Single cell RNA sequencing has emerged as a powerful tool for resolving transcriptional diversity in tumors, but is limited by throughput, cost and the ability to process archival frozen tissue samples. Here we develop a high-throughput 3′ single-nucleus RNA sequencing approach that combines nanogrid technology, automated imaging, and cell selection to sequence up to ~1800 single nuclei in parallel. We compare the transcriptomes of 485 single nuclei to 424 single cells in a breast cancer cell line, which shows a high concordance (93.34%) in gene levels and abundance. We also analyze 416 nuclei from a frozen breast tumor sample and 380 nuclei from normal breast tissue. These data reveal heterogeneity in cancer cell phenotypes, including angiogenesis, proliferation, and stemness, and a minor subpopulation (19%) with many overexpressed cancer genes. Our studies demonstrate the utility of nanogrid single-nucleus RNA sequencing for studying the transcriptional programs of tumor nuclei in frozen archival tissue samples. Single cell RNA sequencing is a powerful tool for understanding cellular diversity but is limited by cost, throughput and sample preparation. Here the authors use nanogrid technology with integrated imaging to sequence thousands of cancer nuclei in parallel from fresh or frozen tissue. Single cell RNA sequencing (RNA-seq) has provided new insights into cancer progression by resolving complex cell types5, developmental hierarchies6, and phenotypic plasticity8. However, initial methods were limited by low-throughput, high costs and extensive technical errors, which inhibited their broad application in cancer research11. Recent technological innovations using microwells14 and microdroplet encapsulation16 have increased the throughput of single cell RNA-seq to thousands of cells and greatly reduced associated costs. However, high-throughput methods do not enable imaging or selection of single cells, leading to high doublet error rates and the inclusion of many unwanted cells, such as dead cells11. Furthermore, the ability to sequence RNA in nuclei instead of whole cells on these platforms has not been demonstrated.The development of single cell sequencing technologies has revolutionized many diverse fields of biology over the last 5 years17. This is logistically challenging and problematic in cancer research, since most archival tissue samples have previously been flash frozen and stored in cryobanks, a process that ruptures the cell membranes. However, previous work has shown that nuclear membranes remain intact during freeze-thaw cycles, and that single nuclei can be isolated from frozen tissues18 that allow nuclear suspension preparation21 and construction of cDNA libraries while avoiding the use of proteases to dissociate whole cells18. Neuroscientists have also shown that RNA-seq of single nuclei is feasible and highly representative of transcriptional profiles from cells, when fresh tissues are dissociated24 and even when postmortem brain stored long term at −80 °C is used18. This is in contrast to whole brain cells, where the use of proteases for whole-cell dissociation has been shown to activate the crucial immediate early genes25. However, to date, no one has investigated the transcriptional profiles of single tumor nuclei, to determine if they are representative of whole tumor cells.A second major challenge for single cell RNA-seq in cancer research is that most methods require fresh tissue to be dissociated into single cell suspensions for analysisTo address these limitations, we developed a nanogrid platform and microfluidic depositing system that enables imaging, selection, and sequencing of thousands of single cells or nuclei in parallel. We applied this nanogrid single-nucleus RNA-seq (SNRS) system to compare the transcriptional profiles of cancer cells and nuclei in cell lines and further applied this method to study phenotypic diversity and subpopulations in a frozen tumor sample from a triple-negative breast cancer (TNBC) patient.rs > 0.9), with only 3–38 discordant genes in each sample -adjusted p value ≥0.05 or |log2(fold change)| < 1) across three biological replicates in each of the four cell lines  and an ER+/PR+ subtype (T47D). Nuclear fractions were purified from cellular suspensions using a detergent to lyse the plasma membrane, followed by three rounds of purification to eliminate residual cytoplasmic RNA (Online Methods). The nuclear suspensions were imaged in bright field and fluorescence using DAPI to ensure that cellular membranes and cytoplasm was no longer present  Fig. . Each weule Fig. . A seconule Fig. . The cDNule Fig. . A detaiule Fig. .Fig. 2OvTo determine if the transcriptional profiles of single nuclei were representative of whole cells, we applied nanogrid SNRS to isolate and sequence nuclei and cells from an isogenic Her2+ breast cancer cell line SK-BR-3) P < 0.05) due to large numbers of nuclei in each distribution, most metrics, including mapped read fractions, 5′ UTR tags, 3′ UTR tags, rRNA reads, and gene coverage showed very low percent changes (2–5%) between nuclei and cells. However, we did observe an 8.5% increase in intron tags and 9.2% decrease in CDS tags in the single nuclei data, which is consistent with the dogma that nuclei contain unprocessed pre-mRNA that have not yet undergone splicing and export from the nucleus29. To compare our data to another platform, we performed Drop-Seq analysis of SK-BR-3 cells and compared the sequencing metrics to downsampled nanogrid RNA data  between nuclei and whole cells, consistent with the bulk experiments  of gene expression values between all cells, and gene dropouts at lower expression levels, justifying for the use of the zero-inflated negative binomial single cell differential expression (SCDE) statistical model |> = 1). Gene ontology and pathway analysis of these genes showed higher levels of LincRNAs, pseudogenes, and nuclear-function genes in the nucleus compared to cells, while conversely the nuclei showed low levels of mitochondrial and transmembrane genes, which showed higher levels in the cells 32, which showed that most signatures (97.35%) were highly concordant, but that 5/189 