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Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multi-color images originating from CH2 and CH3 vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.
With rare exceptions, histopathological techniques cannot be used for in situ diagnosis since they require the tissue to be frozen or fixed, thinly sliced, and stained with devitalizing dyes before the tissue can be observed under a microscope. A real-time, in situ technique for acquiring histopathological images would thus be tremendously advantageous in situations where the tissue removal imposes risk and delay required for fixation, sectioning and staining. Ideally, in situ stain-free histopathology could be used in the setting of tumor surgery to examine the margins of a resection cavity for residual neoplastic tissue.
Coherent Raman imaging (CRI), including coherent anti-Stokes Raman scattering (CARS) microscopy1,2 and stimulated Raman scattering (SRS) microscopy3-6, allows chemical imaging based on intrinsic vibrational properties of the molecules in the tissue and thus does not require staining or labeling. The more recent SRS microscopy techniques offer further advantages: (1) elimination of image artifacts due to non-resonant background, (2) excitation spectra identical to well-documented spontaneous Raman spectra, (3) a linear relationship between signal and concentration of target molecules. Compared to spontaneous Raman scattering, signal levels are amplified by orders of magnitude by virtue of stimulated excitation of molecular vibrations, allowing video-rate image acquisition speeds7,8. Due to nonlinear excitation, CRI enables intrinsically three-dimensional sectioning1,4,9 and does not require physical sectioning of the tissue. Efficient signal detection in reflection of thick samples8,10as well as biocompatibility of laser excitation intensities11,12 have been demonstrated, allowing stain-free, in vivo imaging in mice7,8,13-16 and humans8,17.
The properties of CRI have inspired us18 and others19-21 to image diseases with CARS microscopy and development of miniaturized clinical instrumentation is underway22-25. This work describes the development of multicolor SRS imaging with contrast originating from lipids, protein and red blood cells, aiming to resembles the most widely used stain in histopathology, hematoxylin and eosin (H&E). We demonstrate ex vivo that the key diagnostic features appreciated with H&E staining are well visualized with CRI in tissue from wild type mice as well as mouse models of various diseases of the central nervous system.
CRI requires two synchronized pulse trains to achieve stimulated excitation of the vibrational transition. In narrowband SRS, the frequency bandwidth of such lasers is chosen to be smaller than the typical linewidth of Raman transition and the difference in frequency of the two center frequencies can be tuned to excite a single vibration at a time. Compared to multiplex excitation of multiple vibrations simultaneously using broadband lasers, this approach maximizes imaging speed. We use a passively mode-locked Nd:YVO4 laser (PicoTrain, High Q Laser) at 1064nm (7ps pulse-duration, 76MHz repetition rate) to provide the first beam, known as the Stokes beam. A portion of this beam is frequency-doubled and used to pump an optical parametric oscillator (Levante Emerald, APE GmbH) to provide the second tunable beam (650nm-1000nm tuning range), known as the pump beam. The two pulse trains are then overlapped in space and time, aligned into a laser scanning microscope (FV300, Olympus) and focused into the sample with a high numerical aperture (NA) water-immersion lens (UPlanSApo 60×, Olympus)2,4.
For SRS microscopy we measure the intensity loss of the pump beam due the excitation of molecular vibrations in the focus. Sensitive detection of the signal requires the implementation of a high-frequency modulation transfer scheme to distinguish the SRS signal from linear sample absorption or laser intensity fluctuation. This relies on the fact that SRS can only occur if both pump and Stokes are incident on the sample. By modulating the Stokes beam at a known frequency with an acousto-optic modulator and measuring the modulation transfer to the originally un-modulated pump beam at the same frequency, SRS can be detected specifically. If the modulation frequency is faster than the typical laser noise (e.g. 10MHz), high sensitivity detection is achieved at the moderate laser power required for medical imaging. Experimentally, the transmitted light from the sample is collected with a high NA oil condenser (Nikon), filtered to block the modulated Stokes beam (CARS890/220, Chroma) and detected with a large-area photodiode (FDS1010, Thorlabs). The modulation transfer is measured by a lock-in amplifier (SR844, Stanford Research), whose signal is fed into the microscope to provide the signal for a pixel. All images were sampled with 512 × 512 pixels ~30μs pixel dwell time, which is limited by the speed of the lock-in amplifier. In the meantime we have developed improved detection electronics and achieved imaging speeds up to video-rate (30 frames per second)4,8.
