Cardiovascular diseases (CVDs), many of which are influenced by exposure to environmental xenobiotics, lack physiologically relevant in vitro models for cardiotoxicity assessment. Although some pollutants have established associations with CVD, the effects of a wide range of potential toxicants remains unknown. Here, we developed a three-dimensional recellularized humanized engineered heart tissue (rHHT) platform by integrating decellularized human left ventricular extracellular matrix with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), yielding spontaneously contracting tissues that recapitulate key features of native ventricular myocardium. We also generated a hiPSC line stably expressing the calcium indicator GCaMP6f, enabling real-time and longitudinal monitoring of calcium transients. Using ethanol and rotenone as examples, we demonstrate that the rHHT platform provides a sensitive system for evaluating cardiotoxicity and is more stringent than conventional monolayer approaches. This study presents a scalable platform for xenobiotic cardiotoxicity assessment, with potential applicability to high-throughput screening, mechanistic studies, and future personalized medicine applications.

Sixty-two centres in 16 countries contributed with 2,694 open fractures cases to an international, multi-centric, retrospective cohort study involving different healthcare settings. The INTELLECT study results show that there are significant disparities on the management of open lower limb fractures internationally. A timely, multidisciplinary, guideline-directed care is a protective factor for developing infective complications, non-union and requiring an amputation.

The composite material-like extracellular matrix (ECM) in the sinoatrial node (SAN) supports the native pacemaking cardiomyocytes (PCMs). To test the roles of SAN ECM in the PCM phenotype and function, we engineered reconstructed-SAN heart tissues (rSANHTs) by recellularizing porcine SAN ECMs with hiPSC-derived PCMs. The hiPSC-PCMs in rSANHTs self-organized into clusters resembling the native SAN and displayed higher expression of pacemaker-specific genes and a faster automaticity compared with PCMs in reconstructed-left ventricular heart tissues (rLVHTs). To test the protective nature of SAN ECMs under strain, rSANHTs and rLVHTs were transplanted onto the murine thoracic diaphragm to undergo constant cyclic strain. All strained-rSANHTs preserved automaticity, whereas 66% of strained-rLVHTs lost their automaticity. In contrast to the strained-rLVHTs, PCMs in strained-rSANHTs maintained high expression of key pacemaker genes (HCN4, TBX3, and TBX18). These findings highlight the promotive and protective roles of the composite SAN ECM and provide valuable insights for pacemaking tissue engineering.

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.

The human heart is a durable biological motor, beating approximately 3.4 billion times in an 80-year lifespan. Actual cardiac performance originates in the function of cardiac myocytes, which are electromotile, changing in length in response to a change in membrane potentials. Groundbreaking work by Hugh Huxley and Jean Hanson et al. in the 1950s introduced the “sliding filament theory” that during muscle contraction, myosin and the filamentous actin protein form cross-bridges, allowing myosin to slide along actin, leading to length changes and force generation. Some muscle contraction mechanisms remain unexplained, such as how the sarcolemma accommodates ∼0.2-μm-displacement length changes per sarcomere per cardiac cycle without experiencing significant distortion. Here we invoke and test for the presence and function of a canonical non-conventional motor protein, prestin (Slc26a5), in cardiomyocytes as an amplifier of actin-myosin force generation. Prestin has been thought to be expressed exclusively in outer hair cells (OHCs) of the inner ear. It is a direct voltage-to-force convertor and mediates electromotility of OHCs as part of the molecular element of cochlear sound amplification. We hypothesize that prestin is expressed in mouse and human heart tissue and serves as the cardiac mechanical amplifier. We investigated whether the genetic deletion of Slc26a5 (Slc26a5-/-) produces in vivo and in vitro cardiac contractility changes. Multimodal Second Harmonic Generation (SHG) two-photon fluorescence microscopy and Stimulated Emission Depletion (STED) microscopy analyses were used to substantiate findings of the expression and functional roles of prestin in ventricular myocytes. We conclude that prestin serves to amplify actin-myosin force generation in cardiomyocytes, accounting for the non-linear properties of muscle contraction. The functional significance of prestin is underpinned by alterations of cardiac contractility in Slc26a5-/-mice. Our results suggest that prestin may serve as a broader cellular motor amplifier.