gene sets were differentially expressed  and two pathways that were upregulated in the whole cells (NFKB inflammation and CSR serum starvation response up-regulated). We also examined the expression levels of 40 breast cancer genes that are frequently deregulated in TCGA26 , was found to be significantly lower (2.4-fold) in the nuclei compared to the cells. We also applied the PAM50 gene signature33 to classify single cells into the five major clinical breast cancer expression subtypes  or basal-like). The relative frequency shifted slightly in each group, however, it was not significant by concordance test . These data showed that most of the single cells and nuclei were classified as Her2 positive, consistent with the report that SK-BR-3 is a Her2-positive cell line by immunohistochemistry34. However, we also found that a subset of the single cells were classified as luminal B , basal-like , normal-like , or luminal A  in both the nuclei and cells (frequency concordance P > 0.4), suggesting that the population of cells represented a composite mixture of molecular subtypes  analysisA26 Fig. . Only onpes Fig. .3. In our data, we found that the copy number profiles of nuclei and whole cells were highly concordant (Pearson’s r = 0.91) and identified several large-scale amplifications, including 1q (MDM4), 7p, 8q (MYC), 17q (HER2), 19 and 20 (BCAS1) and deletions of 6q, 11p, 13, 17p (TP53), 18 (DCC) and Xp . Collectively, these data suggest that transcriptome profiles of nuclei are highly representative of whole cells, and can be used to study many cancer genes and signaling pathways.We used the single cell gene expression data to calculate genomic copy number profiles at ~1 megabase resolution (Online Methods). A similar approach was previously applied to single cell RNA-seq data from glioblastoma patients Xp Fig. . However21. The hierarchical clustering of the copy number data identified 5/502 cells with normal diploid copy number profiles representing stromal cells  and 497 cells with aneuploid tumor profiles. The aneuploid tumor cells had amplifications in chromosomes 1q (MDM4), 19q (AKT2), and deletions on chromosome 1p, 3, 4, 11p, 12q (MDM2), 17p (TP53), 19q, 22. These data suggest that the purity of the tumor cells was increased from 41 to 99% using the automated imaging and selection of nanogrid wells with larger nuclei  breast tumor that was cryopreserved for 2 years. Nuclear suspensions were generated from the frozen tumor and 502 nuclei were sequenced using cDNA obtained with the nanogrid platform. In total, we identified 1421 wells with single nuclei during the imaging step and selected 502 wells with larger nuclei (>8 microns) to increase the tumor purity and avoid sequencing normal stromal cells. The tumor purity was estimated by histopathology to be 41%. The data metrics resulted in an average of 975,097 reads, 14,886 UMI counts, and 3619 genes/nucleus ] ≥ 1) between single tumor cells (N = 497), stromal cells (N = 5), and normal breast cells (N = 240). This analysis identified 30/229 known cancer genes (T200 targeted platform) and 11/40 TCGA breast cancer genes that were differentially expressed relative to the normal breast cells, including KRAS, GATA3, CCND1, CDH1, GNAS, and several others. Most of these genes were expressed across all tumor cells, however, a few cancer genes, including AURKA and TOP2A, were restricted to a minor subpopulation  and several Keratin genes . Subpopulation C was the rarest subpopulation (18.8%), consisting of 78 cells, but had the highest number of cancer genes (N = 13) that were overexpressed . Several genes that were differentially expressed in subpopulation C are involved in DNA damage repair , apoptosis  and mitosis or cell cycle regulation, including high expression of Ki-67 (MKI67), a marker of cell proliferation. To determine if the expression of the 13 cancer genes were truly restricted to the minor C subpopulation, we plotted the individual gene expression levels of single cells using Violin plots  and performed principle component analysis (PCA) linear dimension reduction to identify the top variable genes from the first five principal components (49.34% variance explained) for unsupervised clustering using shared nearest neighbor (SNN) modularity optimization37 Fig.  and t-diots Fig. . These d40. Our analysis of the cell cycle data estimate that 18.75% of the cells were in the G2/M stage of the cell cycle, suggesting that we did not bias strongly against other cell cycle stages by the selection of larger nuclei by DAPI staining during the nanowell imaging steps.Due to the high levels of Ki-67 in subpopulation C, we performed gene signatures analysis for the cell cycle stages  in all of the tumor nuclei Fig. . These d33 to investigate the diversity of the five major breast cancer subtypes, and found that most cells were of the basal-like (156/416) subtype, which is expected since this subtype is commonly associated with TNBC patients33. However, we also identified a significant fraction of single tumor nuclei that were Her2 positive (15.6%), luminal A (3.4%), luminal B (26.4%), and normal like (17.1%), suggesting that the tumor was a mixture of different subtypes , but that a few cells were IM (4%) or belonged to the other TNBC subclasses  and individual single cell library construction, the nanogrid system completes imaging of all 5184 wells in only 10 min and requires only a single sequencing library to be constructed for sequencing analysis. This greatly reduces the cost to about $2.20/cell, with the cost per 1800 cell library at $1.10/cell and the sequencing cost at $1.10/cell for achieving 220K reads/cell on a HiSeq4000 system (Illumina). Another study developed a nanowell system that uses barcoded mRNA capture beads to create a portable, low-cost system called Seq-Well43. While very cost effective and high throughput, this system does not allow selection of imaged nanowells, which is an advantage of our platform.In this study we report a nanogrid SNRS technology that performs high-throughput single-nuclei imaging, selection, and sequencing in an integrated platform. Nanogrid sequencing has several technical advantages over existing