For the multi-color imaging, three individual scans per region in the tissue are acquired with one beam fixed at 1064nm and the other at 816.7nm (CH2-stretching vibration), 810.5nm (CH3-stretching vibration) and 700nm (hemoglobin TPA). Fast tuning between these three wavelengths can be achieved within a few seconds by changing the Lyot filter and the cavity length of the optical parametric oscillator. Images are acquired with the microscope scanning software and processed in ImageJ. Pseudo-colors can be assigned arbitrarily, e.g. to mimic H&E stained sections. In most cases we use a red-green-blue (RGB) look-up table. The green and red channels show CH2 and hemoglobin images, respectively. The blue channel is a CH3-CH2 difference image thresholded to show only nuclei (see Results section). To normalize for the curvature of the field of view we calculated a featureless reference image by averaging and smoothing about one hundred SRS images acquired the same day from different regions and dividing the CH2 and CH3 images before subtraction. Linear look-up tables are applied for the RGB coloring. H&E-like colors were applied using the “multiply layers” blend mode in Adobe Photoshop.
All animals were treated in compliance with Harvard IACUC protocols #29-01 and #10-02. Mice were euthanized using CO2 or by an overdose of ketamine and xylazine, followed by cervical spine dislocation. Organs were harvested and imaged immediately. We used a mouse brain matrix (Harvard Apparatus) to produce 1mm thick tissue slices and mounted them stably for imaging between a No.1 coverslip and a coverslide using a ~480μm spacer (Grace Bio-Labs Inc.). For the comparison with H&E-stained micrographs, the brain was coronally sectioned and one half was immediately fixed in 10% formalin for standard histopathological evaluation, while the other was imaged without further preparation as described above. Comparison is shown from mirror-images.
We created primary brain glioma models from immunocompromised mice. The human glioma stem cell lines were derived from Brigham and Women's Hospital patients undergoing surgery according to approved protocols. These xenografts were derived from glioblastoma biopsies and implanted. Dissected xenografts were washed in artificial cerebrospinal fluid (CSF) and manually dissociated to single cells. Red blood cells were removed using Lympholyte-M (Cedarlane). The cells were cultured in DMEM/F12 (with L-glutamine, Invitrogen) medium containing glucose (0.3%), penicillin/streptomycin (50μg/ml), Apo-transferrin (0.1mg/ml), Progesterone (20nM), Sodium selenite (30nM), putrescine (60μM), insulin (25μM/ml), sodium bicarbonate (3mM), HEPES (10 mM), 20ng/ml EGF and 20ng/ml FGF. Live cells were counted using a hemocytometer and Trypan blue exclusion. The Brigham and Women's Hospital IACUC protocol number is 000995.
We created breast cancer metastasis models from nude mice (Harlan Laboratory). MDA-MB-231 breast cancer cells were cultured in Eagle's Minimum Essential Medium (ATCC, containing non-essential amino acids, 2mM L-glutamine, 1mM sodium pyruvate, and 1500mg/L sodium bicarbonate) supplemented with 10% fetal bovineserum, 1% penicillin-streptomycin, and grown in a 5% CO2 incubator at 37°C. Metastatic breast cancer were established by intracranially injecting 1×105 MDA-MB-231 cells (in 2μl PBS)at 1mm posterior to bregma, lateral 2mm and 2.5mm depth from skull surface. The Brigham and Women's Hospital IACUC protocol number is 04662.
We used a transient model of focal brain ischemia in C57/BL6J mice (Charles River lab). Under an operating microscope (Leica), the origin of right common carotid artery (CCA) was ligated with 6-0 silk suture. A loose ligature and a microvascular clip (F.S.T) were placed on the CCA before the origin of the internal carotid artery (ICA). An incision was made between the two sutures through which the silicone coated tip (HeraeusKulzer) of a 7-0 filament was advanced to the origin of the middle cerebral artery (MCA). With a flexible fiber probe fixed to the right skull above the MCA, regional cerebral blood flow was monitored with laser Doppler flowmetry (PF2B, Perimed) to confirm successful MCA occlusion and reperfusion. The filament was withdrawn 30 minutes later. Mice were euthanized at day 3 post-ischemia. The Massachusetts General Hospital IACUC protocol number is 2007N000168.
