandpass filter, DM=dichroic mirror, LP=longpass filter, CM=concave mirror.

As the handheld system contains no visual imaging capabilities, a targeting system was developed to allow accurate identification of measurement location. This targeting system consists of a guiding collar with a removable reticle. The collar attaches to the patient’s skin via adhesive tape, and fits a key in the microscope window. The removable reticle serves both to guide placement of the probe and to allow accurate identification of the Raman measurement location.

3.1 Clinical microspectrometer performance evaluation

Because the handheld system uses the same laser source and detection system as the benchtop system previously described, the spectral resolution of the handheld system is similarly <7 cm-1. Focal position of the handheld system was determined experimentally by placing a thin (6 µm) cellophane film against the silica window and collecting Raman spectra as the objective was scanned through its full range of motion in 1 µm increments. This produced an intensity profile for each Raman peak in the cellophane spectrum with the maximum intensity corresponding to the objective’s focal position at the window surface. The intensity profiles of the Raman peaks at 810, 971, 1167, 1328, and 1458 cm-1 were each normalized to maximum and minimum intensities, then the normalized profiles were averaged and fit to a Gaussian distribution (Fig. 4(a)). The FWHM of this profile corresponds to the axial resolution of the microscope, and was measured to be ~14 µm. This value is very near that predicted by geometric optical theory (13.5 µm), as detailed in [22

22. R. Tabaksblat, R. J. Meier, and B. J. Kip, “Confocal Raman microspectroscopy: Theory and application to thin polymer samples,” Appl. Spectrosc. 46, 60–68 (1992). [CrossRef]

], and improves upon the bench-top system resolution due to the improved illumination beam profile (via SM fiber-coupling), The slight asymmetry in this profile is likely due to refractive effects of the silica window. Lateral resolution of the system was not measured, but theoretically determined to be ~1.2 µm, according to the derivations in [23

23. T. Wilson and A. R. Carlini, “Size of the detector in confocal imaging systems,” Opt. Lett. 12, 227–229 (1987). [CrossRef] [PubMed]

], (Fig. 4(b)). Lateral placement of the probe is accurate to within 400 µm, as determined by the mechanical tolerances of the targeting system.

Fig. 4. (A) Mean intensity (normalized) of cellophane Raman peaks at 810, 971, 1167, 1328, and 1458 cm-1 versus axial position of the objective, Gaussian approximation used for estimation of axial resolution, and theoretical response. FWHM is shown to be ~14 µm. (B) Theoretical lateral response used to determine lateral resolution; FWHM is shown to be ~1.2 µm.

3.2 Clinical microspectrometer example spectra

Example Raman spectra are shown in figure 5. Acetamidophenol (Fig. 5(a)) produces very clear Raman spectra in short integration times (10 seconds), as does the cellophane film used for performance evaluation (Fig. 5(b)). Biological Raman spectra are also easily resolvable, as shown in the raw spectrum of human skin (Fig. 5(c)) measured in vivo, both before and after noise-smoothing with a 2nd order Savitzky-Golay filter and subtraction of intrinsic tissue fluorescence. Silica Raman bands at 440, 490, 600, and 800 cm-1 are evident in the handheld spectra, and are likely due to impurities in the probe window. However, these silica bands do not affect the “biological fingerprint” region of the Raman spectra at ~850–1800 cm-1. The SNR of the in vivo skin measurements is decreased as compared to in vitro skin samples measured by the bench-top system (not shown). This is expected, and is likely due to the innate biochemical and metabolic processes of living tissue, which do not occur in the in vitro samples, causing increased turbidity in the measured sample volume. However, the dominant tissue peaks are still easily resolved upon processing.

Fig. 5. Raman spectra collected by the handheld CRM system: (A) acetaminophen, (B) cellophane film, and (C) human skin measured in vivo before and after noise processing.

4. System comparison

The handheld microspectrometer presented here was developed in-house as a portable system for clinical use. In order to validate the capability of the in-house built systems, spectral quality of measured samples were compared using the systems presented here and a commercially available CRM. Raman spectra were acquired from purified powder of the amino acid tryptophan using a commercial system (System 1000, Renishaw, UK) with an Ar-pumped Ti:sapph laser source, as well as the bench-top and handheld systems described here. Figure 6 shows the spectra measured from the three systems after binning the spectra to 3.5 cm-1 (without any further processing). Similar intensities and relative intensities are observed over the entire measured range of wavenumbers. All of the major tryptophan peaks are clearly resolved in the in-house systems’ spectra, and with higher spectral resolution than the Renishaw system used for comparison.