high-throughput single cell RNA-seq methods that use microwells24. We further applied nanogrid SNRS to study a frozen tumor sample from a TNBC patient that was cryopreserved for about 2 years. On a parenthetical note, we do not expect that length of freezing will have a major effect on the stability of nuclear RNA, however, multiple freeze-thaw cycles may lead to RNA degradation. In the TNBC tumor data, we identified a minor (19%) subpopulation of tumor cells that were highly proliferative and overexpressed many cancer genes. Our data also showed phenotypic heterogeneity in stemness, angiogenesis, and proliferation, as well as the co-existence of multiple breast cancer subtypes in single cells from an individual tumor. These data are consistent with a recent single cell RNA-seq study in glioblastoma that showed variation in EMT and the co-existence of many clinical subtypes within the same patient’s tumors. This striking amount of pre-existing phenotypic variation may explain why TNBC patients evolve rapid resistance to neoadjuvant chemotherapy46.We applied the nanogrid SNRS to study the transcriptional differences of cells and nuclei in a breast cancer cell line, which showed a high concordance in transcript abundance and expression levels. These data challenge the long-standing paradigm that nuclear transcriptomes are not representative of whole cells. However, our data are consistent with recent studies in neural cell types that have reported a high concordance between the transcriptional profiles of nuclei and whole cells48. Our data also show that mitochondrial genes are not expressed at high levels in the nucleus, which is consistent with the localization of the mitochondria in the cytoplasm49. We also found an increased abundance of intronic sequences in the nucleus, which is expected based on our knowledge of alternative splicing of pre-mRNAs in the nucleus29. Importantly, our data suggest that these gene expression differences did not have a major influence on the measurements of most cancer genes and signaling pathways.Although the majority of nuclear and cytoplasmic genes were concordant, we did identify a few differences in LincRNAs, pseudogenes, mitochondrial genes, and nuclear-function genes. These data are consistent with previous data showing that pseudogenes and LincRNAs are transcribed and preferentially located in the nucleus over the cytoplasmIn closing, the SNRS nanogrid system opens up new avenues of investigation into the analysis of single nuclei transcriptomes from frozen tissue sections. In addition to imaging live and dead cells, or nuclear size as estimated from intensity, the nanogrid imaging approach is flexible and can be applied broadly to identify cell types of interest based on fluorescent markers. We expect that the SNRS nanogrid approach will benefit not only cancer research, as demonstrated in this study, but will also benefit many diverse fields of biomedical research, where the analysis of single nuclei from frozen tissue samples can provide new insights into human diseases.The SK-BR-3 breast cancer cell was obtained from Characterized Cell Line Core (CCLC) Facility at the University of Texas MD Anderson Cancer Center, Houston, TX. The cell line was tested and found to be mycoplasma-negative and authenticated with the short tandem repeat method by the CCLC facility. A frozen tumor sample was obtained from an invasive ductal carcinoma patient in collaboration with Dr Hong Zhang and Dr Funda Meric-Bernstam at MD Anderson Cancer Center. The study was approved by the internal review board at the University of Texas MD Anderson Cancer Center and the patient consented to the study. The triple-negative status of this tumor sample was determined by immunohistochemistry for estrogen receptor (<1%) and progesterone receptor (<1%), and fluorescence in situ hybridization analysis of HER2 amplification using the CEP-17 centromere control probe (ratio of HER2/CEP-17 < 2.2).2, 21 mM MgCl2, 0.05% BSA, 0.2% Nonidet P-40)), 200 mL of 106 mM MgCl2, 10 mg of DAPI, and 5 mM EDTA. The frozen tumors were dissociated into nuclear suspensions by mincing with no.11 surgical scalpels in 1 mL of NST-DAPI cytoplasmic lysis buffer at 4C using ice blocks in a plastic Petri dish. Nuclear suspensions were filtered through 37-μm plastic mesh . The final suspension was diluted to 1000 μL of 1 cell/50 nL with 1× PBS and D-RNase free water . For the SKBR3 cell line, a 10 cm cell plate at ~100% confluence was tryspinized and washed two times with 1× PBS. The nuclei were released and stained with DAPI/NST lysis buffer, re-pelleted, suspended in DAPI-NST, and then filtered through a mesh filter. As with the tumor single-nucleus suspension, the final suspension was diluted to 1000 μL of 1 cell/50 nL with 1× PBS and D-RNase free water.Nuclei from frozen tumors were isolated using a NST/DAPI buffer . The whole-cell suspension was dispensed in the same manner as the nuclear suspension.To ensure that the nuclear fractions did not have any evidence of cytoplasmic membranes, we stained the nuclear fractions with Hoechst and Cytotracker Red for imaging at ×40 on the Nikon Eclipse fluorescent microscope. Similarly, we stained cellular fractions with Hoechst and Cytotracker Red to image the cytoplasm and nucleus. Z-stack images were collected in fluorescent channels, in addition to bright field imaging, and images were merged to observe the cytoplasm and nucleas of the cell suspensions.2 aluminum alloy with 5184 nanoliter wells (150 nL) using standard manufacturing processes50. Each nanowell was preprinted with barcoded primers (UMIs) with poly(dT) ends during manufacturing. This chip-based technology has been published previously in targeted sequencing and real-time PCR applications52. The second component is a multisample nanodispenser that uses microsolenoid-control to precisely dispense 50 nL volumes into the nanowells. The third component is an automated imaging system composed of an Olympus BX43 microscope fitted with a ×4 objective, a robotic stage, and a CCD camera that is programmed to take images of all 5184 wells using a customized version of µManager open source software, followed by