Raman Imaging in Biochemical and Biomedical Applications

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Raman Imaging in Biochemical and Biomedical Applications. Diagnosis and Treatment of Breast Cancer Halina Abramczyk* and Beata Brozek-Pluska Laboratory of Laser Molecular Spectroscopy, Institute of Applied Radiation Chemistry, Lodz University of Technology, Wroblewskiego 15, 93-590 Lodz, Poland methods for obtaining images, X-rays imaging, mammography, CT (computer tomography), PET (positron emission tomography), single photon emission computed tomography (SPECT), MRI (magnetic resonance imaging), and ultrasound imaging,10−31 many other methods are “under research” to improve the sensitivity and specificity of the diagnostic methods. Moreover, cancer is a multifactorial disease, and molecular imaging methods must demonstrate various mechanisms and phases of pathogenesis. Thus, the combination of information using different modalities, various imaging agents, and various biomarkers is required. As a result there has been an increased use of imaging of biomarkers to monitor metabolism, cell proliferation, cell migration, receptor expression, gene expression, signal transduction, hypoxia, CONTENTS apoptosis, angiogenesis, and vascular function.1 Imaging systems produce images that must have differences in contrast. 1. Introduction A The differences in contrast can be achieved by exploring 2. Linear Raman Imaging. Confocal Raman Microschanges in the endogenous physical properties of the biological copy B tissue or by the use of exogenous agents. The endogenous 3. Surface-Enhanced Raman spectroscopy D methods need no external labeling and use the properties of the 4. Tip-Enhanced Raman Spectroscopy E biological tissue as a contrast to produce the image. The 5. Raman-Based Spectroscopy as a Detection endogenous mechanisms are based on the following properties Technique for Breast Cancer Diagnostics F of the tissue: radiation absorption, Raman scattering, reflection 6. “Optical Biopsy” of Human Breast Cancer by and transmission, magnetic relaxivity, magnetic susceptibility, Raman Imaging I water molecule diffusion, magnetic spin tagging, oxygenation, 7. Conclusions L spectral distribution, temperature, electrical impedance, acousAuthor Information M tic frequency shifts, mechanical elasticity, and many others.1 Corresponding Author M The exogenous agents are based on similar properties, but in Notes M contrast to the endogenous methods, external labeling with, for Biographies M example, radioactive substances, fluorescent dyes, or nanoAcknowledgments M particles is needed. Practically, it is usually performed with References M contrast agent injection or drinking and waiting until it is distributed in the body to more clearly show the boundaries between organs or between organs and tumors. 1. INTRODUCTION There is strong interest in the development of highly One of the fundamental goals of biochemistry, biophysics, and sensitive imaging optical technologies that would shift medical molecular biology is to understand the complex spatiotemporal diagnosis to the next level of state-of-the-art in biomedical interactions of molecules at biological interfaces. To study these diagnostics, pathogen detection, gene identification, gene interactions, researchers commonly use in vivo cellular imaging mapping, and DNA sequencing. and in vitro assay detection. Molecular images of human cells, The optical imaging systems are ideally suited for early tissues, and organs, and the human body are able to monitor 1−9 detection of intraepithelial diseases, including most cancers, and various structures or mechanisms related to pathology. The to assess tumor margins and response to therapy. images are produced as a result of interaction of energy from Optical methods offer several significant advantages over the the external field with human tissue. The energy can be in the routine clinical imaging methods, including (a) noninvasiveness form of radiation, magnetic or electric fields, or acoustic energy. through the use of safe, nonionizing radiation, (b) display of Most clinical imaging systems are based on the interaction of contrast between soft tissues based on optical properties of the electromagnetic radiation with body tissues or fluids. Ultrasound is an exception as it is based on the reflection, scattering, and frequency shift of acoustic waves. Although many Received: February 14, 2012 radiologists consider for practical applications only a few © XXXX American Chemical Society

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dx.doi.org/10.1021/cr300147r | Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Scheme of the Rayleigh and linear spontaneous Raman scattering.

tissue, (c) a facility for continuous bedside monitoring, and (d) high spatial resolution (less than 0.5 μm lateral resolution in the visible range).32 Confocal, laser-based fluorescence microscopy became a golden standard for optical imaging, offering the construction of 3D images at the spatial lateral resolution determined by the wavelengths of the probing light and the axial resolution corresponding to about twice this value.33−39 Consequently, the typical volume that can be probed in the visible range by fluorescence microscopy is about 500 nm × 500 nm × 1000 nm. At this resolution we can see the distribution and concentration of probes in particular areas of the tissue, although the molecular details of subcellural organization and structure, such as chemical covalent bonds or the structure of the fluorescent chromophores attached to a protein receptor, cannot be monitored. Raman imaging is an emerging field that has generated a lot of interest both for the label-free Raman methods and for the unique properties of nanoparticles applied as contrast agents and Raman reporters of proteins and nucleic acid targets. Raman imaging (RI) has now reached a level of sophistication that makes it competitive with more classical methods of confocal fluorescence microscopy due to the following reasons: (1) RI provides direct biochemical information (vibrational fingerprint). The structural fingerprinting is very effective owing to its narrow and highly resolved bands (0.1 nm compared with a bandwidth of 10−50 nm for fluorescence). (2) RI can monitor biological tissue without any contrast agents, because display of contrast between soft tissues is based on Raman cross section scattering properties. (3) The resonance Raman effect and surface-enhanced Raman spectroscopy (SERS) are able to amplify the probe’s signal to the point that it can be detected. (4) RI combined with contrast-enhancing probes active in Raman spectroscopy and able to cross cell membranes may identify specific gene products, DNA sequences, and intracellular processes. (5) Raman polarization methods provide a tool for visualization of various structures and pathologies localized in tissue that are orientation sensitive. This results from the fact that the laser beam of different polarizations propagates in tissues differently. (6) Raman spectra and autofluorescence of the sample can be directly probed. (7) RI has a high spatial resolution (less than 0.5 μm lateral resolution in the visible range).

