Minbiao Ji

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering microscopy provides a rapid, label-free means of detecting tumor infiltration of brain tissue ex vivo and in vivo. Virtual Histology During brain tumor surgery, precision is key. Removing healthy tissue can cause neurologic deficits; leaving behind tumor tissue can allow cancer to spread and treatment to fail. To help the surgeon clearly see tumor versus normal tissue, Ji and colleagues developed a stimulated Raman scattering (SRS) microscopy method and demonstrated its ability to identify malignant human brain tissue. In SRS microscopy, laser beams are directed at the tissue sample to generate a series of output signals called “Raman spectra.” These spectra depend on the molecular composition of the tissue. Ji et al. implanted human brain cancer (glioblastoma) cells into mice, allowed them to infiltrate and grow into tumors, and then removed slices for SRS imaging. From the resulting spectra, the authors were able to differentiate the two major components of brain tissue—lipid-rich white matter and protein-rich cortex—as well as tumors, which are full of proteins. Intraoperatively, using an imaging window into mouse brains, the authors found that SRS microscopy could locate tumor infiltration in areas that appeared normal by eye, which suggests that this tool could be applied during surgery. Imaging fresh tissue slices ex vivo could also complement or perhaps replace standard hematoxylin and eosin (H&E) staining in the clinic because it avoids artifacts inherent in imaging frozen or fixed tissues. To this end, Ji and colleagues showed that SRS microscopy could identify hypercellular tumor regions in fresh surgical specimens from a patient with glioblastoma. Certain diagnostic features were present in these specimens and readily identified by SRS, including pseudopalisading necrosis and microvascular proliferation. The next step will be to apply SRS microscopy to a large collection of human specimens to see whether this technology may be useful in quickly distinguishing glioblastoma from healthy tissue, both outside and inside the operating room. Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Large angular jump mechanism observed for hydrogen bond exchange in aqueous perchlorate solution.

Wet Twists and Turns When salts dissolve in water, their constituent positively and negatively charged ions are pulled apart and surrounded by shells of H2O molecules (see the Perspective by Skinner). Ji et al. (p. 1003) looked closely at the motion in these shells, using a type of vibrational spectroscopy sensitive to both the orientation and to the neighbors of the targeted molecules. In agreement with recent theoretical predictions, the individual water molecules shifted orientation between an anion and the surrounding liquid in sudden discrete steps, rather than by making smooth incremental rotations. Tielrooij et al. (p. 1006) compared the relative impacts of cations and anions on the rigidity of the wider water network, using spectroscopic techniques sensitive to the role of each ion. Certain cation/anion combinations, such as magnesium sulfate, appeared to act together to restrict water motion beyond the boundaries of individual shells. Water molecules shift orientation between dissolved ions and the surrounding liquid by taking large, sudden steps. The mechanism for hydrogen bond (H-bond) switching in solution has remained subject to debate despite extensive experimental and theoretical studies. We have applied polarization-selective multidimensional vibrational spectroscopy to investigate the H-bond exchange mechanism in aqueous NaClO4 solution. The results show that a water molecule shifts its donated H-bonds between water and perchlorate acceptors by means of large, prompt angular rotation. Using a jump-exchange kinetic model, we extracted an average jump angle of 49 ± 4°, in qualitative agreement with the jump angle observed in molecular dynamics simulations of the same aqueous NaClO4 solution.

Efficient Multiple Exciton Generation Observed in Colloidal PbSe Quantum Dots with Temporally and Spectrally Resolved Intraband Excitation

We have spectrally resolved the intraband transient absorption of photogenerated excitons to quantify the exciton population dynamics in colloidal PbSe quantum dots (QDs). These measurements demonstrate that the spectral distribution, as well as the amplitude, of the transient spectrum depends on the number of excitons excited in a QD. To accurately quantify the average number of excitons per QD, the transient spectrum must be spectrally integrated. With spectral integration, we observe efficient multiple exciton generation in colloidal PbSe QDs.

Multicolored stain-free histopathology with coherent Raman imaging

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 multicolor 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.

Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy

Quantitative SRS microscopy can detect human brain tumor infiltration with high sensitivity and specificity, even in tissues appearing grossly normal. Image-based classifier calls out cancer cells Ji and colleagues used a microscopy technique called stimulated Raman scattering, or SRS, to image cancer cells in human brain tissue. SRS produces different signals for proteins and lipids, which can then be assigned a color (blue and green, respectively), allowing the authors to differentiate brain cortex from tumor from white matter. Biopsies from adult and pediatric patients with glioblastoma revealed not only distinctive features with SRS microscopy but also the presence of infiltrating cells in tissues that appeared otherwise normal with traditional staining. Such infiltrating cells are important to catch early because leaving them behind after surgery nearly always leads to cancer recurrence. To make this SRS microscopy approach amenable to routine use in neuropathology, the authors also created an objective classifier that integrated different image characteristics, such as the protein/lipid ratio, axonal density, and degree of cellularity, into one output, on a scale of 0 to 1, that would alert the pathologist to tumor infiltration. The classifier was built using more than 1400 images from patients with glioblastoma and epilepsy, and could distinguish between tumor-infiltrated and nontumor regions with >99% accuracy, regardless of tumor grade or histologic subtype. This label-free imaging technology could therefore be used to complement existing neurosurgical workflows, allowing for rapid and objective characterization of brain tissues and, in turn, clinical decision-making. Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

Atomic-force-microscope-compatible near-field scanning microwave microscope with separated excitation and sensing probes

We present the design and experimental results of a near-field scanning microwave microscope working at a frequency of 1 GHz. Our microscope is unique in that the sensing probe is separated from the excitation electrode to significantly suppress the common-mode signal. Coplanar waveguides were patterned onto a silicon nitride cantilever interchangeable with atomic force microscope tips, which are robust for high speed scanning. In the contact mode that we are currently using, the numerical analysis shows that contrast comes from both the variation in local dielectric properties and the sample topography. Our microscope demonstrates the ability to achieve high resolution microwave images on buried structures, as well as nanoparticles, nanowires, and biological samples.

Multicolor stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy has opened up a wide range of biochemical imaging applications by probing a particular Raman-active molecule vibrational mode in the specimen. However, the original implementation with picosecond pulse excitation can only realize rapid chemical mapping with a single Raman band. Here we present a novel SRS microscopic technique using a grating-based pulse shaper for excitation and a grating-based spectrograph for detection to achieve simultaneous multicolor SRS imaging with high sensitivity and high acquisition speeds. In particular, we use a linear combination of the measured CH2 and CH3 stretching signals to map the distributions of protein and lipid contents simultaneously.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

Significance Microscopic imaging of DNA has to rely on the use of fluorescent staining, an exogenous labeling in biological and biomedical studies, which often leads to uncertainty with respect to the quality and homogeneity of the staining. Label-free imaging of DNA will enable noninvasive visualization of live cell nuclei in both human and animals. Spontaneous Raman microspectroscopy offers label-free chemical contrast for DNA imaging; however, its slow imaging speed hampers its wide application for in vivo and dynamic studies. Here we developed a novel and simple approach with multicolor stimulated Raman scattering microscopy to evaluate rapid DNA imaging, which can be applied to both in vivo DNA dynamic studies and instant label-free human skin cancer diagnosis. Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator.

Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but it sacrifices chemical specificity in samples with overlapping bands compared to broadband (multiplex) excitation. We develop a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multicolor SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets.

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ5-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.