We created relapsing-remitting EAE (R-EAE) from C57Bl/6 mice (Charles-River). Each mouse was immunized subcutaneously (into the flanks) with 100μg of MOG35-55 (MEVGWYRSPFSRVVHLYRNGK), 400μg of Mycobacterium tuberculosis (strain H37Ra; Difco) in complete Freund's adjuvant (CFA, Difco). 100ng of Bordetella pertussis toxin (List Biological Laboratories via Cedarlane Ltd.) was given intravenously at the time of immunization and 2 days later. The Brigham and Women's Hospital IACUC protocol number is 04576.
Different molecules in the sample can be selectively imaged by tuning the difference frequency of the excitation beams into the target vibrational resonance (see Methods). Fig. 1A shows the vibrational spectra of the three major components of cells and tissue (lipids, protein and water) with characteristic peaks for the CH2 and CH3 stretching vibrations at 2845cm-1 and 2940cm-1, respectively. While imaging of DNA and RNA has recently been demonstrated with CRI in the finger-print region of Raman spectra26, the above 3 species are present in higher concentration and allow for faster imaging speed, as desirable for medical in vivo imaging18.
Figs. 1B-D show SRS images of a C2C12 mammalian cell at these two bands. Consistent with spontaneous Raman spectroscopy27, the CH2 image (Fig. 1B) mainly highlights lipids in the cytoplasm, while lipid-poor nuclei appear dark and featureless. The CH3 image (Fig. 1C) shows signal from both lipids and proteins (Fig. 1A). The same cytoplasmic features can be seen as in the CH2 image, but there is additional detail from within nuclei. We have demonstrated that a quantitative protein image17, which shows a similar distribution of proteins in both the nucleus and cytoplasm, can be extracted from the CH2 and CH3 images based on the Raman ratios in Fig. 1A. Here we aim to generate a vibrational counter-stain to the CH2 image that accentuates nuclei similarly to hematoxylin component of an H&E stain. To do so, we subtract the CH2 from the CH3 image such that the cytoplasmic signal essentially vanishes and then threshold the resulting difference image to only show nuclear signal. This is possible in a robust way because the low CH2signalinside the nucleus. Fig. 1D illustrates the resulting multi-color overlay showing the cell-body information from the CH2 image in green and nuclear information from a thresholded CH3-CH2 difference image in blue. Diagnostically important sub-nuclear features such as nucleoli are visible in the thresholded CH3-CH2 difference image, demonstrating that it is a true nuclear counterstain rather than merely the inverse of the CH2 image.
Figs. 1E-J illustrate similar contrast obtained in fresh mouse brain tissue. Fig. 1E and the green component of Fig. 1H show theCH2 signal from lipid-rich myelinated axons28 and unmyelinated neuropil. Additionally, the vibrationally off-resonant image (Fig. 1G) has contrast due two-color two-photon absorption (TPA)9,16,29 and shows individual red blood cells within capillaries due to hemoglobin absorption. Overlaying these three images results in a single three-color, stain-free histological image shown in Fig. 1H. Since the choice of the color scheme is arbitrary, it is possible to choose the look-up tables to mimic an H&E stained section (Fig. 1J). This allows easy comparison with traditional histopathological techniques (Fig.1I). Comparison of Fig.1I and J reveals that the stain-free image is free of the vacuolization observed in the H&E stained section because the fresh tissue imaging eliminates the need for freezing, fixation or sections and hence associated tissue processing artifacts.