automated image analysis software called CellSelect™ that is used to analyze acquired images and identify single cell or nuclei containing wells. A more detailed technical description of the ICELL8 nanogrid single cell sequencing system and its hardware components is provided elsewhere41.The ICELL8™ single cell nanogrid RNA-seq system consists of three main components. The first component is an ICELL8™ nanogrid chip manufactured in a square layout (72 × 72) in a 41 mm53 (10 nt) and a 30-mer oligo-dT that is subsequently incorporated into the 3′ end of the transcript during WTA using the SMRT-Seq254 chemistry.Disaggregated nuclear or cellular suspensions were diluted to 20 cells/µL in eight wells of a 384-well plate (A1 through D2) and dispensed into the WaferGen ICELL8™ chip resulting in a Poisson distribution with about 30% of nanowells with single nuclei or cells. Poisson distribution of cells or nuclei in the chip nanowells is achieved because each 50 nL dispense on average dispenses a single cell when cells are at 20 cells/µL. Addressing every well of the ICELL8 chip takes 12–15 min. Every nanowell contains an adapter sequence with a well Barcode , UMIg for 5 min to collect cells in a single plane and the nanogrid wells are automatically imaged using an Olympus BX43 fluorescent microscope with a robotic stage. The image acquisition takes about 6 min (3 min/fluorphore). After imaging, the microchip is sealed, placed in freezing chambers, and stored at −80 °C until reverse transcription (RT). Custom CellSelectTM software identifies wells with single cells, and filters cells with no cells or multiple cells based on multiple automatic and user adjustable imaging parameters. The nanowells with single cells or nuclei are then prioritized and the user can manually review images and fluorescent channels to identify live cells or nuclei for selection. A file containing positional information on identified candidate wells (dispense file) instructs the nanodispensor to deposit reagents only in the selected wells for WTA.Following cell dispensing, the microchip is centrifuged at 300×Frozen chips were thawed, and 50 nL of RT solution  was deposited into each selected well using the nanodepositing system. For chips with single nuclei, the 57.2 μL D-RNase free water was replaced with 52.8 μL D-RNase free water and 4.4 μL Triton X-100 to promote lysis of the cellular membrane. After RT, cDNA products from selected wells were pooled together, purified, and underwent exonuclease I treatment  to remove excess, unannealed primers. The pooled barcoded cDNA libraries then underwent PCR amplification  for 18 cycles for cells or 19 cycles for nuclei. The PCR products were purified with 0.6× AMPure XP beads and eluted in 12 μL D-RNase free water. The size distribution of cDNA was QCed using the Qubit dsDNA HS fluorometric assay and Agilent’s high sensitivity DNA chip on the Bioanalyzer system.The pooled cDNA was diluted to 0.2 ng/μL and used to construct Nextera XT (Illumina) DNA libraries with i7 index primers following the manufacturer’s instructions. The final libraries, containing barcoded single nuclei or single cell transcriptomes, were sequenced at 100 paired-end cycles on the HiSeq4000 system (Illumina). Data were processed using the CASAVA 1.8.1 pipeline (Illumina Inc.), and sequence reads were converted to a master FASTQ files.RNA was extracted from the nuclear and cell fractions isolated from four breast cancer cell lines . One 10 cm dish at 60–85% confluence was used for each biological replicate  and was washed with 9 mL of 1× PBS . Cells were then resuspended in 1 mL trypsin and 9 mL cold PBS . 60 μL of the trypsinized cells were set aside to prepare slides for whole-cell imaging and microscopy. The remainder of the cells was spun at 200 rcf for 5 min. The supernatant was subsequently removed, and the cells were resuspended again in 2 mL PBS . One mL of the PBS cellular suspension was removed for whole-cell RNA extraction, spun at 100 rcf for 5 min at 4C, and resuspended in 2 mL trizol. A 20G needle was used to break apart the insoluble trizol pellets, and whole-cell RNA extraction was performed according to the manufacturer’s instructions (Fisher TR 118–100ML). The other mL of the PBS cellular suspension was removed for nuclear RNA extraction, spun at 100 rcf for 5 min at 4 °C, and resuspended in 1 mL DAPI/NST with EDTA. The DAPI/NST solution was incubated at room temperature for 4 min, spun at 8000 rcf for 5 min at 4C, and resuspended in 1 mL DAPI/NST. After a final spin at 8000 rcf for 5 min at 4C, the nuclei were resuspended in 1 mL trizol. A 20G needle was used to break apart the nuclear pellet, and an additional 1 mL of trizol was added to the suspension. Nuclear RNA extraction was performed according to the manufacturer’s instructions (Fisher TR 118–100ML).55, and gene expression levels were summarized into expected count and transcripts per kilobase million (TPM) values using RNA-seq by expectation maximization (RSEM)56. Only uniquely mapped reads were used for analysis. A quality-control step was performed on the tophat257 aligned bam files using RSeQC58 and samtools (0.1.19)59 to summarize distributions of reads that were mapped to rRNAs, mtRNA, introns, CDS, 5′ UTR, 3′ UTR and 10 kb up-/downstream of transcripts. The number of UMIs for each gene was counted by dropping reads that had duplicated UMIs using custom Perl scripts. However, we did not include UMI assays in this study.The master FASTQ file containing total reads was demultiplexed into individual fastq files with each representing one single cell or one population of cells using a Perl script. Sequencing reads in each single fastq file were mapped to the human transcriptome using bowtie214 analysis, which fits individual error models for single cell RNA-seq data using a Bayesian approach based on a zero-inflated negative binomial model process. The differentially expressed genes are defined as FDR-adjusted p value <0.05 and |log2(fold change)|> = 1. We removed genes that were detected with counts <10 in <30 cells. We applied an Empirical Bayes hierarchical model (EBSeq)60 for population RNA-seq differential expression analysis at