Beyond the borders — Biomedical applications of non-linear Raman microscopy

ADR-12784; No of Pages 10 Advanced Drug Delivery Reviews xxx (2015) xxx–xxx

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Beyond the borders — Biomedical applications of non-linear Raman microscopy Martin Josef Winterhalder, Andreas Zumbusch ⁎ Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany

a r t i c l e

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Available online xxxx Keywords: Non-linear Raman CARS SRS Optical microsopcy Biomedical imaging

a b s t r a c t Raman spectroscopy offers great promise for label free imaging in biomedical applications. Its use, however, is hampered by the long integration times required and the presence of autofluorescence in many samples which outshines the Raman signals. In order to overcome these limitations, a variety of different non-linear Raman imaging techniques have been developed over the last decade. This review describes biomedical applications of these novel but already mature imaging techniques. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . Technical background . . . . . . . . . . . . . . . . Materials investigated with non-linear Raman microscopy 3.1. Polymeric samples . . . . . . . . . . . . . . 3.2. Lipids . . . . . . . . . . . . . . . . . . . . 3.3. Nanoparticles . . . . . . . . . . . . . . . . . 3.4. Marker for non-linear Raman microscopy . . . . 3.5. Pharmaceutical compounds . . . . . . . . . . 4. Biomedical applications . . . . . . . . . . . . . . . 4.1. Investigations of cells . . . . . . . . . . . . . 4.2. Tissue experiments . . . . . . . . . . . . . . 5. Conclusion and outlook . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Optical microscopy and cellular biology share a common historical development. In fact, the development of early microscopes was prerequisite to the discovery of the cellular organization of biological material by Robert Hooke in the 17th century. From this early start on, scientific questions from both fields have mutually stimulated their respective progress. With respect to the spatial resolution achievable with light microscopy, the physical limits posed by diffraction have been reached already in the late 19th century by Abbe. This physical barrier has only very recently been broken by two different types of ⁎ Corresponding author. E-mail address: [email protected] (A. Zumbusch).

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super-resolution microscopy, which were rewarded with the Nobel prize in Chemistry in 2014 [1,2]. Since Abbe's time, the main focus of the further development of optical microscopy concerned the introduction of new modes of contrast generation. In the beginning, this involved various staining approaches leading to specific absorption of light by different parts of the sample. Examples are the hematoxylin and eosin (H&E) stains, perhaps the most widely used staining in histology. One should note that already the generation of images using these simple stains involves a combination of absorption spectroscopy with microscopy in that sample regions are discerned based on their different properties with respect to the absorption of visible light. The principle of using a wide range of other well-established spectroscopic techniques has subsequently been a basis for a wide range of different microscopy modalities.

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Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

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During the last three decades, especially fluorescence excitation in combination with optical microscopy has become one of the most important practical techniques for the investigation of biological samples. There are several reasons for its success: i) Fluorescence spectroscopy is exquisitely sensitive. It has been shown in the late 1980s by Orrit and coworkers that fluorescence detection is sensitive enough to detect single molecules [3]. In practice, it is much easier to detect a weak fluorescence signal than a weak absorption signal, because the Stokes shifted fluorescence can be separated from the excitation light by means of a simple optical filter. For a single molecule, this means that a fluorescence signal of typically 108 photons/s can be detected against essentially a zero background, whereas a similar absorption measurement requires detection of a change in intensity of 108 photons/s against the background of the excitation light level of typically 1014 photons/s [4]. ii) Fluorescence imaging can be performed with a very high three-dimensional resolution. Standard wide-field fluorescence microscopy offers a diffraction limited spatial resolution of typically 200–300 nm in the plane perpendicular to the excitation beam propagation direction if high numerical aperture objectives are used. Along the beam propagation direction, the spatial resolution can be improved to be slightly below 1 μm if a confocal microscope is used. In this case, the detection volume is restricted by virtue of a pinhole in the beam path [5]. iii) The importance of fluorescence microscopy greatly increased with the advent of fluorescent proteins as genetically encoded fluorescence labels [6–8], since labeling with fluorescent proteins offers the best target selectivity imaginable. Despite the enormous success of fluorescence microscopy, specific applications still require the development of new microscopic approaches which cannot be tackled with existing methods. Especially medical applications are a realm of such approaches, since here staining techniques can most often not be employed. This has served as a strong motivation for the development of label-free microscopy techniques which should otherwise perform similar to fluorescence microscopy. Especially vibrational spectroscopy techniques have attracted a lot of attention, since in many cases, molecular selectivity is desired. In vibrational microscopy, contrast is generated on the basis of the vibrational spectra of the sample molecules. Since even small molecules exhibit many different vibrational resonances and because in many cases these are spectrally very narrow, an unequivocal identification of pure compounds based on their vibrational spectra is possible. The downside of this multitude of vibrational bands, however, is that mixtures of compounds as they are typically found in most biological applications, will lead to a plethora of vibrational bands which is hard to analyze. While in principle, direct infrared absorption and Raman scattering can be employed for microscopic approaches, for a number of practical reasons the overwhelming majority of experiments especially aiming at investigations of biological samples is based on Raman scattering. Raman scattering is the inelastic scattering of excitation light, i.e., laser light impinging onto the sample is scattered with a transfer of energy between the excitation light and the sample. The scattered light can be higher (anti-Stokes) or lower (Stokes) in energy than the illumination light. In practice, Raman microscopy is nearly exclusively based on the analysis of Stokes-scattered light, since these bands have a much higher intensity. Raman spectroscopy is readily adapted to microscopy, because the excitation light can be chosen to be in the visible or near infrared spectral range, such that high quality objectives can be used. The use of confocal pinholes for high 3D resolution is also possible and widely applied [9]. The major drawback of spontaneous Raman microscopy is the low Raman scattering cross section of molecules. Therefore, Raman microscopy is a rather slow technique which requires long integration times, even if progress towards faster imaging by using line illumination has recently been made [10]. Equally important, the low scattering efficiency together with a weak autofluorescence, which is present in many samples, often prevents the detection of a Raman signal. In order to overcome these limitations, non-linear optical microscopy Raman techniques, namely Coherent anti-Stokes Raman Scattering (CARS) and