(8) Confocal microendoscopes with catheter-based microtransducer advancements and optically fiber coupled spectrometers are extending the in vivo mode of operation in real time. (9) Surface-enhanced Raman scattering has also shown potential to be invaluable in the research field of genomics, such as determining the sequence of DNA,40,41 SERS immunoassays,42 proteomics,43 analysis of hemoglobin,44 enzymes,45 and nucleic acids,46 and multiplex detection of cancer biomarkers.47,48 Regarding these aspects of research, one can state that Raman imaging is a patchwork of interdisciplinary research that combines the fields that have already been preserved for physical scientists and on the other side for molecular biologists. That is, the physical background is needed to understand and adopt the technology by the life science and clinical communities, and on the other side the physicist must learn at least the principles of biochemistry, genetics, and molecular biology to develop the next generation of molecular diagnostic products. This review is our interpretive summary of literature results, with some background research on breast cancer tissue from our laboratory included. There is certainly no shortage of important problems to solve. In the context of this review we ask what obstacles remain, what unanticipated developments might come into play, and which applications will be important in the near future of Raman imaging. We will demonstrate that Raman-based methods offer unsurpassed sensitivity and multiplexing capabilities to the field of molecular imaging that will translate this optical technique into a novel clinical diagnostic tool in biomedical applications.

2. LINEAR RAMAN IMAGING. CONFOCAL RAMAN MICROSCOPY First, we will discuss mechanisms and methods of Raman-based molecular diagnostics that improved resolution, sensitivity, selectivity, and multiplexing, such as confocal microscopy, nanoparticle-enhanced Raman spectroscopy, and Ramantargeted spectroscopy. Raman scattering is inelastic scattering, and measuring the difference between the energy of the incident photons and scattered photons, one can obtain information about the vibrational energy and frequencies (Figure 1). The energy of the scattered photon can be shifted to lower frequencies (Stokes component) when the incident photon gives part of its energy to the environment bath (mainly vibrations) or to higher frequencies (anti-Stokes component) when the incident photon gets the energy from the bath. B


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The levels denoted as E0 and E1 represent electronic energy levels, while the levels numbered with the vibrational quantum number υ represent the vibrational energy levels. If the sample is illuminated with photons of energy ℏωL, smaller than the resonance energy ΔE = E1 − E0, all of the photons that interact with the sample are not absorbed, but cause the potential energy of the interacting molecules to be raised to virtual state ℏωL above the ground state. Almost immediately most molecules return to the ground state through the emission of photons of the same energy as the incident photons. This elastic scattering is called the Rayleigh scattering. A small fraction of the molecules drop back to the first excited vibrational state (υ = 1) instead of to the ground state. Since the energies of the incident and the scattered photons are different, the scattering is inelastic, and the process is known as Stokes Raman scattering, with the scattered radiation observed at lower energy, ℏ(ωL − ωvib). Molecules that are already in the excited vibrational state (υ = 1) will undergo an analogous effect when illuminated with a laser light. When the excited molecules drop back to the ground vibrational state (υ = 0), the scattered radiation will be observed at higher energy, ℏ(ωL + ωvib). This scattering is known as anti-Stokes Raman scattering, The frequency ωvib denotes the frequency of a given vibrational mode of the molecule. To describe Raman scattering, a fully quantum-mechanical theory is required, but some intuitive description can also be obtained from a classical picture. The electric field E0 cos(k·r − ωLt) drives the electron displacements that induce the polarization P in a medium modulated in time that in turn generates a wave at the same frequency, ωL (Rayleigh scattering). When the dipole oscillations are modulated additionally by the molecule vibrations at frequency ωvib, the waves at ωL − ωvib (Stokes Raman scattering) or ωL + ωvib (anti-Stokes Raman scattering) are generated.49,50 Raman microscopy has many advantages over fluorescence microscopy. First, Raman spectroscopy needs no external labeling and uses the induced polarizability (polarizability tensor derivative with respect to the normal coordinates) as a contrast in generating the vibrational spectra. Thus, the labelfree Raman images rely on the contrast provided by the different vibrational spectra in various regions of the biological tissue. Second, biochemical signatures of the molecules are much richer as each component of the tissue, such as nucleic acids, lipids, biological chromophores, and proteins, provides its own pattern of vibrational behavior in different spectral regions. Second, the new generation of Raman microscopes can offer a powerful nondestructive and noncontact method of sample analysis. One of the greatest benefits is the use of a confocal Raman microscope design.51,52 This enables a very small sample area or volume to be analyzed down to the micrometer scale. Combining this micro Raman analysis with automated focusing, XYZ movement, makes possible production of “chemical” images of a sample with a higher spatial resolution than ever before. Raman scattering can be observed microscopically using instrumentation presented in Figure 2, which is very similar to that of laser fluorescence microscopy, but instead of the fluorescence signal, the Raman scattering signal is detected. Light from a laser enters through a small pinhole and expands to fill the pupil area of a microscope objective lens. The incident light is focused on the biological sample by means of a high numerical aperture (NA) objective lens to the resolution corresponding to the diffraction limit. The diffraction limits for

Figure 2. Confocal Raman microscope: 1, laser; 2, single-mode fiber; 3, objective; 4, scan table; 5, filter; 6, multimode fiber; 7, monochromator; 8, CCD detector; 9, white light illumination; 10, Z-stage for focusing.

lateral and axial spatial resolution, δlat and δax, are determined by δ lat = 0.61λ/NA