Cardiac cells generate and amplify force in the context of cardiac load, yet the membranous sheath enclosing the muscle fibers-the sarcolemma-does not experience displacement. That the sarcolemma sustains beat-to-beat pressure changes without experiencing significant distortion is a muscle-contraction paradox. Here, we report that an elastic element-the motor protein prestin (Slc26a5)-serves to amplify actin-myosin force generation in mouse and human cardiac myocytes, accounting partly for the nonlinear capacitance of cardiomyocytes. The functional significance of prestin is underpinned by significant alterations of cardiac contractility in Prestin-knockout mice. Prestin was previously considered exclusive to the inner ear's outer hair cells; however, our results show that prestin serves a broader cellular motor function.

HomeCirculation: Arrhythmia and ElectrophysiologyVol. 14, No. 12Making Heads or Tails of the Large Mammalian Sinoatrial Node Micro-Organization Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBMaking Heads or Tails of the Large Mammalian Sinoatrial Node Micro-Organization Regan L. Smithers, MA, Hillary K.J. Kao, MA, Sarah Zeigler, MA, Sergey Yechikov, PhD, Jan A. Nolta, PhD, James W. Chan, PhD, Nipavan Chiamvimonvat, MD and Deborah K. Lieu, PhD Regan L. SmithersRegan L. Smithers Department of Internal Medicine, Division of Cardiovascular Medicine (R.L.S., H.K.J.K., N.C., D.K.L.), University of California, Davis. Institute for Regenerative Cures and Stem Cell Program, University of California Davis Health Systems (R.L.S., H.K.J.K., S.Z., J.A.N., D.K.L.). , Hillary K.J. KaoHillary K.J. Kao https://orcid.org/0000-0002-0849-8507 Department of Internal Medicine, Division of Cardiovascular Medicine (R.L.S., H.K.J.K., N.C., D.K.L.), University of California, Davis. Institute for Regenerative Cures and Stem Cell Program, University of California Davis Health Systems (R.L.S., H.K.J.K., S.Z., J.A.N., D.K.L.). , Sarah ZeiglerSarah Zeigler Institute for Regenerative Cures and Stem Cell Program, University of California Davis Health Systems (R.L.S., H.K.J.K., S.Z., J.A.N., D.K.L.). Bridges to Stem Cell Research Program, California State University (S.Z.). , Sergey YechikovSergey Yechikov Department of Biomedical Engineering (S.Y.), University of California, Davis. , Jan A. NoltaJan A. Nolta Institute for Regenerative Cures and Stem Cell Program, University of California Davis Health Systems (R.L.S., H.K.J.K., S.Z., J.A.N., D.K.L.). , James W. ChanJames W. Chan Department of Pathology and Laboratory Medicine (J.W.C.), University of California, Davis. , Nipavan ChiamvimonvatNipavan Chiamvimonvat https://orcid.org/0000-0001-9499-8817 Department of Internal Medicine, Division of Cardiovascular Medicine (R.L.S., H.K.J.K., N.C., D.K.L.), University of California, Davis. Department of Veterans Affairs, Northern California Health Care System, Mather, CA (N.C.). and Deborah K. LieuDeborah K. Lieu Correspondence to: Deborah K. Lieu, PhD, Department of Internal Medicine, Division of Cardiovascular Medicine, Institute for Regenerative Cures, University of California, Davis, No. 1616, 2921 Stockton Blvd, Sacramento, CA 95817. Email E-mail Address: [email protected] https://orcid.org/0000-0002-3765-3203 Department of Internal Medicine, Division of Cardiovascular Medicine (R.L.S., H.K.J.K., N.C., D.K.L.), University of California, Davis. Institute for Regenerative Cures and Stem Cell Program, University of California Davis Health Systems (R.L.S., H.K.J.K., S.Z., J.A.N., D.K.L.). Originally published19 Nov 2021https://doi.org/10.1161/CIRCEP.121.010465Circulation: Arrhythmia and Electrophysiology. 2021;14Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: November 19, 2021: Ahead of Print The sinoatrial node (SAN) is endowed with protective mechanical and electrical properties that allow the pacemaking cardiomyocytes (PCMs) to sustain robust automaticity. The leading pacemaking site is known to be located at the superior (head) and inferior (tail) SAN1; however, it remains an enigma why these regions are the leading sites and how they are protected from a source-sink mismatch. Here, we discovered a unique micro-organization at the head and tail regions of the large mammalian SAN that may restrict the leading pacemaking sites to these origins through electrical and mechanical insulations, enabling