Fig. 6. Raman spectra of powdered tryptophan measured by the bench-top and handheld CRM systems developed in-house and with a commercially available CRM.

5. Summary and outlook

Most conventional Raman microscopes are designed for bench-top operation and cannot be easily translated for clinical application. A bench-top Raman microscope was developed in-house, to provide a prototyping platform for optical components and design of a clinical system. Based on this system’s layout, a handheld Raman microspectrometer with portable measurement system was developed for clinical studies. This system provides operating characteristics similar to the bench-top prototype, and enables collection of biological Raman spectra in clinically feasible measurement times. Furthermore, the spectra obtained from both systems were shown to be comparable to those acquired via much more expensive commercial systems.

Spatial resolution of the bench-top microscope was compromised due to residual astigmatism in the ECDL. Thus the anamorphic prism pair and cylindrical lens do not achieve diffraction-limited circularization and collimation of the beam. An alternative approach would be to couple the bench-top ECDL into a single-mode fiber, as was done for the handheld system. The single-mode fiber coupling of the laser in the clinical system allows diffraction-limited illumination, resulting in higher axial resolution than the bench-top system. While this resolution is still not considered truly confocal, it is within the intended range of multiple-cell measurement in tissue. Additional studies are planned to evaluate alternative stabilization and targeting methods (pinholes, reticles, different window materials, etc.) to maximize system performance.

The clinical system is currently being used in an ongoing study to assess the feasibility of Raman microspectroscopy for clinical skin cancer diagnosis. Using the results of the analysis of system performance described here, a 3rd generation Raman microscope is currently being designed to incorporate imaging capabilities both for guidance as well as additional diagnostic value. This next-generation probe will use conventional optics as well as micro-opto-electromechanical systems (MOEMS) to yield a penlight sized probe for future studies. The end product will be a micro-probe ideal for clinical application of Raman spectroscopy in a variety of tissue sites.

Acknowledgements

The authors thank Dr. Nicholas Stone for providing the tryptophan spectrum using the Renishaw system. Funding for this project was provided by the National Institutes of Health (CA86283, CA95995).

References and links

1.

Y. E. Gorbaty and G. V. Bondarenko, “High-pressure high-temperature Raman cell for corrosive liquids,” Rev. Sci. Instrum. 66, 4347–4349 (1995). [CrossRef]

2.

D. Schiferl, S. K. Sharma, T. F. Cooney, S. Y. Wang, and K. Mohanan, “Multichannel Raman spectrometry system for weakly scattering materials at simultaneous high pressures and high temperatures,” Rev. Sci. Instrum. 64, 2821–2827 (1993). [CrossRef]

3.

D. L. Dickensheets, D. D. Wynn-Williams, H. G. M. Edwards, C. Schoen, C. Crowder, and E. M. Newton, “A novel miniature confocal microscope/Raman spectrometer system for biomolecular analysis on future Mars missions after Antarctic trials,” J. Raman Spectrosc. 31, 633–635 (2000). [CrossRef]

4.

H. G. Edwards, E. M. Newton, D. L. Dickensheets, and D. D. Wynn-Williams, “Raman spectroscopic detection of biomolecular markers from Antarctic materials: evaluation for putative Martian habitats,” Spectrochim. Acta A 59, 2277–2290 (2003). [CrossRef]

5.

A. Wang, L. A. Haskin, and E. Cortez, “Prototype Raman spectroscopic sensor for in situ mineral characterization on planetary surfaces,” Appl. Spectrosc. 52, 477–487 (1998). [CrossRef]

6.

E. B. Hanlon, R. Manoharan, T. W. Koo, K. E. Shafer, J. T. Motz, M. Fitzmaurice, J. R. Kramer, I. Itzkan, R. R. Dasari, and M. S. Feld, “Prospects for in vivo Raman spectroscopy,” Phys. Med. Biol. 45, R1–59 (2000). [CrossRef] [PubMed]

7.