δax = 2λn/(NA)2

(1)

where λ is the wavelength of the exciting light, n is the refractive index, and NA is the numerical aperture of the objective. Light scattered back from the illuminated spot on the sample is collected by the objective, propagates through the dichroic mirror, and is directed to a pinhole placed in front of the spectrometer. As was mentioned, placing a pinhole aperture in the emission light path at a conjugate location of the focal volume in the specimen allows 3D resolution to be attained. Indeed, only photons generated inside this volume will be focused at the pinhole aperture and can be transmitted to the detector in contrast to photons from outside this focal volume, which are defocused at the aperture plane and will be blocked. This ability to reject light from above or below the focal plane enables the confocal microscope to perform depth discrimination and provides a solution for optical tomography. Scanning of the specimen is achieved by moving the laser beam along the specimen’s surface or a moving the microscope table in a raster pattern. A true 3D image can be processed by taking a series of confocal images at successive planes into the specimen. Scanning along the x and y axes provides mapping of the sample, while scanning along the z axis provides image sectioning of the sample, without the need to section it physically. In the nonconfocal Raman image, a specimen plane outside the focal plane deteriorates the information of interest from the focal plane. In the confocal image, specimen details blurred in nonconfocal imaging become distinctly visible, and the image throughout is greatly improved in contrast. To create Raman images, the following steps have to be implemented: (1) The microscope has to be coupled to the Raman spectrometer. (2) Raman spectra of spatially resolved points have to be collected by a raster-scanning of the focused laser beam over the sample or by moving the sample through the laser focus in a raster pattern via a high-resolution microscope stage. The resulting data sets of several thousand individual spectra can be converted into spectral images. C


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(3) Spectral images have to be improved with different statistical methods, such as principal component analysis (PCA), vertex component analysis (VCA), and hierarchical cluster analysis (HCA).53−55 Optical Raman imaging56−59 has emerged from the Raman spectroscopy technique60−90 as a new modality which enables real time, noninvasive, high-resolution imaging of epithelial tissue, with a particular focus in this review on breast cancer diagnosis.56−64 The method has a potential to replace the conventional biopsy and histopathological analysis by optical Raman biopsy. The in vivo mode of operation in real time becomes possible due to confocal microendoscopy advancements, optically fiber coupled spectrometers, and semiconductor technology. Raman microscopy for biological and medical applications generally uses near-infrared (NIR) lasers that reduce the risk of damaging the specimen and lower fluorescence by applying higher wavelengths. However, the intensity of NIR Raman is low (owing to the 1/λ4 dependence of the Raman scattering intensity), and most detectors require very long collection times. Recently, more sensitive detectors have become available, making the technique easily applicable.

3. SURFACE-ENHANCED RAMAN SPECTROSCOPY One of the most serious limitations of conventional Raman spectroscopy is its low sensitivity, much lower than that of fluorescence spectroscopy. To compare, the effective fluorescence cross sections can be as high as 10−16 cm2/molecule for high quantum yield fluorophores. In contrast, the cross sections for Raman scattering are several orders of magnitude smaller, ranging from 10−30 to 10−25 cm2/molecule. The inherently small Raman scattering cross section results in a small intensity of Raman signals and long spectral acquisition times per point. However, Raman scattering efficiency can be enhanced by factors of >108 when a substance is adsorbed on or near rough metal surfaces. The Raman scattering amplification associated with this phenomenon is known as surface-enhanced Raman spectroscopy.91 These huge increases in Raman scattering are primarily due to the increased intensity of the electromagnetic fields present at the surface of these metals. SERS preserves the essential features of Raman scattering, yet the surface enhancement by the metal nanoparticle allows unique spectra to be acquired from a variety of adsorbed species. The enhancement can further increase even up to 11 orders and alimit of detection of 10−16 M in surface-enhanced resonance Raman spectroscopy (SERRS),92,93 13 orders in surface-enhanced Raman scattering−scanning near-field optical microscopy (SERS−SNOM),43 or even 14−15 orders of magnitude and detection down to 10−10 to 10−14 M in nonlinear optical microscopy (e.g., coherent anti-Stokes Raman scattering (CARS)).44−46,94−97 Therefore, with further advancements of SERS combined with highly sensitive SERS-active probes, Raman techniques may open up a new direction for high-sensitive high-throughput signal detection, even down to the single-molecule level, in nanomedicine and bioimaging.98−102 The enhancement mechanism for SERS comes from intense localized fields arising from surface plasmon resonance in metallic (e.g., Au, Ag, Cu) nanostructures with sizes on the order of tens of nanometers. The sizes, shapes, and compositions of metal nanoparticles can be systematically varied to produce materials with distinct light-scattering properties.103 Figure 3 shows nanoparticles of Ag generated

Figure 3. Nanoparticles of Ag generated according to the procedure described in ref 102 and their absorption spectra (results from the Laboratory of Laser Molecular Spectroscopy).

according to the procedure described in ref 104 and their absorption. One can see that localized surface plasmon resonances can be tuned in the spectral range from the visible up to the far-IR in the case of silver nanostructures. The amplification of Raman scattering in the SERS effect is generally generated by two mechanisms: (i) electromagneticfield enhancement through the localization of optical fields in metallic nanostructures and (ii) chemical or electronic enhancement due to the increase of the Raman cross section when the molecule or lattice is in contact with metal nanostructures. Despite the numerous advantages, SERS shows several limitations, such as (i) the susceptibility of the SERS signal to dramatic changes in the intensity owing to colloidal aggregation, (ii) possible side reactions owing to the catalytic activity of the metal nanoparticles, (iii) signal enhancement of the analyte overlapping the nanoparticle spectrum, and (iv) promotion of toxic reactions.105 There are many types of hybrid systems that exploit the SERS effect: SERRS, FT near-IR−SERS microspectroscopy, SERS−SNOM, or tip-enhanced Raman spectroscopy (TERS).106−111 SERRS combines the SERS effect and the resonance Raman spectroscopy (RRS) enhancement. The great enhancement of the Raman scattering intensity signal in RRS occurs when the frequency of the laser beam is tuned to be near an electronic transition (resonance) of molecules, usually dyes. The signal is D


