Significance: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm.Aim: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms.Approach: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected.Results: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%.Conclusions: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify “indeterminate” nodules and other cytologically challenging cases.
Raman microscopy is well recognized as a nondestructive, label-free biomedical imaging method that provides abundant chemical information of the specimen. Excitation wavelengths in deep near-infrared (e.g., 1064 nm) are used in certain situations, such as when analyzing photosensitive/photolabile specimens to suppress the strong fluorescence and to avoid photodamage. However, the speed and quality of 1064 nm Raman imaging suffers from the low scattering efficiency at this long excitation wavelength and the high noise level of InGaAs detectors. In this study, we investigated a multifocal patterned approach for 1064 nm Raman imaging. A 2-D Hadamard-coded multifocal array generated with X-Y scanning galvomirrors is used to excite and collect multiple Raman spectra simultaneously. The individual spectrum at each focus is retrieved and reconstructed from the superimposed spectra of the multifocal patterns. We demonstrate that the multifocal approach improves both the signal-to-noise ratio (SNR) and the imaging speed of Raman microscopy. Compared to the traditional point scan, at optimal detector conditions, the multifocal approach can be two-times faster for achieving the same image quality and SNR, or provides spectra with three-times higher SNR while applying the same energy dose at the focus. Such improvements of imaging speed and SNR increase up to one or two orders of magnitude under higher noise conditions, such as higher readout rate and higher detector temperatures. The multifocal approach presents advantages for certain imaging situations, such as when heating related damage limits the excitation energy dose that can be applied to the sample.
Hirschsprung disease (HD) is a congenital disorder in the distal intestine and is characterized by the absence of nerve ganglion cells (aganglionosis). HD affects newborns by causing severe constipation. Surgical management is needed and consists of the accurate identification and removal of the aganglionic segment and the reconstruction of the intestinal tract. The gold standard for the definitive diagnosis of the aganglionic segment is the histologic evaluation of rectal biopsies through stained sections. However, it is a time-consuming procedure, and recognized factors for inaccurate diagnoses have been commonly reported. In recent years there has been much interest in the use of optical techniques to improve diagnostics in health care. Here, for improving the identification of ganglion cells, we propose an ex-vivo study to evaluate a combination of label-free optical modalities: second harmonic generation (SHG); two-photon autofluorescence; and Raman spectroscopy. SHG and autofluorescence images have been used to locate regions of interest in the tissue for Raman analysis, which acquires a molecular fingerprint of the ganglion cells, without needing any stains or labels. Multivariate statistical analyses of the Raman spectral data have been used for objective identification of the ganglion cells in the tissue samples with high accuracy.
The cost of taking a drug to market can exceed $2 billion dollars. The escalating cost of drug discovery is a major motivating factor for seeking new methods to predict the safety and efficacy of new compounds as early as possible in the drug development process to avoid drug attrition during late phases of clinical trials or even the withdrawal of approved drugs. Cardiotoxicity accounts for nearly 30% of US post-marketing drug withdrawal and remains a major concern to the point where the US Food and Drug Administration (FDA) is focused on in vitro cardiotoxicity screening to minimize cardiac risks associated with drugs. A technique that can directly quantify interactions between drugs and cardiomyocytes without the interference from exogenous genetic or chemical labels would be highly beneficial for directly screening these new drugs. Our group has previously shown that second harmonic generation (SHG) signals generated from myosin filaments in cardiomyocytes can be used as a robust label-free optical technique for recording cell shortening dynamics at high spatial and temporal resolution due to the ability of the myosin rod domains in heart muscle cells to emit the frequency-doubled light. The dynamics is recorded without adding any fluorescent labels that may otherwise affect and modify the natural cell contractility of the cell. In this study, we investigated the use of SHG microscopy for measuring drug-induced changes in cardiac cell contractility and discuss its feasibility as a tool for screening drugs and evaluating cardiotoxicity.
Thyroid nodules are very common, and their incidence increase with age. The majority of thyroid nodules are benign, but approximately 5-15% are thyroid cancer. The cornerstone to evaluation of most thyroid nodules is a neck ultrasound followed by fine-needle aspiration (FNA) to evaluate for malignancy. Unfortunately, approximately 15-30% of FNAs are considered “indeterminate”. In these cases, cytopathologist cannot determine if the thyroid nodule is benign or malignant based on FNA alone. Although the majority of these “indeterminate” nodules are ultimately benign, majority of these patients require thyroidectomies to rule out cancer. This puts the patient at unnecessary risk of surgical complications and increases health care costs. A better method is needed to help physicians determine the risk of malignancy in patients with indeterminate thyroid nodules. In recent years there has been much interest in the use of optical diagnostic in cancer detection. Recent investigations potentially suggest that Raman spectroscopy (RS) can be used as a clinical tool that could confer great patient advantage with minimally invasive, non-destructive, rapid and accurate diagnosis. In this study, we investigate the use of line-scan RS and imaging in combination with multivariate statistical analysis of the spectral data for objective identification and classification of single cells isolated from frozen samples of different types of human thyroid nodules. Preliminary results indicate a high sensitivity and specificity for identifying different cell types.
Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an unlimited ex vivo supply of heart cells for cardiac applications. The establishment of pure iPSC-CMs populations is crucial for downstream medical applications such as human disease modeling, patient-specific stem cell therapy, human transplantation, and drug development. However, a significant challenge is the lack of an established purification method to isolate populations of iPSC-CMs by their phenotype, maturity, and subtype due to the lack of specific iPSC-CM markers. The ability to remove potentially teratoma forming pluripotent stem cells, arrhythmia inducing immature and pacemaking cells, and other non-CMs is extremely important for engineering tissues with desired cell compositions that are both safe for human transplantation and that can accurately replicate cardiac functions. Contemporary purification techniques have either low specificity or require genetic modification. We have proposed that second harmonic generation (SHG) signals, which are known to originate from the sarcomeric myosin filaments in cardiomyocytes, can be a highly specific, labelfree marker for identifying iPSC-CMs. Here, we demonstrate the use of SHG microscopy for characterizing iPSC-CMs and their subtypes.
KEYWORDS: Raman spectroscopy, Eye, Retina, In vivo imaging, Imaging systems, Scanning laser ophthalmoscopy, Luminescence, Signal detection, Eye models, Signal to noise ratio
The use of Raman spectroscopy in biochemistry has been very successful, particularly because of its ability to identify elementary chemical species. However, application of this spectroscopic technique for in vivo assessment is often limited by autofluorescence, which make detection of Raman signatures difficult. The mouse eye has been used as an optical testbed for investigation of a variety of disease models and therapeutic pathways. Implementation of in vivo Raman spectroscopy in mice retina would be valuable but needs to be examined in context of the intrinsic auto-fluorescence artifact and potential light damage if high probing beam powers were used. To evaluate feasibility, a Raman system was built on a custom SLO/OCT platform allowing mouse positioning and morphological data acquisition along with the Raman signal from a desired retinal eccentricity. The performance of the Raman system was first assessed with a model eye consisting of polystyrene in the image plane (retina), using excitation wavelengths of 488 nm, 561 nm, and 785 nm to determine whether auto-fluorescence would be reduced at longer wavelengths. To improve the SNR, the combined system is featured with the optical compatibility for these three excitations such that their corresponding spectra from a typical region of interest can be acquired consecutively during single imaging run. Our results include emission spectra acquired over 10 s with excitation energy less than 160 J.s-1.m-2 for all wavelengths and corresponding retinal morphology for different mouse strains including WT, BALB/c and ABCA4-/-.
KEYWORDS: Calcium, Harmonic generation, Luminescence, Heart, Two photon imaging, Second-harmonic generation, Microscopes, Control systems, Line scan image sensors, Signal detection
The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.
Oftentimes cells are removed from the body for disease diagnosis or cellular research. This typically requires fluorescent labeling followed by sorting with a flow cytometer; however, possible disruption of cellular function or even cell death due to the presence of the label can occur. This may be acceptable for ex vivo applications, but as cells are more frequently moving from the lab to the body, label-free methods of cell sorting are needed to eliminate these issues. This is especially true of the growing field of stem cell research where specialized cells are needed for treatments. Because differentiation processes are not completely efficient, cells must be sorted to eliminate any unwanted cells (i.e. un-differentiated or differentiated into an unwanted cell type). In order to perform label-free measurements, non-linear optics (NLO) have been increasingly utilized for single cell analysis because of their ability to not disrupt cellular function. An optical system was developed for the measurement of NLO in a microfluidic channel similar to a flow cytometer. In order to improve the excitation efficiency of NLO, a scanned Bessel beam was utilized to create a light-sheet across the channel. The system was tested by monitoring twophoton fluorescence from polystyrene microbeads of different sizes. Fluorescence intensity obtained from light-sheet measurements were significantly greater than measurements made using a static Gaussian beam. In addition, the increase in intensity from larger sized beads was more evident for the light-sheet system.