gene levels. The differentially expressed genes are defined as those had posterior probability of being DE >0.95 and |log2(fold change)|> = 1. Clustered heat maps of gene expression were generated with R package “heatmap3”20 based on log2(count + 1), log2(TPM + 1), or z-scores. Differentially expressed genes were analyzed with Ingenuity IPA for pathway analysis and cellular organelle localization annotations. To identify differentially expressed cancer genes in TNBC tumor cells, we combined the three subpopulations of single nuclei that had aneuploidy copy number aberrations (CNAs) profiles as tumor cell populations and combined the predicted matched normal nuclei and normal nuclei from another patient as the normal cell populations. We then performed differential gene expression analysis between the two groups of single nuclei using MAST36. The differential genes were defined as having FDR-adjusted p value <0.05 and |log2 (fold change)| ≥ 1. Finally, differentially expressed cancer genes in tumor cells were identified by intersecting the DE gene list with T200 clinical gene panel and with a 40-gene TCGA breast cancer gene list.We compared the gene expression in single nuclei to whole cells using a Bayesian method for SCDE61 to classify single cells and nuclei into five established intrinsic breast cancer molecular subtypes  using “genefu” package62 with log2(TPM + 1) data matrix. Single cells or nuclei with low prediction confidence (<0.7) are set as undefined. In a separate analysis, single nuclei were classified into six TNBC subtypes, including two basal-like (BL1 and BL2), an IM, a mesenchymal (M), a MSL, and a LAR subtype63. The ER+ cells were excluded from the TNBC subtype prediction.We used the intrinsic gene centroids signature (PAM50)31 to determine the molecular phenotypes of single cells and nuclei using log2(TPM + 1) data. We first obtained GSVA scores for 189 oncogenic gene sets (MSiDB version 5.2)32 for each single nuclei or whole-cell sample, and then compared the nuclei enrichment scores to whole cells by using R package “limma”64. Differentially enriched gene sets were defined as FDR-adjusted p value <0.05 and |score difference|> = 0.1.We applied single-sample GSVA (ssGSVA)67 with annotations of “G1 phase of mitotic cell cycle” for G1 phase genes, “S phase of mitotic cell cycle” for S phase genes, “M phase of mitotic cell cycle” for M phase genes were used. A G2 phase gene list that was previously defined in synchronized HeLa cells was also used68. We then defined the four cell phase  scores as the average expression [log2(TPM + 1)] of curated cell cycle genes and defined the cell cycle phase by hierarchical clustering of centered phase scores using R package “heatmap3”20.Cell cycle genes from gene ontology set (version 5.2 MSiDB)2(count + 1) matrix was centered and scaled to z-scores to perform PCA using “prcomp” function in R (www.r-project.org). Genes that were detected in <30 cells were excluded from the analysis. The first five components were selected based on “elbow” principle and top 20 loading genes were sent for clustering using a SNN modularity optimization-based method37 and then marker genes for each detected clusters were selected using normalized log2(count + 1) matrix with the “bimodal”-based gene expression analysis69 that was built in the “FindAllMarkers” function in R package “Seurat”70. The t-SNE38 method was applied based on the pair-wise Euclidean distances of the log2(count + 1) matrix. Hierarchical clustering with “ward.D2” linkage and “Euclidean” distance was performed on log2(count + 1) matrix or z-scores using R package “heatmap3”20.The normalized log2(TPM + 1) matrix using a “moving average” approach that was adapted from a previous study3. We use the log2(TPM + 1) values as gene expression values and we further scaled the total expression of all cells to 100,000 to normalize gene expressions within each single cells to comparable scales and avoid floating the variance among highly expressed genes. We sorted the analyzed genes by their genomic coordinates that were annotated by University of California Santa Cruz gene list containing a total of 23,346 genes. We excluded genes that have expression values <0.15 on average, and ended up with ~3000 genes across the genome and ~130 genes/chromosome on average (except that Y chromosome had only one or two genes). To define the copy number baseline, we also sequenced a set of 380 normal breast tissue single cells, and took their average expressions of each gene as the normal copy number base line. We normalized single cell gene expression to this baseline to obtain a relative gene expression for each gene location. To mitigate the bias caused by extreme gene expression levels, we replaced the relative gene expression values that are >3 with 3 and relative expressions <−3 with −3. We then obtained “moving average” of adjacent 50 gene relative expression values to represent the log2(copy number ratio) of the genomic location. We normalized the log2(copy number ratio) to their mean values for each cell to center around zeros. Last, we performed hierarchical clustering20 of all tested single cell CNAs with the normal breast tissue cells to identify aneuploid tumor and normal diploid cell populations.Single cell and nuclei copy number was calculated from the logThe data from this study have been deposited into the Sequencing Read Archive and are available for download under accession SRP095350.Supplementary Information