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distance from the specimen, the radiation experiences significant diffraction and enters the far-field regime. A schematic of the main modules of an inverted optical microscope-based SNOM system is presented in Figure 5. The

further amplified by the SERS effect for these molecules in contact with a metal. A number of different metals in different formats have been used to provide SERRS, usually roughened metal surfaces or metal nanostructures are employed. To obtain the SERRS signal from the appropriate surface covered with a dye, the laser excitation frequency must be close to that of the absorption maximum of the dye and the surface plasmon of the metal. As silver and gold have plasmon frequencies in the visible region, they are suitable for use with standard Raman instruments. Many important components of biological tissue (e.g., DNA) do not meet the requirements for SERRS due to the lack of a suitable visible chromophore. To overcome this disadvantage and to make a similar approach for DNA SERRS, we need to add a label either as a non-sequence-specific intercalator or as a specific label covalently attached to a unique probe sequence. There are commercially available fluorophores and specifically designed dyes which can be used to label DNA strands.112 In the case of nucleic acid detection, it has been demonstrated that SERRS provides femtomolar sensitivity and multiplexed detection of DNA and RNA targets.112 SERS combined with SNOM enables us to not only improve the sensitivity, but also increase the optical and spatial resolution as well. In contrast with conventional microscopy with far-field scanning, in near-field microscopy both the radius of the illuminating source and the separation distance between the sources of radiation are smaller than the wavelength of the incident light. As a consequence, imaging radiation illuminating a specimen is confined by the dimensions of a subwavelength diameter aperture. By scanning the specimen point-by-point at a distance less than the aperture diameter, one can achieve the image with an optical resolution greater than the diffraction limit, which can be lower than 100 nm in the visible. The typical resolutions for most SNOM instruments range around 50 nm, which is 5 or 6 times better than that achieved by the conventional Raman confocal microscopes working in the diffraction limit of the visible spectral range. To go beyond the diffraction limit in the near-field regime, the separation distance between the probe and specimen surface must be much lower than the excitation wavelength, typically on the order of a few nanometers. A schematic illustration of the SNOM method is presented in Figure 4. The main difference between the near-field and far-field regimes is related to the fact that radiation near the source is highly collimated within the near-field region and the mode of light propagation is primarily evanescent (and parallel to the specimen surface). After propagation of a few wavelengths

Figure 5. Schematic modules of an inverted optical microscope-based SNOM system.

laser excitation source is coupled into a fiber optic probe for specimen illumination, with the probe tip movement being monitored through an optical feedback loop incorporating a second laser focused on the tip. The motion of the probe tip, translational stage movement, and acquisition and display of optical and topographic (or other force) images is controlled through additional electronics and the system computer. Several different techniques have been employed to monitor the z-position of the probe tip and its instantaneous separation from the specimen surface. To date, the two most commonly employed mechanisms of tip positioning have been optical methods that monitor the tip vibration amplitude (usually interferometric) and a nonoptical tuning fork technique.113 The greatest advantage of SNOM probably rests in its ability to provide optical and spectroscopic data at high spatial resolution, in combination with simultaneous topographic information. Combining atomic force measurements and near-field scanning optical microscopy has proven to be an extremely powerful approach in many areas of research, providing new information about a variety of specimen types that is simply not attainable with far-field microscopy. However, SNOM needs further development, and more research is needed toward improving probe fabrication techniques and more sensitive feedback mechanisms.

4. TIP-ENHANCED RAMAN SPECTROSCOPY TERS has many advantages over its parents, SERS and SNOM. In TERS, a metallic tip (usually silver/gold-coated) is used to enhance the electric field intensity of the excitation beam. TERS can be carried out on metallized atomic force microscopy (AFM) tips. During the measurements, the tip is illuminated and localized surface plasmons are excited at the tip apex, resulting in the enhanced field at close vicinity of the tip. Since the electric field is enhanced at the tip end, the Raman signal is predominantly generated at the tip end, which can be utilized to create a Raman image. To obtain the Raman enhancement, the tip is placed at a close proximity to the surface (usually a few nanometers). The Raman signal from the tip is enhanced

Figure 4. Schematic representation of the SNOM technique. E


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Figure 6. Schematic presentation of the TERS technique and the separation distance R (Å) between the atomic centers in the TERS technique.

by (E/E0)4, where E is the enhanced electric field and E0 is the excitation electric field, achieving a 107-fold amplification in the Raman signal.107 TERS has unprecedented sensitivity and spatial resolution. The spatial resolution, approximately of the size of the tip apex (typically 20−30 nm), is by far better than Abbe’s diffraction limit of λ/2. TERS has been shown to have sensitivity down to the single-molecule level. Optical microscopy with such an excellent spatial resolution and high sensitivity has a very promising future. Since the enhancement is mainly provided by the near-field mode excited at the apex of a suitable tip, forces between the probe tip and the atoms of the surface are crucial to understand TERS. The interaction can be conceptualized by envisioning the dependence of the forces (Lennard-Jones, van der Waals, dipole interactions) on the separation distance R (Å) between the atomic centers (Figure 6). In the near-field scanning microscopy configuration the most important interactions between the probe tip and the specimen are the van der Waals forces. The van der Waals contribution to the total tip− specimen force varies with the electronic properties of the particular atoms involved. The potential energy between the tip and the specimen can be expressed as a function of the distance R (Å) between the lowest atom on the tip and the nearest specimen surface atom. This interaction is utilized to control the distance between the tip and the surface in the loop feedback, which is crucial to maintain the near-field mode. TERS is currently still in its infancy, and more research is needed toward developing many aspects, such as (a) the factors affecting the TERS enhancement factor, (b) how to measure and improve the spatial resolution in TERS imaging, and (c) how to apply the technique to understand the structure− property relationship of nanoscale structures and chemical characterization of organic surfaces and biosurfaces at the nanoscale. After presentation of the instrumental strategies we will focus on the practical applications of the main directions of Raman microscopy experiments in selected fields of molecular biology.