Stimulated Raman Scattering (SRS) microscopy have been developed in the last two decades. After an initial period of mainly methodological development, both schemes have meanwhile found a lot of applications. Recently published review articles give an excellent overview over the methodology and basic research in this field [11–16]. The focus of this article, by contrast, will be to review the current state of the art of biomedical applications of CARS and SRS microscopy. 2. Technical background In the following, we will give a very basic description of the technical background of non-linear Raman microscopy with the main aim of highlighting the specific features of CARS and SRS microscopy which need to be known in order to apply them properly. A more extensive practical guide to the implementation of these experiments has recently been published [17]. The basic idea of both techniques is that vibrational spectroscopy is used to generate contrast for microscopic images. In both approaches this is typically achieved by coupling two laser beams with intensities Ipump and IStokes into a multiphoton fluorescence microscopy experiment (Fig. 1). The generated contrast is strongest, if a resonance condition between the excitation light and the sample molecules under investigation is met. Ideally, a simple relation connects the signal intensity with the number N of molecules of interest which are probed. Both SRS and CARS are non-linear optical effects. This means that the signal intensity does not depend linearly on the excitation intensity, but quadratically (SRS) or cubically (CARS): ISRS ∝N σ Raman Ipump IStokes and 2 ICARS ∝ χð3Þ I2pump IStokes : Here, σRaman is the Raman scattering cross section and χ(3) is the third order non-linear optical susceptibility. Since χ(3) is proportional to the number of molecules, the two equations point out an important difference between SRS and CARS: whereas SRS has a linear dependence on the concentration of probed molecules, CARS has a quadratic dependence. In addition, χ(3) contains a non-resonant component, which gives rise to a background signal. In practice this has two consequences. Firstly, a quantitative interpretation of SRS signals as the number of molecules in the probe volume is much easier than that for CARS signals. Secondly, CARS signals can only be detected easily where the resonant signal is much stronger than the non-resonant background. To recover weak resonances in CARS microscopy, sophisticated excitation and data analysis schemes have to be employed [18,19]. In practice this means that CARS microscopy has mostly been limited to imaging in the spectral range of CH-stretch vibrations, whereas SRS increasingly is also applied in the vibrational fingerprint region between 600 and 1800 cm−1. Yet, CARS microscopy in many cases is still the preferred choice. The reason for this is that it offers the advantage that the detection of CARS signals is much easier than that of SRS signals. In a CARS process, the signal has the frequency ωCARS = 2ωpump − ωStokes which is different from that of the excitation light. By contrast SRS signals are detected as a loss or gain in the intensity of one of the two exciting lasers. This means that whereas, just as in the case of fluorescence microscopy, the CARS signal can be separated from the excitation light by means of a simple optical filter [20], the detection of a SRS signal requires more demanding lockin detection [21,22]. The detection in CARS microscopy is therefore similar to other multiphoton techniques such as two-photon excited fluorescence emission and/or second (third) harmonic generation signals. For multimodal implementations, only the addition of the respective detection channels is required, since the lasers used for CARS signal generation can also be used to drive the other excitation processes [23]. This

Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

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Fig. 1. Schematic representation of the CARS (left) and the SRS (right) processes. The CARS signal is generated at a frequency ωCARS which is different from the frequencies of the exciting lasers. It is easily separated by an optical filter. SRS is detected as a small intensity change ΔIS or ΔIP of the exciting lasers. SRS excitation pulses must arrive at the same time and are displaced only for clarity. For CARS as well as for SRS microscopy, detection in the transmission and backscattering direction is possible.

opens interesting perspectives especially in investigations of tissues, since all of the processes generate a different type of contrast [24]. Similar multimodal approaches are possible with SRS microscopy, which, however, requires a fast modulation of the excitation intensity and use of lock-in amplification for SRS detection. From what has been described above, it is possible to sum up the practical differences between non-linear and conventional, spontaneous Raman microscopy. The first advantage of non-linear Raman imaging is its comparatively low sensitivity against autofluorescent background from the sample, because the signal and fluorescence spectra are not coinciding. This allows investigations of most types of samples, many of which cannot be accessed by spontaneous Raman microscopy. The second important difference concerns the image integration times. Compared to spontaneous Raman microscopy, non-linear Raman imaging is several orders of magnitudes faster, with video-rate imaging being achievable [25–27]. However, the fast imaging capability comes at the expense of significantly reduced spectral information of one image, because most non-linear Raman microscopy implementations probe only one vibrational resonance at a time. However, simultaneous multicolor and broadband approaches have been developed for CARS and SRS microscopy [18,28–36] and show how to overcome this limitation. 3. Materials investigated with non-linear Raman microscopy CARS microscopy was first reported in the early 1980s [37] and has found widespread application after 1999 [20]. SRS microscopy, by contrast, was first reported in 2007 [38]. For this reason, to date many more reports of applications employing CARS microscopy have been published, but SRS microscopy is increasingly finding its way into the laboratories. The range of applications covers many different areas from material science to medicine. Due to their relevance for pharmaceutical applications, this chapter will also briefly cover some of the more material scientific applications, such as investigations of polymeric

samples and nanoparticles. In general, in the vast majority of experiments aliphatic and aromatic CH-stretch vibrations have been monitored. This is owed to fact that CH-bands of lipid and proteins give very strong third-order non-linear signals [39] and are easy to detect with CARS microscopy, because they are spectrally well separated from any other vibrational resonance, and because lipids in many cases appear in high local concentrations. 3.1. Polymeric samples Already in the early phases of its development, CARS microscopy has been applied to investigations of polymeric systems both because of their broad practical importance and also because of their welldefined spectral properties. While the vibrational spectra of polymers can be very rich in information, it is commonly easy to distinguish two different polymers by vibrational bands specific to the one or the other. Thus, polymer beads composed of either polymethylmethacrylate (PMMA) or polystyrene (PS) are commonly used as calibration standards in non-linear Raman microscopy. In this case, the resonances for aliphatic CH-stretch vibrations (found in PMMA and PS) are well separated from aromatic CH-resonances (only in PS) [40]. The same principle has been used to visualize the distribution of polymers in thin films of polymer blends [41–43]. Interestingly, the molecular vibrations do not only exhibit different spectral resonances but also different vibrational lifetimes [31,44], which in a manner similar to fluorescence lifetime imaging can also be exploited for generating contrast in the imaging of polymer blends [45]. CARS microscopy of polymers has soon been used for monitoring chemical changes induced by photochemical or polymerization reactions. In the first experiment of this type, the photochemical conversion of the photoresist poly(tert-butyloxycarbonyloxystyrene) was monitored [46]. It was possible to image sub-micrometer illumination patterns in thin polymer films. Similar experiments have recently been

Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

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performed with the inorganic material hydrogen silsesquioxane [47]. In both of these experiments, resonances with vibrational frequencies well below 1000 cm− 1 have been exploited for the contrast generation, whereas broadband CARS microscopy monitoring of CH-stretch vibrations were recently used to follow two photon polymerization of an acrylic resin [48,49].