intratissue and intertissue current source-sink balance.The SAN from 6-month-old market hogs was sectioned parallel to the epicardial surface and immunostained for the pacemaking HCN4 (hyperpolarization-activated cyclic nucleotide-modulated 4) channel, TNT (troponin T), and VIM (vimentin) to identify the PCMs, CMs, and fibroblasts, respectively (n=3). Over 400 confocal images were tiled from each SAN cryosection at ≈1.1 to 1.4 mm from the epicardial surface to reveal the histology from the SAN head to tail (Figure [A]). Here, we report for the first time the distinct regional micro-organization in the SAN. Specifically, PCMs were arranged in randomly distributed clusters at the head and tail sections, in stark contrast to those in the midsection that aligned in the direction of the head-to-tail axis. The clustering pattern in the SAN head and tail may contribute to an optimal intratissue current source-sink balance between the PCMs and fibroblasts that facilitates the pacemaking function, whereas the higher length-to-width aspect ratio in the aligned midsectional PCMs could enable fast conduction of the excitation wavefront to the opposite end, thereby restricting the pacemaking initiation to a single leading region for synchronized excitation.Download figureDownload PowerPointFigure. Porcine sinoatrial node (SAN) micro-organization. A SAN cryosection immunostained for HCN4 (hyperpolarization-activated cyclic nucleotide-modulated channel 4) and TNT (troponin T) showing cellular micro-organization from the head to tail (A) and extracellular matrix (ECM) protein distribution for COL (collagen) I (B), COL III (C), and ELN (elastin; D). A magnified image of a SAN conduction pathway (SACP) is shown (A). Magnified images for the central regions of the head to tail are also presented (B–D). Immunostaining of HCN4 and TBX (T-box) 18 in the SAN regions and the respective regional normalized expression quantification are shown (E; HCN4 head, midsection, and tail: n=8 images quantified for each section; TBX18 head, midsection, and tail: n=406, 437, and 385 nuclei, respectively, quantified from 3 images each of HCN4-positive cells). An illustration summarizing the SAN micro-organization (F). The intratissue electrical coupling between the CM and fibroblast (FB) clusters creates a source-sink balance in the head and tail sections, which is connected by an aligned midsection for fast conduction connecting the two SAN ends. The ECM at the SAN-atrial border—mainly ELN (elastin)—electrically and mechanically insulates the SAN, minimizing intertissue electrical coupling except at the SACPs. ACM indicates atrial cardiomyocyte; PCM, pacemaking cardiomyocyte; VIM, vimentin; and DAPI, 4',6-diamidino-2-phenylindole. *P<0.05, **P<0.01, and ****P<0.0001.The SAN is electrically insulated from the neighboring atrial myocardium (the intertissue current sink), except for several specialized SAN conduction pathways that allow the excitation wavefront to exit2. A SAN conduction pathway was observed in the tail region, with PCM clusters extending from the SAN to the atrial myocardium (Figure [A], inset). Major ECM proteins—COL (collagen) I, COL III, and ELN (elastin)—showed a similar organizational pattern in the central region of the SAN as what we had reported3, with ELN spanning the tensile-bearing COL I- and COL III-surrounded PCM clusters, forming an ECM with a composite-like material property (magnified images in Figure [B and D]). No sectional difference in the ECM organization was observed among the central region in the head, middle, and tail sections within the SAN. At the SAN-atrial peripheral border, COL I and III similarly exhibited no sectional difference from the tissue head to tail. Contrariwise, ELN formed a thick barrier at the border of the SAN head and tail but less so in the midsection. This elastic border is anticipated to undergo deformation during cyclic contractions to dissipate the mechanical stress imparted on the PCMs, thus acting as a mechanical insulator in minimizing the cellular strain within the protective border. Furthermore, it may also serve as an electrical insulation to prevent the atrial sink from draining current from the PCM source, consequently facilitating pacemaking in the head and tail sections. While the midsection lacking an insulating border could increase coupling to