RCP − ILCP) in transmission at wavelength ~1.5 μm reaches 97% and the extinction ratio (ER = IRCP/ILCP) is as high as 345:1. Experimental fabrications and measurements of the proposed all-dielectric metasurface are implemented and found to be in excellent agreement with the simulations. The proposed all-dielectric chiral metasurface is of advantages of high-dichroism, easy-fabrication and standard semiconductor fabrication techniques compatible, which could lead to enhanced security in fiber and free-space communications, as well as imaging and sensing applications for circularly polarized light with a highly integrated photonic platform.We propose and experimentally demonstrate a high efficient circularly polarizing dichroism waveplate (CPDW) using a Si-based all-dielectric 2Dchiral metasurface. We demonstrate that the CPDW exhibits a unique dichroism in that it functions as a transmissive quarter waveplate for one of either left-or right-handed circularly polarized incident lightand a reflective mirror for the opposite polarization. The circular polarization dichroism (CPD = I Circularly polarizing dichroism (CPD) has attracted much attentions recently due to its wide applications in various optical techniques and devices12345et al. proposed and demonstrated a 3-dimensioanl (3D) helical nano-wire circular polarizer in the IR rangeRCP − ILCP with IRCP and ILCP being the transmission of right-handed and left-handed polarization) was achieved. The physical mechanism of CPD behind the 3D helical nano-wire structure is the phase matching between the polarized electric vector along the propagating direction and surface plasmonics along the spatial spiral direction of the 3D helical nano-wires. Following the concept of the 3D helical wires, different versions of the helical wires based CPD devices were investigated, which include tapered gold-helix metamaterials1112et al. also proposed a stacked nanorod metamaterials arrays with a tailored rotational twist, from which the CPD reaches 70% theoretically, but 30% experimentally due to losses through multilayer metalsOver the past few years, advances in surface plasmonic polaritons opened an avenue for designing compact functional circularly polarizing device on subwavelength scale. In 2009, Gansel et al. reported that asymmetric transmission of a circularly polarized light through a planar metal nanostructure consisting of continuous chiral “fish-scale” elements can be observed and a maximum CPD of 25% in the visible to near-IR spectral region can be achieved16et al. demonstrated a chiral circular dichroism metamaterial consisting of a periodic array of ‘Z’-shaped silver (Ag) chiral metamolecules, from which a CPD of 90% in the reflection mode was achieved due to chiral metal-atom anisotropic absorptionThe CPD with planar plasmonic structures was also investigated. Fedotov et al. designed and fabricated a GaAs-based semiconductor chiral gammadion photonic crystal incorporated with an InAs quantum dot (QD) layer that produces circularly polarized photoluminescence (PL). Due to interaction between the chiral crystal and the photons radiated from the QDs, a strong circular anisotropy from the chiral nanostructure was observed in both simulation and experiment, and a maximum experimental circular polarization degree of 26% was achieved (circular polarization degree = (IRCP − ILCP)/(IRCP + ILCP), ILCP and IRCP are the emission intensities of the left and right circular polarization)et al. improved the single layer chiral gammadion structure with multilayer (~20 layers) chiral gammadion structure of different materials, from which a circular polarization degree of 81% in experiment was obtainedet al. further proposed a chiral nanostructure in which gammadions are replaced by four rectangles, each rotated by 90° with respect to its nearest neighborset al. proposed a 2D chiral silicon based metasurface for circularly polarized infrared radiation in the wavelength band of 3–5 μm All-dielectric based nano devices have been attracting much attention because of their low losses in the visible and near-infrared spectral range and compatible with standard semiconductor fabrication techniques. In 2011, Konishi 2 substrate with a very small aspect ratio (depth/width ~0.7). It is shown that the CPDW exhibits a unique dichroism in that it functions as a transmissive quarter waveplate for one of either left- or right-handed circularly polarized incident light and a reflective mirror for the other. The CPD in the transmission at wavelength ~1.5 um reaches 97% and the extinction ratio is as high as 345:1. Experimental fabrications of the proposed all-dielectric metasurface structure are implemented with electron beam lithography and inductively coupled plasma etching. Experimental demonstrations of the CPDW are consistent with the simulations. The proposed all-dielectric 2D chiral metasurface is of advantages of high-dichroism, easy-fabrication and able to use standard CMOS-compatible semiconductor fabrication techniques, which could provide useful devices for remote sensing and imaging applications for circularly polarized light with a highly integrated photonic platform.In this paper, we propose and experimentally demonstrate a high efficient circularly polarizing dichroism waveplate (CPDW) using an all-dielectric 2D chiral metasurface that is based on CMOS-compatible materials: silicon and its oxide, in which a simple Z-shaped arrays are etched into an ultra-thin silicon layer (~200 nm) on top of a SiO1, L2, W and P stand for the perpendicular beam, horizontal beam, width of the slot and the period of the unit cell, respectively.The proposed chiral metasurface  is an al1 on the transmission of LCP and RCP incidences. It is seen that the valley positions of both RCP and LCP transmission remainalmost unchanged. In contrast to the effect of L1, the horizontal beam L2 plays an important role on the operating bandwidth of the transmission dichroism. As shown in 2 increases, while the valleys at 1.4–1.45 μm of LCP incidence and ~1.5 μm of RCP incidence shift very slightly. The phenomena shown in 1and L2 do not change the longitudinal waveguide structure significantly (both the waveguide dimension and effective refractive index of core and cladding formed by substrate-metasurface (Si)-air), i.e., both valleys at 1.4–1.45 μm of LCP incidence and at ~1.5 μm of RCP incidence remain almost unchanged, changes in L2 do change the chiral transverse waveguide mode in which the width of chiral Si ridges  and the corresponding width of the effective cladding formed by the Z-slot along Y-axis change as L2 changes, which results in a significant dichroism valley shift of LCP incidence as shown in RCP − ILCP) and an extinction ratio (IRCP/ILCP) of 345:1 have been achieved with the structural parameters: P = 0.98 μm, L1 = 0.22 μm, L2 = 0.50 μm W = 0.32 μm and H = 0.215 μm.The effects of different parameters of the structure on the performance of different circular polarizations in the wavelength range of 1.3–1.7 μm are firstly investigated. 