Apart from their normal physiological function, LDs have proven to be also important in infectious diseases, such as hepatitis C. The strong LD signals observed in CARS microscopy allowed time-lapse studies of the infection process under a variety of different conditions [80–82]. 3.3. Nanoparticles

3.2. Lipids As has been pointed out above, CH-stretch vibrations have been the main target for contrast generation when using non-linear Raman microscopies [15]. Of course this means that primarily lipids are being imaged. It is, however, also possible to use the CH-stretch vibrational resonances to distinguish between saturated and unsaturated lipids [50,51], as well as between lipids and proteins [52]. In cellular imaging, this allows for the clear distinction between nuclei and cytoplasm [53]. The strong CARS signals of lipids have prompted a number of CARS microscopy studies on multilamellar [18,29,54–56] vesicles. Here, especially multiplex CARS microscopy has been used to study phase transitions and packing of lipids. The natural extension of these experiments has been the investigation of unilamellar vesicles either free or spread on a cover slip, where CARS microscopy was used to study phase separations and structural dynamics [57–61]. While these experiments give important insights into lipid dynamics in membranes, they also demonstrate the detection limits of CARS microscopy which is roughly the detection of CARS signals from single lipid bilayers, e.g., in a GUV. In order to properly interpret the sensitivity in these measurements, one should keep in mind that in point scanning methods like CARS or SRS microscopy, the signal is gathered from a volume of approximately 1 μm3. The relative concentration of a compound in this volume and not in the whole sample therefore is relevant for determining the sensitivity of the method. It turns out that the detection limit is in the order of some tens of thousands lipid molecules within this volume or approximately 1 mM of diluted similar compounds [62]. Apart from these test systems, non-linear Raman microscopy has very soon been used to also investigate lipids in living cells and living model organisms, such as yeast, Drosophila melanogaster and Caenorhabditis elegans. One important focus of these investigations is gaining an understanding of the dynamics of lipid droplets (LDs). Since it has been realized that LDs in cells are highly dynamic and actively participate in a large number of cellular processes, they have increasingly been seen as full blown cellular organelles [63–65]. CARS microscopy has turned out to be an ideal tool for investigating LDs in living cells and animals, since only moderate excitation intensities are necessary and no potentially cytotoxic staining agents have to be employed. Thus, after an initial demonstration that LDs can be visualized by CARS microscopy in their cellular environment [66–68] — similar SRS experiments have also been reported lately [69], many research efforts have been dedicated to using CARS microscopy to unravel the biological functions of LDs. Here, an especially interesting point has been to understand the growth mechanism of LDs in human adipocytes, which was largely unknown until recently. Long-term CARS microscopy experiments have shown that LDs indeed grow by fusion of smaller LDs [70,71]. Due to the slow fusion dynamics, fluorescence microscopy could not reveal these processes as the long observation times necessary ultimately lead to strong photobleaching of the fluorescent stains. Also the involvement of LDs in lipid metabolism has been investigated using CARS microscopy. For this purpose, it is possible to analyze the size distribution of LDs which can be obtained from 3D image stacks [72–76]. However, the spectroscopic nature of non-linear Raman microscopy additionally allows the assignment of resonances to specific compounds and thus the investigation of the content of LDs. It is thus possible to determine relative amounts of e.g., saturated and unsaturated lipids and cholesterol in the LDs as a function of the dietary conditions using multiplex CARS [75,77,78] or SRS microscopy [79].