the atrial sink, the alignment of the PCMs parallel to the atrial myocardium may minimize the transverse electrotonic conduction to the atrium if the gap junctions mainly localize at the intercalated disks.For pacemaking-associated genes, HCN4, responsible for the membrane clock, was 1.7-fold and 1.4-fold higher in PCMs at the head and tail regions, respectively, compared with the midregion (Figure [E]), consistent with the leading pacemaking site reported at either end of the SAN but a faster frequency of automaticity if led by the superior end.1 Remarkably, transcription factor TBX (T-box) 18, important for the SAN head formation,4 was more abundant in PCMs in the head and tail regions. Although TBX18 is reportedly present only during cardiac development,4 it could be detected in the nucleus of HCN4-positive PCMs and VIM-positive fibroblasts. TBX18 was also observed in the atrial tissue but the expression was restricted to the fibroblasts (data not shown). Quantification of nuclear TBX18 fluorescence in PCMs showed the highest expression in the SAN head, at ≈450% and ≈21% higher than the middle and the tail regions, respectively (Figure [E]). The presence of TBX18 in the PCMs agrees with a publication suggesting that a TBX18-positive tertiary heart field is the origin of PCMs5.Our findings provide unique intratissue structural insights for the SAN head and tail, with an electrical and mechanical intertissue insulation formed by an ELN-rich border (Figure [F]). The PCMs in these sections are clustered among the fibroblast aggregates and expressed higher HCN4 and TBX18, distinct from the aligned midsectional PCMs. Collectively, the differential micro-organization and gene expression in the SAN are critical for restricting the pacemaking initiation to either the head or tail region and a faster pacemaking frequency at the superior SAN.Data are available on request from the authors.Article InformationSources of FundingThis work is supported by the California Institute for Regenerative Medicine (CIRM DISC2-10120 to Dr Lieu). H.K.J. Kao is supported by the NIH T32HL0863500 fellowship. Dr Chan is supported by the NSF grant 1264776. Dr Chiamvimonvat is funded by NIH R01 HL085727, NIH R01 HL085844, NIH R01 HL137228, VA Merit Review Grant I01 BX000576, and I01 CX001490.Disclosures Dr Lieu is a scientific consultant for Novoheart, Ltd. The other authors report no conflicts.Footnotes*R.L. Smithers and H.K.J. Kao contributed equally.For Sources of Funding and Disclosures, see page 1089.Correspondence to: Deborah K. Lieu, PhD, Department of Internal Medicine, Division of Cardiovascular Medicine, Institute for Regenerative Cures, University of California, Davis, No. 1616, 2921 Stockton Blvd, Sacramento, CA 95817. Email [email protected]eduReferences1. Brennan JA, Chen Q, Gams A, Dyavanapalli J, Mendelowitz D, Peng W, Efimov IR. Evidence of superior and inferior sinoatrial nodes in the mammalian heart.JACC Clin Electrophysiol. 2020; 6:1827–1840. doi: 10.1016/j.jacep.2020.09.012CrossrefMedlineGoogle Scholar2. Fedorov VV, Glukhov AV, Chang R, Kostecki G, Aferol H, Hucker WJ, Wuskell JP, Loew LM, Schuessler RB, Moazami N, et al.. Optical mapping of the isolated coronary-perfused human sinus node.J Am Coll Cardiol. 2010; 56:1386–1394. doi: 10.1016/j.jacc.2010.03.098CrossrefMedlineGoogle Scholar3. Gluck JM, Herren AW, Yechikov S, Kao HKJ, Khan A, Phinney BS, Chiamvimonvat N, Chan JW, Lieu DK. Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix.PLoS One. 2017; 12:e0185125. doi: 10.1371/journal.pone.0185125CrossrefMedlineGoogle Scholar4. Wiese C, Grieskamp T, Airik R, Mommersteeg MT, Gardiwal A, de Gier-de Vries C, Schuster-Gossler K, Moorman AF, Kispert A, Christoffels VM. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3.Circ Res. 2009; 104:388–397. doi: 10.1161/CIRCRESAHA.108.187062LinkGoogle Scholar5. Bressan M, Liu G, Mikawa T. Early mesodermal cues assign avian cardiac pacemaker fate potential in a tertiary heart field.Science. 2013; 340:744–748. doi: 10.1126/science.1232877CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails December 2021Vol 14, Issue 12Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCEP.121.010465PMID: 34794338 Originally publishedNovember 19, 2021 Keywordselectrophysiologycardiac myocytessinoatrial nodemammalsPDF download Advertisement SubjectsBasic Science Research

Hirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.

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.

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c, a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18, and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

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.

Medullary thyroid carcinoma (MTC) is a rare form of thyroid malignancy that can be diagnostically challenging on fine needle aspiration (FNA) cytology. Ancillary tests such as elevated serum or immunohistochemical positive calcitonin have been helpful, yet they can occasionally provide false positive results. In search for an alternative method to improve diagnostic accuracy (DA), we applied hyperspectral Raman spectroscopy to characterize the biochemical composition of single cells from MTC and compared their spectral information to cells from other types of thyroid nodules. Hyperspectral Raman images of 117 MTC single cells from digested tissue were obtained with a line-scan hyperspectral Raman microscope and compared to 127 benign and 121 classic variant of papillary thyroid carcinoma (CVPTC) cells. When principal component analysis and linear discriminant analysis were used to classify the spectral data, MTC cells were differentiated from benign and CVPTC cells with 97% and 99% DA, respectively. In addition, MTC cells exhibited a prominent Raman peak at 1003 cm-1, whose intensity is 84% and 226% greater on average than that observed in benign and CVPTC cells, respectively. When specifically utilizing only this peak as a spectral marker, MTC cells were separated from benign and CVPTC cells with 87% and 95% DA, respectively. As this peak is linked to phenylalanine, which is known to be associated with calcitonin release in thyroid parafollicular cells, the increased intensity further suggests that this Raman peak could potentially be a new diagnostic marker for MTC. Furthermore, preliminary data from MTC cells (n=21) isolated from a simulated FNA procedure provided similar Raman signatures when compared to single cells from digestion. These results suggest that "Raman-based cytopathology" can be used as an adjunct technique to improve the diagnostic accuracy of FNA cytopathology at a single cell level.

Soluble, small amyloid-β oligomers (AβO) are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid-β (Aβ) capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between Aβ peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600cm-1 and 2900cm-1 can be used to monitor the molecular interactions between SLF and Aβ. This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti-Aβ therapeutic agents.

We show that multifocal 1064 nm Raman microscopy based on Hadamard-coded multifocal arrays is useful for imaging carbon nanotubes (CNTs) that would otherwise be damaged if a conventional single focus microscope design is used. The damage threshold for CNTs, dependent on laser power density and exposure time, limits the spectral detection sensitivity of single focus Raman imaging. With multifocal detection, the signal-to-noise ratio of the Raman spectra were improved by more than a factor of three, allowing for the G and D Raman bands of CNTs to be detected while avoiding specimen damage. These results lay the foundation for developing multifocal 1064 nm Raman microscopy as a tool for in situ imaging of CNTs in plant material.

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.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have many promising applications, including the regeneration of injured heart muscles, cardiovascular disease modeling, and drug cardiotoxicity screening. Current differentiation protocols yield a heterogeneous cell population that includes pluripotent stem cells and different cardiac subtypes (pacemaking and contractile cells). The ability to purify these cells and obtain well-defined, controlled cell compositions is important for many downstream applications; however, there is currently no established and reliable method to identify hiPSC-derived cardiomyocytes and their subtypes. Here, we demonstrate that second harmonic generation (SHG) signals generated directly from the myosin rod bundles can be a label-free, intrinsic optical marker for identifying hiPSC-derived cardiomyocytes. A direct correlation between SHG signal intensity and cardiac subtype is observed, with pacemaker-like cells typically exhibiting ~70% less signal strength than atrial- and ventricular-like cardiomyocytes. These findings suggest that pacemaker-like cells can be separated from the heterogeneous population by choosing an SHG intensity threshold criteria. This work lays the foundation for developing an SHG-based high-throughput flow sorter for purifying hiPSC-derived cardiomyocytes and their subtypes.

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, label-free marker for identifying iPSC-CMs. Here, we demonstrate the use of SHG microscopy for characterizing iPSC-CMs and their subtypes.

A rapid widely accessible spectroscopic analysis was developed for quantification of carbon nanotubes in plant tissues.