2 substrate and Si metasurface), Z = 0.10 μm  and Z = 0.215 μm . As can be seen, significant differences in the electric field distribution can be seen in the RCP and LCP incidences at both chiral resonant wavelengths of 1.50 μm (the 1st and 2nd column) and 1.56 μm (the 3rd and 4th column). In the case of LCP incidence at both resonant wavelengths (2nd and 4th column), it is seen that very strong light localization happens within the chiral transverse waveguide formed by the Si ridged core along Y axis cladded by Z-shaped air slots along Y axis due to the strong coupling interaction when the handedness of incident circular polarization and the handedness of structure matches. It is also noticed that major part of the energy in the transverse waveguide are near the interfaces at Z = 0 and Z = 0.215 μm. In contrast, in the case of RCP incidence, negligible light localization can be seen in the transverse waveguide (Si-ridge core) at all the different cross sections, which implies that no strong coupling interaction occurs in the case of opposite handedness of the chiral structure and polarized incidence, so that all the RCP incidence transmits through the structure, as witnessed in The “Z-shaped” transverse waveguide is chiral in structure . The chiral structure will result in circular dichroism, i.e., different optical responses  for left-handed circularly polarized (LCP) incident light and right-handed circularly polarized (RCP) incident light, which has been observed and reported1418The strong dichroism in transmission of the two circular polarization states through the proposed 2D chiral dielectric metasurface makes it very distinct from 2D chiral metallic metasurfaces that rely on either Ohmic dissipation or symmetry-breaking substrate effects15The detailed polarization state of the transmission and reflection from the proposed 2D chiral metasurface can be further examined both numerically and analytically. A detailed quantitative analysis regarding the transformation of the polarization state of a RCP and LCP incidences is shown in RCP − ILCP)/(IRCP + ILCP)] and polarization azimuth angle θ = [arg(ARCP) − arg(ALCP)]/2, where ARCP and ALCP denote the complex transmission coefficients of RCP and LCP waves, and arg represents the phase angleRCP ≈ 1) while LCP component in the reflection is nearly zero (ILCP ≈ 0) at the wavelength of 1.5 μmin the case of a LCP incidence. This results in a circular polarized light in the reflection with ellipticity and azimuth angle being 44.2 and −8.5°, respectively, compared to η = 45 and θ = 0° of a circular polarization theoretically, which is again consistent with that numerically observed in where A and B are the polarization components along X and Y axis, respectively, in Cartesian coordinate system. It is then clear that a linear polarization can be obtained at the wavelength of ~1.50 μm, where the intensity of RCP and LCP components in transmission are equal, which is consistent with that numerically observed in The unique behavior of the proposed circular dichroism device can be further understood with a linearly polarized incident lightof different azimuth angles . 1 = 0.237 μm, L2 = 0.491 μm and W = 0.319 μm, which are very close to the designed parameters as that in Experimental verifications of the proposed all-dielectric 2D chiral metasurface are performed with both left and right-handed chiral metasurface arrays of silicon film, The detailed polarization state of the transmitted and reflected light when a RCP and LCP polarized light is incident on the left-handed chiral metasurface is also measured and analyzed. In summary, we have proposed and experimentally demonstrated a high efficient circularly polarizing dichroism waveplate (CPDW) using CMOS-compatible all-dielectric chiral metasurface structure. Polarizing behaviors of the proposed structure are analyzed and examined both theoretically and experimentally, in which the structure functions as a transmissive quarter waveplate for one of either left- or right-handed circularly polarized incident light and a reflective mirror for the other at the designed wavelength. Further examinations show that a linearly polarized transmission light with invariable polarizing azimuthal angle and a circularly polarized reflection light can be simultaneously obtained for a linearly polarized incident light with arbitrary polarizing azimuthal angle. These unique functions along with the simple all-dielectric structure make the device distinctive from all the previously reported ones. The demonstrated ultracompact device could be useful inquantum measurements, quantum telecommunications, remote sensing and polarizing imaging with a highly integrated photonic platform.The performance of the circular polarized dichroism of the proposed structure can be characterized using finite difference time domain method (FDTD) . The perfectly matched layers (PMLs) along Z direction and the periodic boundary conditions along X and Y directions owing to the periodicity of the chiral structures are assumed in the simulation. The dielectric properties of Si given by Palik are adoptedA 250 nm silicon film is grown on a silicon dioxide with PECVD technology. Only the top silicon layer is etched into the designed Z-shaped slot arrays. The nano patterns were first made with an electron beam lithography system using PMMA resist . After post baking and development, inductively coupled plasma etching was used to obtain the final nanostructures and it ended with an additional 10% over etch to ensure that Si was removed completely in the patterned regions.The optical transmission behaviors of the fabricated all-dielectric 2D chiral metasurface were measured at normal incidence with both rightand left circularly polarized light in the wavelength range between 1.45 μm to 1.70 μm using a supercontinuum laser source  and an optical spectral analyzer . The broadband light was firstly directed to a monochromator and a collimator. A linear polarizer and a tunable quarter-wave