5. RAMAN-BASED SPECTROSCOPY AS A DETECTION TECHNIQUE FOR BREAST CANCER DIAGNOSTICS Raman information can be obtained at different levels of complexity of the biological structure, on whole organisms and tissues, at the cellular level, and at the level of biomolecules. At the microscopic level of biomolecules it is especially important to identify the DNA sequence and specific gene products, whereas Raman monitoring of intracellular processes occurring in blood or tissue that are associated with cancer and whose measurement or identification is of key importance in patient diagnosis or clinical management. We will spend a moment summarizing the main directions of Raman microscopy experiments on single-biomolecule detection, SERS nucleic acid-, gene-, and protein-based diagnostic tests, and DNA mapping and sequencing,40,114−116 before we continue to review work reported on medical diagnostics for a more complex task of human tissue. We feel that such a discussion may help to design and perform radically new Raman experiments that employ the great potential of Raman spectroscopy for molecular monitoring of cancer. The Raman systems can assess both native optical contrast of the biological system without any labeling117−119 (color-coded image from intrinsic molecular markers of the tissue) and exogeneous optically active contrast agents.120−128 The typical concentrations of oncogene biomolecules produced by the organism in response to the cancer’s presence are from micro- to picomolar levels. Because so small concentrations are involved, novel strategies must be created to amplify the probe’s signal to the point that it can be detected. We will demonstrate that Ramanbased spectroscopy and imaging is one of the best modern strategies to achieve this goal. Regarding low concentrations of probe biomolecules, the effort must concentrate on amplification of Raman signals with optically active contrast agents and targeted biomarkers that monitor the functional features of cancer, where minimizing the size and the concentration of probe molecules is essential, because they must overcome biocompatibility, vascular, interstitial, and cell membrane barriers to deliver the imaging probes to their molecular target. Many nanoparticles have been investigated for biomedical applications targeting cancer (Figure 7). F


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are enhanced, thereby resulting in a composite spectrum of all species at the metal surface. There have been several approaches to solve the SERS specificity problem in optical labeling.132 To achieve specificity of interactions in biological systems, several approaches may be involved, such as DNA hybridization, aptamer recognition, supramolecular host−guest complexation, or antibody−antigen pairing. The last method is the most popular, because nature itself provides the solution to reach specificity. An antigen is a protein molecule that triggers antibody generation. An antibody is a protein molecule (also called immunoglobulin) that is produced by the organism in response to the pathology presence due to a mutation in specific genes of the DNA molecules. An elevated level of expression may be regarded as a prognostic biomarker of the pathology. The antibody has a unique ability to bind with high specificity to the antigen. Each antibody binds to a specific antigen; the interaction is similar to a lock and key. The most known test to monitor antigen−antibody interactions is an assay called ELISA (enzyme-linked immunosorbent assay). It uses chromogenic reporters, which are enzymatic reporters that produce some kind of observable color change to indicate the presence of antigen or analyte. Raman spectroscopy offers many serious advantages over the standard enzymatic methods in terms of sensitivity and multiplexing capabilities. There are a number of formats used to provide a Raman signal based on antibody−antigen pairing. Currently, a promising way to catch cancer lesions early is to use Raman reporters coupled with nanoparticles and antibodies that recognize and bind to cancer cells. When conjugated with biomolecular targeting ligands such as monoclonal antibodies, peptides, or small molecules, these nanoparticles can be used to target malignant tumors with high specificity and affinity. This approach is illustrated in Figure 9. Various antibodies are produced by the organism in response to the cancer’s presence due to a mutation in specific genes of the DNA molecules called oncogenes. They are often called tumor markers. A schematic illustration of SERS detection based on antigen−antibody interaction is presented in Figure 10. It has been shown that mutations of EGF (epidermal growth factor) result in cancer, so the target carcinoma cancer cells must contain antigen EGFR (epidermal growth factor receptor).133 Cell-targeting molecules, such as antibodies, directed against EGFR are attached to the outside of the nanoparticles (HAuNs, hollow gold nanoparticles). Zhang et al. used antibodies (C225, human−mouse chimerized monoclonal antibody) directed against EGFR that target human epidermal growth factor receptor 2 (HER2) on breast cancer cells.134 The experiments showed that the targeted nanoparticles did bind to breast cancer cells with HER2 and were easily spotted using Raman spectroscopy.134 A contrast agent with biological specificity consists usually of three parts: (a) an optically active label, (b) a probe molecule which provides molecule-specific recognition of cancer biomarkers (e.g., monoclonal antibodies against cancer-specific biomarkers or peptides which bind selectively to cancer-specific biomarkers), (c) an outside protecting layer (e.g., silica encapsulation). The typical Raman-active labels are organic fluorescent dyes, quantum dots, and metal nanoparticles.135,136 Modern SERS contrast agents consist typically of a metal core or nanoshell (silver or gold) for optical enhancement (plasmons), a Raman reporter molecule (dye) for spectroscopic activity, and a protein (antibody) for spectroscopic recognition of cancer. Figure 11 shows the main parts of the contrast agent: (a) an optically active label (gold nanoparticle), (b) a probe,

Figure 7. Nanoparticles used for biomedical applications.

Raman spectroscopy combined with nanoparticles is an optical technique that offers unsurpassed sensitivity and multiplexing capabilities (that is, a laboratory procedure that simultaneously measures multiple analytes) to the field of molecular imaging (Figure 8).

Figure 8. Instrumental setup for SERS multiplexing tasks.

The modern SERS methods are based on contrast agents that strongly enhance the Raman signal. The use of SERS-based contrast agents (known as SERS nanotags or biotags) as a direct replacement for the “golden” standards provides the opportunity for more sensitive tests, objective results, and the capability of running multiple tests on a single sample. The design of proper SERS-based biotags requires a complex interplay of biological interactions for an increasing number of fluorescent proteins or nanoparticles, which is only beginning to be understood.129−131 The valuable contrast agent should be both an optical enhancer of the Raman signal and a specific biological reporter monitoring some of the molecular mechanisms of carcinogenesis to image the expression of specific molecular signatures of cancer. Conventional SERS is largely nonspecific because the Raman signals from any molecule species encountering the nanoparticle metal surface G


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Figure 9. Antibody−phthalocyanine−gold nanoparticle bioconjugate.