CARS microscopy has also been found to be suited for investigating the uptake and distribution of nanoparticles in cells and organisms. Here, two cases can be distinguished. Firstly, CARS microscopy has been used to investigate the biological impact of metal or metal-oxide nanoparticles. Many of these particles exhibit a very strong purely electronic contribution to χ(3). Even if CARS microscopy can be used to visualize such nanoparticles in a biological context, this means that no vibrational resonance is exploited. Instead the purely electronic process is used for image generation in a manner similar to third harmonic generation microscopy [83]. This approach has first been exploited to visualize the uptake of sub-100 nm diameter metal oxide nanoparticles by fish gills [84] and marine worms [85]. On a cellular level, similar studies on particle uptake have been performed using Au nanoparticles [86,87]. While the aforementioned investigations were aiming at the assessment of nanoparticle toxicity, nanoparticles have also found use for the enhancement of CARS signals for analytical purposes. Thus, the basal cell protein p63 has been detected in tissues using surface enhancement of CARS signals on Au/Ag nanoshells [88]. The standard vibrational contrast mechanisms have been employed for the second type of CARS experiments on nanoparticles, in which particles suited as drug carriers were investigated. Similar to previous fluorescence based microscopy experiments [89,90], CARS microscopy based single particle tracking has been used to evaluate the receptormediated uptake of polystyrene containing liposomes with a diameter of approximately 200 nm by epidermal cells [91]. CARS microscopy has also allowed monitoring the distribution of polymeric nanoparticles in animals after oral feeding [92,93]. The latter experiments were performed on tissue sections. The nanoparticle work cited above shows the great potential of CARS microscopy for investigations of nanoparticles in cells, tissues, and organisms. One can state, that for metal and metal oxide particles, CARS microscopy can be used to track particles with diameters well below 100 nm. It should be noted that similar experiments can be done with third harmonic generation microscopy which has a slightly different contrast mechanism but is conceptually simpler in that only one excitation laser is necessary [94]. For applications in which polymeric particles are studied, CARS microscopy requires minimum particle diameters between 100 and 200 nm. 3.4. Marker for non-linear Raman microscopy As has been pointed out in the introductory section of this article, a main motivation for the development and application of non-linear Raman microscopy is the needlessness to stain the sample. Nevertheless, specific Raman active stains increasingly find use. In order to rationalize this, one has to remember that the spectral region between approximately 1800 cm− 1 and 2700 cm− 1 in most materials is devoid of any strong bands in the vibrational spectra. However, both the CD-stretch vibrations of deuterated compounds and covalent triple bonds as found in alkynes have vibrational resonances around 2100 cm− 1. Since in addition neither deuterium nor alkynes are abundant in biological materials, they are ideally suited for the development of staining reagents. In comparison to fluorescent stains, the use of Raman labels is much less invasive, since deuterated compounds behave chemically nearly exactly as their hydrogenated counterparts. Alkynes, by contrast, are much smaller than typical fluorophores such that alkyne modified molecules can also be expected to behave similar to natural occurring compounds.

Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

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Both types of modifications are readily available, deuterated compounds because they are widely employed in mass spectrometry and alkynes because a lot of synthetic routes to alkyne modified molecules for bioorthogonal labeling via click chemistry have been published lately. The use of Raman labels for non-linear Raman microscopy is thus attractive mainly because it allows imaging of the biological fate of compounds administered either to cells in culture or to organisms [95]. This strategy has been used in a number of recently published experiments, in which exclusively SRS microscopy was employed for imaging (Fig. 2). The first report of this type has described the monitoring of overall protein synthesis in two different cell lines by incorporation of deuterated amino acids [96]. Soon after, similar experiments were published which reported the detection of choline metabolites after feeding deuterated choline [97], of lipid metabolism after administration of deuterated fatty acids [98], and of lipogenesis after addition of deuterated glucose [99]. One should keep in mind that an important factor of the success of these experiments is the fact that all target compounds appeared in high local concentrations in the sample. The great potential of these experiments also motivated the introduction of isotopic editing schemes different from deuteration. Deuteration is easily detected, because the large relative mass difference between both hydrogen isotopes leads to a drastic shift in the vibrational resonance. These are much smaller if, e.g., 13C is exchanged against 12 C. Nevertheless, the viability of 13C labeling for SRS microscopic imaging was demonstrated. In one experiment, proteome degradation was monitored by using 13C marked phenylalanine and subsequent

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ratiometric imaging [100]. A second experiment finally combined alkyne labeling with 13C isotopic exchange. For this purpose, the authors synthesized alkyne groups with the three different combinations of 12C and 13C which resulted in three distinguishable vibrational resonances. These could be exploited in a multicolor labeling experiment in which DNA, RNA and fatty acids were imaged simultaneously in live mammalian cells [101]. 3.5. Pharmaceutical compounds Imaging of pharmaceutical compounds is a typical application for spontaneous Raman microscopy. However, also here, their fast imaging speed makes non-linear Raman microscopy techniques attractive. The reduced spectral information is usually not a problem, since the samples consist of a limited and well defined number of different compounds, the spectra of which are well known. If single frequency images are nevertheless needed, broadband approaches can be employed which are still much faster than spontaneous Raman microscopy. Consequently, non-linear Raman imaging has been used both for investigating the distribution of compounds in pharmaceutical preparations as well as the uptake of active ingredients. The image acquisition time advantage of SRS over spontaneous Raman microscopy was explored in investigations of commercially available tablets from different manufacturers. Here it was found that results of similar quality require 104 times less acquisition time when SRS microscopy was used [103]. CARS microscopy based single frequency imaging has been performed on a variety of other pharmaceutical products including protein loaded lipid solid

Fig. 2. Live cell SRS microscopy of 5-ethynyl-2′-deoxyuridine (EdU), an alkyne carrying a thymidine analogue used to visualize DNA at the alkyne vibrational resonance at 2120 cm−1. a) Spontaneous and SRS spectra of EdU. b) and c) Live cell SRS imaging of DNA (2120 cm−1), background (2180 cm−1) and lipids (2850 cm−1) after addition of EdU to the culture medium. The last row then shows respective overlays of the images. d) SRS images of DNA as a function of EdU concentration. Image integration times 2.62 s (256 × 256 pixels). Reproduced with permission from [102].

Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

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particles [104] and loaded silica particles [105], lipid based solid dosage forms [106] and micronized particles in liquid dosage forms [107]. As has been explained, the fast imaging capabilities of non-linear Raman microscopy is among other factors owed to the reduced spectral information collected, but new developments in the field of broadband CARS microscopy also allow for rapid image acquisition over extended spectral ranges, as was demonstrated in similar experiments. Under these conditions, CARS microscopy was about 100 times faster than conventional Raman microscopy [108]. Further improvement on the imaging speeds achievable with non-linear Raman microscopy using ultrashort excitation pulses [31] has been demonstrated on tissues and will also be applicable to investigations of solid materials [109]. Compared to this relatively large number of studies of the drugs themselves, relatively little work has been published on their uptake and distribution in an organism. This is certainly due to the low local concentrations, which are often below the detection limits of non-linear Raman microscopy. On a cell culture basis, uptake of tyrosine-kinase inhibitors has been investigated with broadband SRS microscopy [110]. In organisms, similar studies, however, have been limited either to monitoring indirect effects like drug induced LD accumulation [111] or to following lipid based drug carriers [92]. 4. Biomedical applications 4.1. Investigations of cells Since the driving force behind the development of non-linear Raman microscopy techniques were biological applications, nearly all papers published in this field have reported imaging of cells, often as a proof of principle for possible future use of the methods. With the more recent shift of focus from methodological development to applications, many reports have now appeared, in which non-linear Raman imaging is used to answer a biological question. An obvious aim of the application of non-linear Raman imaging is the identification of cellular organelles and the distinction between different cell types based on their vibrational spectra. Ideally this is based on a full vibrational spectrum which is subsequently analyzed with appropriate chemometric approaches [112]. With the exception of broadband experiments, non-linear Raman images are, however, recorded at single frequencies. Subsequent recording of images at different vibrational resonances can be used to distinguish specific features, if specific marker bands are known. With this approach, it was possible to distinguish between nucleus and cytoplasm using CARS microscopy [113]. The vibrational resonances suited for DNA imaging are in a crowded spectral region between 1000 and 1100 cm−1. Due to the accompanying strong non-resonant background in CARS microscopy under these conditions, much better results have been obtained with SRS microscopy with which it is possible to follow the structural rearrangements during the cell cycle [114]. So far, the identification of cellular identity during stem cell differentiation has been the main cellular biological question which was tackled using non-linear Raman imaging. Initial reports on the monitoring of embryogenic stem cells with CARS microscopy still suffered from poor image quality for imaging in the fingerprint region [115]. Nevertheless it was possible to distinguish between osteoblasts and adipocytes differentiated from adipose-derived stem cells [116]. Much improvement over these single frequency imaging results is obtained if broadband CARS microscopy is employed instead. Under these circumstances, also spectral components in the fingerprint region can be recovered and analyzed. The quality of the data thus obtained is high enough to allow distinction between osteoblasts, chondrocytes and adipocytes from human mesenchymal stem cells with an excellent statistical significance despite the fact that the cell populations exhibit pronounced heterogeneities [117]. Lipid enrichment is accompanying aggressive growth in certain tumors. CARS microscopy in the CH-stretch vibration region therefore