To further demonstrate the universal utility of this multi-color contrast we imaged a variety of organs of a wild-type mouse (Fig. 2). The myocardium image (Fig. 2B) shows myocytes with numerous lipid droplets, central nuclei and a rich vascular network. The typical morphology of the renal glomerulus inside Bowman's capsule with tufts of capillaries and surrounding renal tubules is seen in the image of the kidney (Fig. 2C). Variable lipid storage within hepatocytes is seen in the liver (Fig. 2D). In the lung, the vast capillary network of the alveolar walls is highlighted in the red channel (Fig. 2E). Characteristic cross striations and capillaries around the myocytes are easily identified in the skeletal muscle image (Fig. 2F). Clusters of germinal cells and larger blood vessels are seen in the ovary (Fig. 2G). Fig. 2H shows two different layers of mouse skin including the stratum corneum layer (top) with a cobblestone arrangement of cells and a protein-rich hair shaft in blue. A deeper image of the epidermis (bottom) collected simply by changing the level of focus highlights many nuclei in the stratum basale. The germinal centers with abundant green cytoplasm surrounded by mature lymphocytes with higher nuclear-to-cytoplasmic ratio, as well as scattered red blood cells, are observed in the white pulp of the spleen (Fig. 2I).
Imaging of gray matter (Fig. 3A) demonstrates neuronal cell bodies in blue, reflecting a strong signal in the CH3-CH2 channel within in a background of the lipid-rich neuropil. Linear processes in green, due to high CH2 signal, are axons (Figs.3A to E). The white matter from the corpus callosum is dominated by densely packed axons with interspersed oligodendrocytes (Fig. 3E).The cytoarchitecture of various regions including the thalamus (B), the dentate gyrus (C) and the granular cell layer of the cerebellar cortex (D) is easily distinguished, as compared with the mirror images stained with Luxol-H&E (Figs. 3F to J)
In addition to demonstrating image quality and histopathological information in healthy tissue, we imaged mouse models of four relatively common brain pathologies including glioblastoma, metastases, demyelination and stroke. We aimed at recognizing tumor from normal tissue, tumor margins, inflammatory infiltration and evidence of demyelination and cell death. For this reason, we imaged mouse models of invasive, high-grade glioma derived from human brain tumor stem cells, and breast cancer metastasis as well as demyelination and stroke models. Fresh tissue samples were bisected to acquire corresponding stain-free and paraffin-embedded, H&E-stained images from the same region of the same animal (Fig. 4).
In comparison to healthy brain, increased cellularity, a fundamental histopathological feature of neoplasia, is made obvious in SRS and H&E images for both primary brain tumors (Figs. 4A, B, E, and F) and brain metastases (Figs. 4C, D, G, and H). The remaining myelinated axons interspersed between tumor cells are visible in the center of the infiltrating glioma (Fig. 4A) in contrast with the solid, expansile metastatic tumor (Fig. 4C). Images of tumor margins illustrate the diffusely invasive nature of glial cells along white matter tracts of the corpus callosum (Fig. 4B), whereas a sharply defined margin is seen between metastasis and adjacent normal brain tissue (Fig. 4D). A three-dimensional image stack of the tumor margin further highlights this critical diagnostic feature (Video S1).
Among other pathologies that can mimic brain cancer on MRI and CT are tumefactive demyelination and subacute stroke30. Fig. 5 and Videos S2 and S3 show multicolor SRS images of mouse models of experimental autoimmune encephalitis (EAE) and ischemic stroke. The comparison with corresponding H&E images shows similar diagnostic features. Lipid-laden macrophages and perivascular lymphocytes (Fig. 5A and C) were present in areas of demyelination. Tissue vacuolization due to cerebral edema was seen as black holes (Fig. 5B and D), which correlated well with the presence of clear vacuoles on H&E-stained sections. Foamy macrophages and condensation of nuclei correlated with apoptosis.
We report the successful acquisition of microscopic, multi-colored images of diagnostic quality on various fresh tissues, without the need for tissue fixation, processing or staining. We acquired images of multiple normal mouse organs and compared them with routine histopathology. Recognition of various tissue types of different organs was easy and characteristic histological features were demonstrated with a clarity that approaches that of traditional H&E staining. In addition, all major histologic features demonstrated in H&E-stained sections of cerebral cortex, white matter, diencephalon and cerebellum were visualized with CRI. Given the great variety of histopathological features in various conditions, we also imaged multiple regions of the normal mouse brain as well as models of glioma, metastasis, stroke and demyelination.