and visualized by microscopy. Although the SERS signal does not originate from the protein, but from the Raman reporter, molecular information is provided. Raman scattering immunohistochemistry analysis that has potential to replace standard immunohistochemistry offers the benefit of selectivity and sensitivity by the detection of ultralow concentrations (i.e., 10−4 mol/m3).137,138 Additionally, the characteristic vibrational, biochemical fingerprint of the Raman reporters offers multiplexing capabilities. Silica-shell-coated SERS-active nanotags have opened new possibilities in using SERS for Raman immunochistochemistry of multiple biomarkers in tissue samples.139 The encapsulation makes it possible to capture distinct Raman labels attached to biofunctionalized antibodies or oligonucleotide chains, leading to multiplexing functions of the molecular assays. SERS spectra of individual proteins, and the successful design of nanoparticle probes, have promoted great progress of SERSbased immunoassays. SERS offers many serious advantages over the standard methods of immunochistochemistry based on the enzymatic reporters (ELISA), including sensitivity and multiplexing. Newer ELISA-like techniques utilize fluorogenic, electrochemiluminescent, and real time polymerase chain reaction (PCR) reporters to create quantifiable signals. In technical terms, newer assays of this type are not strictly ELISAs, as they are not “enzyme-linked” but are instead linked to some nonenzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs. Recent advances in development of new molecular assays in breast cancer pathology, such as in situ hybridization (ISH) for the topoisomerase II α (TOP2A) gene and for the aberrations in the copy number of the centromeric region of chromosome 17, Oncotype DX, MammaPrint, and Breast Cancer Profiling (BCP or H/I ratio),135,140 offer significant potential to improve the prognostic accuracy, treatment choice, and health outcomes. Although these new reporters are better than the traditional enzymatic reporters, Raman SERS still has various advantages

Figure 10. Schematic presentation of the concept of SERS detection using antibody−antigen interactions. Adapted from ref 134. Copyright 2008 American Chemical Society.

e.g., antibodies immobilized on the surface of the nanoparticle, (c) a unique SERS reporter. The outside layer (silica encapsulation) protects the core from degradation in a physiologically aggressive environment and can form covalent and/or electrostatic bonds with positively charged agents or biomolecules that have basic functional groups such as amines and thiols.137 The nanoparticles exist as a monodispersed suspension. However, to obtain maximum enhancement in near-IR the particles need to be aggregated into discrete clusters. Sometimes the analyte plays a role of aggregating medium. The nature of the aggregating agent depends on the chemical adsorption properties of the analyte chosen as the target in the surface enhancement studies. There are various methods of preparation of SERS tags, but the main steps are similar. First, the gold nanoparticles exist as a monodispersed suspension. A biomarker-specific antibody and a Raman reporter (i.e., an organic dye with an isothiocyanate group) are next adsorbed onto the gold nanoparticle surface. Once the antibody and the Raman reporter are bound to the nanoparticles, a silver ionic solution and a reducing agent are applied to form a silver shell around the gold nanoparticles to achieve SERS signal enhancement. After the antibody−Raman dye complex binds to its target, the SERS signal can be detected H


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Figure 11. Schematic representation of SERS-based biotags.

and specificity of 99%.163 The results demonstrate the capability of Raman spectroscopy to determine the HER2 status in cells and promise for application in the diagnosis of HER2-positive breast cancer in clinical practice. In summary, Raman-based methods are beginning to demonstrate their potential as ubiquitous detection tools in clinical diagnostics. Multiple architectures exist in the field, but all exhibit the robustness, sensitivity, and flexibility necessary for imaging, diagnostic tests, and therapeutic applications.

over ELISA-like techniques, including higher sensitivities and multiplexing. There are many papers demonstrating that breast cancer oncology may benefit from SERS-active nanoparticles. They have been used in assays for prostate-specific antigen (PSA), BRCA1, and HER2, along with examples of nucleic acid detection.141−161 It is well-known that breast cancer cell lines have overexpression of the estrogen receptor, progesterone receptor, and ERBB2. The antibody-conjugated SERS biotags were successfully applied to the targeting of HER2 and CD10 on cellular membranes and exhibited good specificity.127,145 The specificity and selectivity of the nanoprobes in the detection of a single-nucleotide polymorphism (SNP) in the breast cancer BRCA1 gene in a homogeneous solution at room temperature have been successfully demonstrated.145 The single-nucleotide polymorphism in the breast cancer BRCA1 gene can be identified also by an SERS-active dye (label) attached to an oligomer containing a BRCA-1 sequence and hence has the potential for being used as a gene probe to identify the BRCA-1 gene.145 Another application with the SERS-active dye has been reported for the in vivo targeted imaging of cancer, where the Raman reporters were stabilized by thiolated PEG and gave large optical enhancements.162 In this other study, SERS nanoparticles composed of a gold core, a Raman-active molecular layer, and a silica coating were used for Raman imaging in vivo.162 The overexpression of HER2 in breast cancer has also been studied on cell lines such as BT474 (HER2-overexpressing breast cancer cell), MCF-10A (human breast epithelial cell), and MCF-10A overexpressing HER2 using a confocal Raman system. Current diagnosis of HER2-positive breast cancer is a time-consuming procedure with an estimated inaccuracy of 20%. Raman spectroscopy was able to differentially identify BT474 breast cancer cells with an overall sensitivity of 100%

6. “OPTICAL BIOPSY” OF HUMAN BREAST CANCER BY RAMAN IMAGING In this section we will describe Raman-based techniques that can potentially be used in clinical practice.56−62,164 A topic of particular importance is to find correlation between the vibrational Raman spectra and progression stages in cancer disease. We have found that the fatty acid derivative composition in the cancerous breast tissue from the tumor mass is markedly different from that of the surrounding breast tissue from the safety margin. The results are of potential importance in clinical assessments, because knowing the type of margin of the removed tissue helps in making the right treatment decisions. This is especially important in deciding whether additional surgery is needed. If the margins are negative, you probably do not need more surgery. If the margins are positive, more surgery is needed.56−62,164 It has been demonstrated that Raman spectroscopy has a potential to distinguish progression stages in breast cancer.162 Sensitive assays for rapid quantitative analysis of histologic sections, resected tissue specimens, or in situ tissue are highly desired for early disease diagnosis. Stained histopathology is the gold standard, but remains a subjective practice on processed tissue taking from hours to days. I


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Figure 12. (A) Stitching image (2 × 2 mm) of a thin layer (2 μm) of the normal tissue prepared as for the histopathological analysis, but without staining, composed of hundreds of single video images. (B) Raman image (1.75 × 1.75 mm) of the region marked in (A) obtained by basis analysis from the average spectra shown in (D). (C) Histological image. (D) Averaged spectra from three different areas in the sample. The colors of the spectra correspond to the colors in the image. Mixed areas are displayed as mixed colors. The integration time was 0.2 s. An alpha 500 R confocal Raman microscope, a 532 nm laser for Raman imaging, and a 50× objective with an NA of 0.50 were used. Reprinted with permission from ref 57. Copyright 2012 Elsevier.