also found use as a diagnostic tool to identify circulating tumor cells both in a mouse model [118] and in blood samples from prostate cancer patients [119]. In both cases, CARS microscopy was employed in a multimodal experiment, in which results from two photon fluorescence microscopy complemented the CARS microscopy data. 4.2. Tissue experiments Similar to the cell based experiments, non-linear Raman imaging was also used to investigate tissues either as sections or in vivo. An important difference to experiments on tissues as compared to cultured cells is that unless thin sections are used, the transmission type detection geometries employed commonly for both CARS and SRS microscopy cannot be used. For CARS microscopy, it has been shown that due to multiple scattering in the sample, a backscattering detection geometry is efficient despite the fact that most CARS signal is generated in the forward direction [25]. The situation is more complicated in the case of SRS microscopy, but recent advances show that also here back-scattered signals can be detected using specific detector geometries [27]. Especially imaging of tissues from the nervous system has become an important application of non-linear Raman imaging. This is due to the fact that myelin sheaths which are forming layers around the neurons, are very rich in lipids and thus give rise to very strong signals. First CARS microscopy experiments in this field were performed on myelin figures as models and exploited polarized CARS scattering in order to unravel the composition of the structures [120]. Subsequently, a number of ex vivo and in vivo CARS studies on nerves were published. The aim in these investigations was to follow myelin degradation which was either drug-induced [121,122] or disease related [123,124]. CARS microscopy was also used to follow healing of injuries of the spinal cord [24] or the sciatic nerve [125]. While myelin structures are clearly separable from the surroundings [126], the situation is more complicated if microscopic techniques shall be used to delineate tumor boundaries. The strategy here is to generate sets of images at specific vibrational resonances and perform image analysis for the distinction of different regions. A typical example is the calculation of volumes of nuclei as compared to that of the cytoplasm in a specific region [53]. Based on similar schemes, SRS microscopy was used for imaging brain tumors in mice [127]. These results and results from CARS microscopy experiments on tissue sections [128] suggest that both non-linear Raman microscopy techniques give results of the same quality than standard H&E histological staining (Fig. 3). Here, the major advantage of the non-linear Raman techniques clearly is the largely reduced sample preparation time due to the unnecessary staining and their in vivo application potential. Meanwhile, all sorts of other tissues have also been investigated. In general one can state that either facile access or the presence of lipid rich structures have qualified systems as suitable for nonlinear Raman microscopy. Optical access is no problem in case of investigations of skin. For this reason, imaging of sebaceous glands has early on served as a test for live animal imaging using CARS and SRS microscopy [22,25]. The high imaging speed of CARS microscopy has recently also been exploited to monitor structural changes of sebaceous glands during sebum secretion [129]. In order to compare healthy skin and pathological changes, a number of experiments have been performed mapping skin with multimodal approaches including CARS microscopy [129–132]. Another example for lipid rich structures which are well suited for non-linear Raman imaging are atherosclerotic deposits. After imaging of arterial tissue using CARS microscopy was demonstrated, a number of investigations proved its potential for the monitoring of atherosclerotic changes. First investigations using broadband CARS microscopy showed that it was possible to correlate the morphologies of atherosclerotic lipid deposits and their chemical composition [133]. Experiments based

Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

M.J. Winterhalder, A. Zumbusch / Advanced Drug Delivery Reviews xxx (2015) xxx–xxx

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Fig. 3. Broadband CARS microscopy of murine liver tissue. a) Spectral image of a portal triad within the murine liver tissue with the nuclei in blue, collagen in orange and protein content in green. A, portal artery; B, bile duct; V, portal vein; Ep, epithelial cell and En, endothelial cell. b) SHG and two photon excited fluorescence images highlighting the fibrous collagen network (the respective spectra are shown in c). d–f) Spectral images of individual vibrational modes represented by the color channels in a) at 785 cm−1 (d, DNA); 855 cm−1 (e, collagen) and 1004 cm−1 (f, phenylalanine). g) Single-pixel spectra from the nucleus (DNA), collagen fiber, arterial wall and a lipid droplet. h–l) Additional spectral channels providing histochemical contrast: 1302 cm−1 (h); 1665 cm−1 (i); 2884 cm−1 (j); 3228 cm−1 (k); elastin (l), 1126 and 1030 cm−1 but not 677, 817 and 1302 cm−1. Scale bars, 20 μm. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.) Reproduced with permission from [109].

on broadband CARS or a combination of CARS and two photon fluorescence microscopy later highlighted the contribution of cholesterol on the formation of the deposits [134,135]. Other examples for investigations of lipid rich tissues are experiments on liver tissue. Also here, CARS microscopy was first used to characterize healthy tissue [136]. Subsequently, the influence of high fat diet or changes due to steatosis were monitored [74,137,138]. Both, broadband CARS microscopy and

SRS microscopy were recently used to investigate drug-induced liver injuries [138,139]. It has been pointed out that especially the possibility to quantify parameters like liver lipid content, number and size of LDs, and their chemical composition and packing are important diagnostic factors which would otherwise require the application of a variety of different techniques on stained tissue samples. Non-linear Raman microscopy therefore appears to have a high clinical diagnostic potential in these

Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024

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cases. Consequently, first studies using CARS microscopy as a tool for basic clinical research are now being published. An example is an investigation on the liver-protective mechanisms of uridine administration together with tamoxifen [140]. 5. Conclusion and outlook Non-linear Raman microscopy, especially CARS and SRS microscopy, is becoming a widely used addition to the standard microscopy techniques. While the methodological development still is a very active area with new non-linear vibrational imaging methods being put forward [141,142], a large number of reports on their application is now available. The main strengths of non-linear approaches are the short image acquisition times and their robustness against background fluorescence from the sample. This opens new possibilities for investigations of biomedical problems which cannot be tackled with conventional spontaneous Raman spectroscopy. Among these applications are CARS microscopy based endoscopic approaches [124,143–145] and non-linear Raman based cytometry [146–148]. Also in both of these cases, the fast imaging capabilities which are realized without the use of previous staining will allow a whole range of new experiments. In conclusion it is clear that non-linear Raman microscopy will be an important complement to existing fluorescence techniques. Especially in biomedical applications, it will allow many experiments for which the use of fluorescence microscopy is prohibited because of the need to stain the sample and spontaneous Raman microscopy is too slow. References [1] T.A. Klar, S.W. Hell, Subdiffraction resolution in far-field fluorescence microscopy, Opt. Lett. 24 (1999) 954–956. [2] E. Betzig, G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, H.F. Hess, Imaging intracellular fluorescent proteins at nanometer resolution, Science 313 (2006) 1642–1645. [3] M. Orrit, J. Bernard, Single pentacene molecules detected by fluorescence excitation in a para-terphenyl crystal, Phys. Rev. Lett. 65 (1990) 2716–2719. [4] W.E. Moerner, L. Kador, Optical-detection and spectroscopy of single molecules in a solid, Phys. Rev. Lett. 62 (1989) 2535–2538. [5] J.B. Pawley (Ed.) Springer, Berlin, 2006. [6] R.Y. Tsien, Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture), Angew. Chem. Int. Ed. Engl. 48 (2009) 5612–5626. [7] O. Shimomura, Discovery of green fluorescent protein (GFP) (Nobel Lecture), Angew. Chem. Int. Ed. Engl. 48 (2009) 5590–5602. [8] M. Chalfie, GFP: lighting up life (Nobel Lecture), Angew. Chem. Int. Edit. 48 (2009) 5603–5611. [9] G.J. Puppels, F.F.M. Demul, C. Otto, J. Greve, M. Robertnicoud, D.J. Arndtjovin, T.M. Jovin, Studying single living cells and chromosomes by confocal Raman microspectroscopy, Nature 347 (1990) 301–303. [10] M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, K. Fujita, Labelfree Raman observation of cytochrome c dynamics during apoptosis, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 28–32. [11] J.X. Cheng, X.S. Xie, Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications, J. Phys. Chem. B 108 (2004) 827–840. [12] M. Müller, A. Zumbusch, Coherent anti-Stokes Raman scattering microscopy, ChemPhysChem 8 (2007) 2156–2170. [13] C.L. Evans, X.S. Xie, Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine, Annu. Rev. Anal. Chem. 1 (2008) 883–909. [14] C.Y. Chung, J. Boik, E.O. Potma, Biomolecular imaging with coherent nonlinear vibrational microscopy, Annu. Rev. Phys. Chem. 64 (64) (2013) 77–99. [15] A. Zumbusch, W. Langbein, P. Borri, Nonlinear vibrational microscopy applied to lipid biology, Prog. Lipid Res. 52 (2013) 615–632. [16] J.P. Pezacki, J.A. Blake, D.C. Danielson, D.C. Kennedy, R.K. Lyn, R. Singaravelu, Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy, Nat. Chem. Biol. 7 (2011) 137–145. [17] A. Alfonso-Garcia, R. Mittal, E.S. Lee, E.O. Potma, Biological imaging with coherent Raman scattering microscopy: a tutorial, J. Biomed. Opt. 19 (2014). [18] H.A. Rinia, M. Bonn, M. Muller, E.M. Vartiainen, Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition, ChemPhysChem 8 (2007) 279–287. [19] M.T. Cicerone, K.A. Aamer, Y.J. Lee, E. Vartiainen, Maximum entropy and timedomain Kramers–Kronig phase retrieval approaches are functionally equivalent for CARS microspectroscopy, J. Raman Spectrosc. 43 (2012) 637–643. [20] A. Zumbusch, G.R. Holtom, X.S. Xie, Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering, Phys. Rev. Lett. 82 (1999) 4142–4145. [21] P. Nandakumar, A. Kovalev, A. Volkmer, Vibrational imaging based on stimulated Raman scattering microscopy, New J. Phys. 11 (2009) 033026.

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Please cite this article as: M.J. Winterhalder, A. Zumbusch, Beyond the borders — Biomedical applications of non-linear Raman microscopy, Adv. Drug Deliv. Rev. (2015), http://dx.doi.org/10.1016/j.addr.2015.04.024