A. Mahadevan-Jansen and R. Richards-Kortum, “Raman spectroscopy for the detection of cancers and precancers,” J. Biomed. Opt. 1, 31–70 (1996). [CrossRef]

8.

N. Stone, C. Kendall, N. Shepherd, P. Crow, and H. Barr, “Near-infrared Raman spectroscopy for the classification of epithelial pre-cancers and cancers,” J. Raman Spectrosc. 33, 564–573 (2002). [CrossRef]

9.

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol. 116, 434–442 (2001). [CrossRef] [PubMed]

10.

L. Chrit, P. Bastien, G. D. Sockalingum, D. Batisse, F. Leroy, M. Manfait, and C. Hadjur, “An in vivo randomized study of human skin moisturization by a new confocal Raman fiber-optic microprobe: assessment of a glycerol-based hydration cream,” Skin pharmacology and physiology 19, 207–215 (2006). [CrossRef] [PubMed]

11.

H. P. Buschman, G. Deinum, J. T. Motz, M. Fitzmaurice, J. R. Kramer, A. van der Laarse, A. V. Bruschke, and M. S. Feld, “Raman microspectroscopy of human coronary atherosclerosis: Biochemical assessment of cellular and extracellular morphologic structures in situ,” Cardiovasc. Pathol. 10, 69–82 (2001). [CrossRef] [PubMed]

12.

H. P. Buschman, E. T. Marple, M. L. Wach, B. Bennett, T. C. Schut, H. A. Bruining, A. V. Bruschke, A. van der Laarse, and G. J. Puppels, “In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy,” Anal. Chem. 72, 3771–3775 (2000). [CrossRef] [PubMed]

13.

R. Erckens, M. Motamedi, and W. March, “Raman spectroscopy for non-invasive characterization of ocular tissue: potential for detection of biological molecules,” J. Raman Spectrosc. 28, 293–299 (1997). [CrossRef]

14.

P. J. Caspers, G. W. Lucassen, H. A. Bruining, and G. J. Puppels, “Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin,” J. Raman Spectrosc. 31, 813–818 (2000). [CrossRef]

15.

L. Chrit, C. Hadjur, S. Morel, G. Sockalingum, G. Lebourdon, F. Leroy, and M. Manfait, “In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe,” J. Biomed. Opt. 10, 44007 (2005). [CrossRef] [PubMed]

16.

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol. 119, 64–69 (2002). [CrossRef] [PubMed]

17.

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J. 85, 572–580 (2003). [CrossRef] [PubMed]

18.

M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: Advances in instrumentation and comparison with histology,” J. Invest. Dermatol. 113, 293–303 (1999). [CrossRef] [PubMed]

19.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995). [CrossRef] [PubMed]

20.

L. D. Swindle, S. G. Thomas, M. Freeman, and P. M. Delaney, “View of normal human skin in vivo as observed using fluorescent fiber-optic confocal microscopic imaging,” J. Invest. Dermatol. 121, 706–712 (2003). [CrossRef] [PubMed]

21.

J. P. Kelly, A. H. Weiss, Q. Zhou, S. Schmode, and A. W. Dreher, “Imaging a child’s fundus without dilation using a handheld confocal scanning laser ophthalmoscope,” Arch. Ophthalmol.-Chic. 121, 391–396 (2003).

22.

R. Tabaksblat, R. J. Meier, and B. J. Kip, “Confocal Raman microspectroscopy: Theory and application to thin polymer samples,” Appl. Spectrosc. 46, 60–68 (1992). [CrossRef]

23.