Here, we present the Raman images of infiltrating ductal carcinoma in human breast tissue. The total number of patients was 220. Two types of samples were examined: the tissue sample from the tumor mass and the tissue from the safety margins outside the tumor mass (that is, noncancerous tissue if the margin is negative) of the same patient. To explain the term “negative safety margin”, let us recall that when a tumor is removed, some tissue surrounding it is also removed. The safety margin, also known as a “margin of resection”, is an area within the distance between a tumor and the edge of the surrounding tissue that is removed along with it in the surgery. A pathologist checks the tissue under a microscope to see if the margins are free of cancer cells. Depending upon what the pathologist sees, the margins of a tumor can be classified as follows: (1) Positive margins: Cancer cells extend out to the edge of the tissue.

To illustrate the advantages of Raman-based methods in cancer diagnosis, we will demonstrate that Raman imaging gives new hope for breast cancer diseases in routine clinical practice. We will present the Raman-based results performed in our laboratory that produce sensitive and accurate color-coded Raman images from intrinsic molecular markers for the complex task of human breast cancerous and noncancerous tissues.56−64,164 To obtain color-coded Raman images, 2D arrays have been prepared of thousands of individual Raman spectra that were evaluated by the basis analysis or by the cluster method. In the basis analysis method each measured spectrum of the 2D spectral array is compared to the basis spectra (BS) using a least-squares fit that tries to minimize the fitting error D described by the equation ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ ⎯⎯⎯→ ⎯⎯⎯→ D = ([Recorded Spectrum] − a × BSA − b × BSB 2 ⎯⎯⎯→ − c × BSC − ...)

(2) Negative margins: No cancer cells are found. (3) Close margins: Any situation that falls between positive and negative is considered “close”. The typical Raman images of the noncancerous and cancerous human breast tissues are presented in Figures 12 and 13. One can see that the Raman images reveal an inhomogeneous distribution of different compounds in the samples, and

(2)

by varying the weighting factors a, b, c, ... of the basis spectra ⎯→ BS.164 The basis spectra are created by averaging over various areas of the scanned surface or by using the cluster analysis. The weight factor in each point is represented as a 2D image of the corresponding color. J


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Figure 13. (A) Stitching image (2 × 2 mm) of a thin layer (2 μm) of the cancerous tissue prepared as for the histopathological analysis, but without staining, composed of hundreds of single video images. (B) Raman image (1.75 × 1.75 mm) of the region marked in (A) obtained by basis analysis from the average spectra shown in (D). (C) Histological image. (D) Averaged spectra from three different areas in the sample. The colors of the spectra correspond to the colors in the image. Mixed areas are displayed as mixed colors. An alpha 500 R confocal Raman microscope, a 532 nm laser for Raman imaging, and a 50× objective with an NA of 0.50 were used. Reprinted with permission from ref 57. Copyright 2012 Elsevier.

carotenoids at 1158 and 1518 cm−1 in the cancerous tissue in Figure 13D that are the most intense peaks for the noncancerous breast tissue from the safety margin, and they are clearly visible in Figure 12D. To make the differences more clear, we present a comparison of the typical Raman spectra for the noncancerous and cancerous human breast tissue (Figure 14). The important observation that one makes when confronting the results presented in Figure 14 is marked differences between the vibrational Raman spectra demonstrated by four characteristic Raman biomarkers, carotenoids, lipids, proteins, and water, which discriminate the noncancerous tissue from the safety margin and cancerous tissues from the tumor mass. Detailed analysis by Raman imaging77−85,164 showed that the noncancerous breast tissue from the safety margin contains a markedly higher concentration of fatty acids and carotenoids as compared to that of the tumor mass tissue dominated by proteins. Moreover, the noncancerous breast tissue and the cancerous tissue have different Raman profiles in the 2800− 3000 cm−1 region, indicating a distinct composition of fatty acids and proteins. These results correlate with the fact that high protein production is linked to cell division, migration, and increased cell proliferation in tumors.165,166 Protein expression profiling in high-risk breast cancers has been identified in much research based on HER2 and BRCA1.167−169

they resemble the conventional histological images. The advantage of the “Raman biopsy” is that it provides direct biochemical information (a vibrational fingerprint) in real time, is not prone to subjective interpretations, and monitors biological tissue without any external agents, in contrast to histopathological assessment. The detailed analysis of Raman image B and Raman spectra D of the noncancerous breast tissue presented in Figure 12 leads us to the following conclusions: the blue areas in the Raman image correspond to the adipose cells because Raman spectra from this area are almost identical to those observed for monounsaturated oleic acid and its derivatives as we have reported recently.58 The intense Raman peaks at around 1158 and 1518 cm−1 seen in Figure 12D which correspond to carotenoids confirm that the adipose cells are reservoirs for natural antioxidants. The green areas have a profile similar to that of the blue regions; the noticeable difference concerns only the intensity of the carotenoid and lipids. The region 2800−3000 cm−1 in Figure 12D shows signals from lipids with maxima at 2854, 2888, and 2926 cm−1. Markedly different vibrational behavior can be observed for the Raman image of the cancerous breast tissue from the tumor mass which is presented in Figure13. The yellow, red, and green areas in Figure 13 are characterized by the vibrational peaks at 1080, 1259, 1444, 1660, and 2940 cm−1. The most surprising finding is the lack of peaks typical for K


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Figure 14. Comparison of the Raman spectra of the noncancerous and cancerous breast tissues of the same patient. Reprinted with permission from ref 57. Copyright 2012 Elsevier.