A major limitation of spontaneous Raman scattering is its intrinsically weak signals, which makes Raman analysis or imaging of biological specimens slow and impractical for many applications. To address this, we report the development of a novel modulated multifocal detection scheme for simultaneous acquisition of full Raman spectra from a 2-D m × n multifocal array. A spatial light modulator (SLM), or a pair of galvo-mirrors, is used to generate m × n laser foci. Raman signals generated within each focus are projected simultaneously into a spectrometer and detected by a CCD camera. The system can resolve the Raman spectra with no crosstalk along the vertical pixels of the CCD camera, e.g., along the entrance slit of the spectrometer. However, there is significant overlap of the spectra in the horizontal pixel direction, e.g., along the dispersion direction. By modulating the excitation multifocal array (illumination modulation) or the emitted Raman signal array (detection modulation), the superimposed Raman spectra of different multifocal patterns are collected. The individual Raman spectrum from each focus is then retrieved from the superimposed spectra using a postacquisition data processing algorithm. This development leads to a significant improvement in the speed of acquiring Raman spectra. We discuss the application of this detection scheme for parallel analysis of individual cells with multifocus laser tweezers Raman spectroscopy (M-LTRS) and for rapid confocal hyperspectral Raman imaging.
Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free
biochemical analysis of individual cells and small biological particles in suspension. The integration of the two
technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in
physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations,
both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing
longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to
carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss
the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that
the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug
discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate
experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical
tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal,
sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on
this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood
cell diseases.
Imaging the structure and correlating it with the biochemical content of the retina holds promise for fundamental
research and for clinical applications. Optical coherence tomography (OCT) is commonly used to image the 3D
structure of the retina and while the added functionality of biochemical analysis afforded by Raman scattering
could provide critical molecular signatures for clinicians and researchers, there are many technical challenges to
combining these imaging modalities. We present an ex vivo OCT microscope combined with Raman spectroscopy
capable of collecting morphological and molecular information about a sample simultaneously. The combined
instrument will be used to investigate remaining technical challenges to combine these imaging modalities, such
as the laser power levels needed to achieve a Raman signal above the noise level without damaging the sample.
The Raman analysis of common, non-absorbing gases was performed using an 18@1 fiber-optic probe coupled to a
multi-pass capillary cell (MCC) for signal enhancement. The MCC is fabricated by metal-coating, using silver or other
highly reflective metals, the inside of a 1-2 mm diameter glass capillary using commercially available silvering solutions
and provides enhancements up to 30-fold over measurements using the fiber-optic probe alone. The design of the MCC
is simple and the device is easy to incorporate into an experimental setup making it suitable for remote and <i>in-situ</i>
analysis. Although the MCC is functionally similar to liquid-core waveguides that have been previously described in the
literature, the MCC is not based on total internal reflection and so the refractive index of the analyte is not important to
the operation of the device. The principle of operation of the MCC is similar to mirror-based multiple pass Raman cells,
however, the MCC is not expensive, alignment is trivial and an optical path length up to several meters in length is
possible. With our first-generation silver-coated MCCs, limits of detection were determined to be 0.02% and 0.2% for
CH4 and CO2 respectively. In this talk we will discuss optimization of the MCC and issues involved in its use.
The use of fs lasers to directly write phonic structures inside a glass has great potential as a fabrication method for three-dimensional all-optical integrated components. The ability to use this technique with different glass compositions --specifically tailored for a specific photonics application -- is critical to its successful exploitation. Consequently, it is important to understand how glass composition effects waveguide fabrication with fs laser pulses and how different glasses are structurally modified after exposure to fs laser pulses. We have used confocal laser spectroscopy to monitor the changes in glass structure that are associated with waveguide fabrication. Using a low power continuous wave (cw) Ar laser as excitation source we have measured both Raman and fluorescence spectra of the modified regions. Raman spectroscopy provides us with information on the network structure, whereas fluorescence measurements reveal the presence of optically active point defects in the glass. In this paper we review our work on fs-laser fabrication and characterization of photonic structures in glass and discuss the effect of glass composition on processing parameters and structural modification.
Refractive index changes have been induced inside bulk fused silica by using femtosecond (fs) laser pulses tightly focused inside the material. Waveguides have been fabricated inside the glass by scanning the glass with respect to the focal point of the laser beam. The refractive index change is estimated to be ~ 10-4. Other more complex three-dimensional structures have also been fabricated (curved waveguides, splitters, and interferometers). We also report on fluorescence spectroscopy of the fs-modified fused silica using a confocal microscopy setup. Using a 488 nm excitation source, a fluorescence at 630 nm is observed from the modified glass, which is attributed to the presence of non-bridging oxygen hole center (NBOHC) defects created by the fs pulses. The fluorescence decays with prolonged exposure to the 488 nm light, indicating that the defects are being photobleached by the excitation light.
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