T. Wilson and A. R. Carlini, “Size of the detector in confocal imaging systems,” Opt. Lett. 12, 227–229 (1987). [CrossRef] [PubMed]

OCIS Codes
(120.3890) Instrumentation, measurement, and metrology : Medical optics instrumentation
(170.1870) Medical optics and biotechnology : Dermatology
(170.4580) Medical optics and biotechnology : Optical diagnostics for medicine
(170.6510) Medical optics and biotechnology : Spectroscopy, tissue diagnostics
(300.6450) Spectroscopy : Spectroscopy, Raman

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: June 11, 2007
Revised Manuscript: August 16, 2007
Manuscript Accepted: August 25, 2007
Published: September 4, 2007

Virtual Issues
Vol. 2, Iss. 10 Virtual Journal for Biomedical Optics

Citation
Chad Lieber and Anita Mahadevan-Jansen, "Development of a handheld Raman microspectrometer for clinical dermatologic applications," Opt. Express 15, 11874-11882 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-15-19-11874


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References

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  8. N. Stone, C. Kendall, N. Shepherd, P. Crow, and H. Barr, "Near-infrared Raman spectroscopy for the classification of epithelial pre-cancers and cancers," J. Raman Spectrosc. 33, 564-573 (2002). [CrossRef]
  9. P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, "In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles," J. Invest. Dermatol. 116, 434-442 (2001). [CrossRef] [PubMed]
  10. L. Chrit, P. Bastien, G. D. Sockalingum, D. Batisse, F. Leroy, M. Manfait, and C. Hadjur, "An in vivo randomized study of human skin moisturization by a new confocal Raman fiber-optic microprobe: assessment of a glycerol-based hydration cream," Skin pharmacology and physiology 19, 207-215 (2006). [CrossRef] [PubMed]
  11. H. P. Buschman, G. Deinum, J. T. Motz, M. Fitzmaurice, J. R. Kramer, A. van der Laarse, A. V. Bruschke, and M. S. Feld, "Raman microspectroscopy of human coronary atherosclerosis: Biochemical assessment of cellular and extracellular morphologic structures in situ," Cardiovasc. Pathol. 10, 69-82 (2001). [CrossRef] [PubMed]
  12. H. P. Buschman, E. T. Marple, M. L. Wach, B. Bennett, T. C. Schut, H. A. Bruining, A. V. Bruschke, A. van der Laarse, and G. J. Puppels, "In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy," Anal. Chem. 72, 3771-3775 (2000). [CrossRef] [PubMed]
  13. R. Erckens, M. Motamedi, and W. March, "Raman spectroscopy for non-invasive characterization of ocular tissue: potential for detection of biological molecules," J. Raman Spectrosc. 28, 293-299 (1997). [CrossRef]
  14. P. J. Caspers, G. W. Lucassen, H. A. Bruining, and G. J. Puppels, "Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin," J. Raman Spectrosc. 31, 813-818 (2000). [CrossRef]
  15. L. Chrit, C. Hadjur, S. Morel, G. Sockalingum, G. Lebourdon, F. Leroy, and M. Manfait, "In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe," J. Biomed. Opt. 10, 44007 (2005). [CrossRef] [PubMed]
  16. A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, "Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy," J. Invest. Dermatol. 119, 64-69 (2002). [CrossRef] [PubMed]
  17. P. J. Caspers, G. W. Lucassen, and G. J. Puppels, "Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin," Biophys. J. 85, 572-580 (2003). [CrossRef] [PubMed]
  18. M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Anderson, and R. H. Webb, "In vivo confocal scanning laser microscopy of human skin II: Advances in instrumentation and comparison with histology," J. Invest. Dermatol. 113, 293-303 (1999). [CrossRef] [PubMed]
  19. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Invest. Dermatol. 104, 946-952 (1995). [CrossRef] [PubMed]
  20. L. D. Swindle, S. G. Thomas, M. Freeman, and P. M. Delaney, "View of normal human skin in vivo as observed using fluorescent fiber-optic confocal microscopic imaging," J. Invest. Dermatol. 121, 706-712 (2003). [CrossRef] [PubMed]
  21. J. P. Kelly, A. H. Weiss, Q. Zhou, S. Schmode, and A. W. Dreher, "Imaging a child's fundus without dilation using a handheld confocal scanning laser ophthalmoscope," Arch. Ophthalmol. (Chicago). 121, 391-396 (2003).
  22. R. Tabaksblat, R. J. Meier, and B. J. Kip, "Confocal Raman microspectroscopy: Theory and application to thin polymer samples," Appl. Spectrosc. 46, 60-68 (1992). [CrossRef]
  23. T. Wilson, and A. R. Carlini, "Size of the detector in confocal imaging systems," Opt. Lett. 12, 227-229 (1987). [CrossRef] [PubMed]

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