The next important problem in clinical practice is development of new diagnostic techniques allowing noninvasive tracking of the progress of therapies used to treat a cancer. Raman imaging of the distribution of photosensitizers may open new possibilities of photodynamic therapy (PDT) to treat a wide range of neoplastic lesions with improved effectiveness of treatment through precise identification of malignant areas.60 Raman imaging has been employed to analyze human breast cancer tissue that interacts with the phthalocyanine photosensitizer (AlPcS4) used in the photodynamic therapy of cancer.60 PCA has been employed to analyze localization of the photosensitizer in various areas of the noncancerous and cancerous breast tissues. The analysis is related to many fundamental questions in terms of application of photosensitizers in molecular biology, such as whether the photosensitizer enters the cell and, in case it does, what the mechanism of uptake is, where in the cell it resides, and whether its presence leads to changes in the cellular biochemistry. Figure 15 presents Raman images and Raman spectra of the noncancerous breast tissue and the cancerous (infiltrating lobular carcinoma) tissue stained with AlPcS4. It has been demonstrated that the emission spectra combined with the Raman images are very sensitive indicators to specify the localization and distribution of phthalocyanines in the cancerous and noncancerous breast tissues.60 To summarize, Raman imaging may play an important role as a method of particular significance in differentiation of normal and tumorous (cancerous or benign pathology) breast tissues and has a potential to be a novel and effective photodynamic therapeutic method with improved selectivity for the treatment of breast cancer.

Figure 15. Microscopy images, Raman images, and Raman spectra of the breast tissue from the safety margin (250 × 250 μm2) (a) and the tissue of the tumor mass (infiltrating lobular carcinoma) (300 × 250 μm2) (b) of the same patient stained with AlPcS4. The Raman images from the regions marked in the microscopy images were obtained by basis analysis from the averaged spectra shown in the Raman spectra. The two spectra were averaged from two different areas in the sample. The colors of the spectra correspond to the colors in the image. Mixed areas are displayed as mixed colors. The integration time was 32 ms. Reprinted with permission from ref 60. Copyright 2012 Adenine Press.

7. CONCLUSIONS Raman-based methods have found a wide range of applications in biochemical and biomolecular diagnostics. For instance, SERS has shown great promise for basic research in genomics and proteomics. The SERS-based DNA label systems enhance the utility of gene-probe technology for gene diagnostics. The multiple SERS probes can also be employed for detection of a single gene, DNA, proteins, and lipids that are difficult to be detected when the conventional fluorescent or chemiluminescent labeling is used. Very recently, the SERS technique

incorporated with near-field optical techniques (e.g., AFM, SNOM) and nonlinear optical microscopy (e.g., CARS) can be recorded with enhanced factors of up to 1012 to 1015 and down to concentrations of 10−10 to 10−14 M. Therefore, with further advancements in fabrication of highly sensitive SERS-active probes, the SERS technique may open up new directions for L


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very sensitive signal detection in biomedical applications, such as chemical sensors, biological sensors, nanomedicine, drug delivery, and bioimaging. Raman imaging gives new hope for cancer diagnosis, because it offers unsurpassed sensitivity, multiplexing capabilities, and biochemical selectivity and specificity to the field of molecular imaging. Researchers have already been able to notice its unique properties for biomolecular diagnostics and have already begun to translate Raman spectroscopy and Raman imaging into a novel clinical diagnostic tool using various endoscopic strategies.

committees (12), an editor of conference proceedings (3), and an organizer of conferences, schools, and workshops (4). She serves as a reviewer for many journals. She has been invited to deliver plenary, keynote, and invited lectures at international conferences and universities (around 40).

AUTHOR INFORMATION Corresponding Author

*Phone: +48 42 6313188. Fax: +48 42 684-00-43. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Beata Brozek-Pluska is an assistant professor in the Institute of Applied Radiation Chemistry at the Lodz University of Technology in Lodz, Poland. She received her Master of Science degree in physical chemistry in 1998 and Ph.D. degree in chemistry in 2002 from the Lodz University of Technology. After spending a postdoctoral period in the Laboratoire d’Optique Apliquee, Ecole PolitechniqueENSTA, France, she has been working in the Laboratory of Laser Molecular Spectroscopy at the Lodz University of Technology, Poland. Her research interests are focused on molecular spectroscopy, photochemistry, and laser spectroscopy, with special emphasis on mechanisms of chemical and physical processes occurring on the picosecond and femtosecond time scales and applications of Raman spectroscopy and imaging in cancer diagnostics.

ACKNOWLEDGMENTS We express our gratitude for the support of this work by The National Science Centre (Grant 2940/B/T02/2011/40).

Halina Abramczyk is a full professor of Chemistry at the Lodz University of Technology in Lodz, Poland. She received her Master of Science degree in physics from the University of Lodz and Ph.D. degree in physical chemistry in 1982 from the Lodz University of Technology. She worked as a research associate at the Chemistry Faculty of the Lodz University of Technology from 1982 until 1989. After spending a postdoctoral period at the University of Bielefeld, Germany, she completed her habilitation in molecular and laser spectroscopy at the Lodz University of Technology. Since 1989, Halina Abramczyk has been a director of the Laboratory of Laser Molecular Spectroscopy (LLMS) of the Lodz University of Technology. In the period 2007−2009 she held a joint appointment with the Max-Born Institute, Berlin, where she headed the Marie Curie Chair. She has supervised around 40 Ph.D. and M.Sc. theses on molecular spectroscopy, medical diagnostics of cancer, fiber optical transmission, and laser-oriented chemical dynamics. Her work led to a series of substantial grants in the United States, Poland, and Europe. She was a coordinator of the Polish-American Joint project between the Polish Ministry of Education and National Science Foundation (MEN-NSF; 2002) and 11 projects financed by the Ministry of Science. She has received the Fulbright (United States), DAAD (Germany), International Federation of University Women (Spain), and Zentrum fur Interdisziplinare Forschung (Germany) awards. In 2007 she received the prestigious award from the EUMarie Curie Excellence Chair. She has been a member of the editorial board of the Journal of Molecular Liquids since 1994 and a member of the International Executive Committee of the European Molecular Liquid Group since 1991, as well as a member of many scientific organizing

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