The goal of this study was to demonstrate medical utility of CRI stain-free histology using a research type microscope in ex vivo tissue to motivate the design of a clinical prototype instrument in the future. We used the original instrumentation of SRS microscopy 4; The imaging speed was limited by the speed of the commercial lock-in amplifier and signal was collected in sample transmission. Since completion of the data acquisition, we have developed faster detection electronics and an improved signal collection scheme, which enable SRS at video-rate and in reflection from thick tissue samples, and demonstrated SRS in vivo imaging8. For this study we also used temporal multiplexing to acquire the individual color channels sequentially. Development of real-time multicolor imaging is underway17,31 and will be available for the clinical prototype. TPA can also be probed at 827nm, so more robust and inexpensive fiber lasers with a limited tuning range over the CH-region of Raman spectra can be used in future. Because imaging depth in CRS is limited to about 150 microns, the most immediate applications are in imaging exposed tissue such as ex vivo pathological samples, skin, and in vivo intra operative margins. The development of a fiber-coupled hand-held scanner for intra operative use is underway22-25. Imaging of hollow organs inside the living patient will require further development of endoscopic instrumentation in the future.
Other label-free microscopy techniques used for in situ stain-free histopathology such as Infrared (IR) absorption microscopy32 allow multi-color imaging, however the spatial resolution is limited due to the long wavelength used inIR microscopy21. Alternative high-resolution techniques include confocal reflection (CR), optical coherence tomography (OCT), two-photon excited auto-fluorescence (TPAF), second harmonic generation (SHG) and third harmonic generation (THG) microscopy33-37. They either lack the capability for multi-color, chemical imaging (CF, OCT & THG) or have contrast limited to a few, specific molecules (TPAF & SHG)38-40. The latter techniques are more similar to specific functional stains (e.g. antibody stains) in traditional histopathology, which can be used after abnormalities have been recognized on H&E sections. TPAF has also been demonstrated to allow identification of nuclear and cytosolic regions41,42 because of absence of TPAF signal inside the nucleus, analogous to the dark nuclei seen in the CH2 CRI scans. Multi-color CRI can additionally provide a “counter-stain” image with positive nuclear contrast for diagnostically important sub-nuclear features, which makes multicolor CRI images an ideal foundation for versatile in situ histopathological tissue diagnosis.
In conclusion, we have demonstrated the use of CRI for microscopic tissue imaging in a label-free manner. Moreover, CRI can be used to generate a degree of contrast, based on the intrinsic vibrational spectrum of the molecular components of biologic tissues that exceeds other label-free optical imaging methods. Our data indicates that CRI can be used for generating high quality histological images without the need of routine tissue processing. Additional work will be required to comprehensively compile an atlas of CRI images of normal tissue and various disorders. However, a preliminary sampling of key neurologic diseases supports the diagnostic capability of CRI. Although, technical hurdles in creating practical intraoperative instrumentation remains, these findings reinforce the possibility that CRI can become an invaluable clinical tool for intraoperative distinction of tumor from normal tissue and immediate margin assessment of in situ tissue before surgical closure.
We thank Dr. John Sims for the mouse stroke model, Chrissy Rooney for the invasive glioma model, Dr. Fa-Ke Lu and SrinjanBasu for the cell images, and Dr. Anita Huettner for early pathology work.
Funding: C.W.F. and B.G.S. thank Boehringer Ingelheim Fonds for a Ph.D. Fellowship and the Army Research Office for a National Defense Science and Engineering Graduate Fellowship, respectively. L.O. acknowledges the Italian Society of Multiple Sclerosis (FISM, Cod 2008/B/5). P.L.D. is a Harry Weaver neuroscience scholar of the National Multiple Sclerosis society and C.W. acknowledges NIH (R01NS049263). This work was supported by the NIH Director's Pioneer Award (to X.S.X.) and NIH/NIBIB T-R01 (grant 1R01EB010244-01).
Disclosure of Competing Interest: Harvard University has filed a patent application based on coherent Raman imaging instrumentation.