plate  were then used to generate the desired circular polarization light. The quality of the generated circular polarization was checked with an additional polarizer. The light was then focused onto the sample by an objective. The polarization state of the transmitted light was measured with an additional polarizer put after the sample. The polarization state of the reflected light was measured with a reflective optical system.How to cite this article: Hu, J. et al. All-dielectric metasurface circular dichroism waveplate. Sci. Rep.7, 41893; doi: 10.1038/srep41893 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Phil. Trans. R. Soc. B373, 20170162. (Published Online 26 February 2018) (doi:10.1098/rstb.2017.0162)It has been brought to our attention that our article ‘Genetics of biologically based psychological differences’ contained some minor errors and ambiguities that we would like to correct. A list of corrections are as follows.‘Although there is no universally accepted framework of personality, the proposed factors (such as those suggested by Eysenck and in the five-factor model (FFM)) provide a good starting point when investigating the genetic architecture of these individual differences [4–7].’In the Introduction (paragraph 2), sentence 2 should read: In Box 1 (paragraph 2), sentence 3 should read: ‘Estimates calculated according to these family-based models can be thought of as ‘true’ heritability.’In the same paragraph, sentence 6 should read: ‘These individuals are thus assumed to share a great deal of their environment, but if the equal environments assumption is violated this can impact on heritability estimates; for example, more similar treatment of identical (MZ) twins could lead to false inflation of heritability estimates.’In Box 1 (paragraph 3), sentence 1 should read: ‘Genome-wide methods estimate heritability using SNP-based approaches.’In the same paragraph, sentence 4 should read: ‘These approaches generally estimate heritability using unrelated individuals, which should minimize confounding due to shared environment, and follow the same experimental design as GWASs [26], but do not give an estimate of true narrow sense heritability because they do not include all additive genetic variance.’In Box 1 (paragraph 4), sentence 4 should read: ‘Any discrepancies between In section 2a (paragraph 1), sentence 6, we should refer to reference [16] not [13].In the same section, paragraph 3, sentence 2 should read ‘The authors, Vukasović & Bratko, find evidence for a heritable component to individual differences in personality, with heritability estimated at approximately 40%.’In section 2b (paragraph 1), sentence 6 should read: ‘These techniques use information on the correlation or linkage disequilibrium (LD) structure across the genome to estimate the SNP heritability In the same section, paragraph 1, sentence 9 should read ‘This approach appears to provide unbiased estimates of In table 1, the study by Power & Pleuss should be included:In section 5 (paragraph 5), sentence 4 should read ‘This is likely due to favourable characteristics that are linked to some genetic vulnerability for the disorder in those who do not succumb to the outcome.’In section 6 (paragraph 2), sentence 1 should read: ‘Although there is ongoing discussion about the best model of individual differences in personality, the factor structures suggested are a useful starting point when investigating the genetic architecture of these traits [4–7].’While these amendments do not change the conclusions of our article, we are grateful for the opportunity to correct them.


Type I, II & III). In a silver-containing zinc phosphate glass, direct laser writing additionally proceeds via the formation of silver clusters at the periphery of the interaction voxel. In this paper, we introduce a novel type of refractive index modification based on the creation of the photo-induced silver clusters allowing the inscription of a new type of optical waveguides. Various waveguides as well as a 50–50 beam splitter were written inside bulk glasses and characterized. The waveguiding properties observed in the bulk of such silver-containing glass samples were further transposed to ribbon shaped fibers made of the same material. Our results pave the way for the fabrication of 3D integrated circuits and fiber sensors with original fluorescent, nonlinear optical and plasmonic properties. The universality of these new findings should further extend in any silver-containing glasses that show similar laser-induced behavior in terms of silver cluster production.Direct laser writing in glasses is a growing field of research in photonics since it provides a robust and efficient way to directly address 3D material structuring. Generally, direct laser writing in glasses induces physical modifications such as refractive index changes that have been classified under three different types ( In the past decades, laser-matter interaction has occupied the scientific community due to its wide range of applications. More specifically, direct laser writing (DLW) in transparent glasses has attracted the interest of many research groups. This technique is simple and allows to directly address 3D micro-structuring inside bulk transparent materials. DLW exhibits obvious advantages over lithography techniques which is limited to 2D structuring and involve multiple steps, making DLW highly compatible for future technological transfer to advanced industrial manufacturing.Davis et al. in 19961. The refractive index changes induced by laser-glass interaction are classified as three distinct types following a progressive energy scale2: type I, type II & type III. Type I modifications are smooth isotropic changes of the refractive index allowing the formation of waveguides9. Type II modifications result from a birefringent change in the refractive index associated with nano-structuration of the glass and self-organized nano-gratings formation due to the local production of moderate plasma12. Finally, type III modifications result in voids or disorganized damage features in the glass matrix, due to an excessive plasma production and subsequent Coulomb explosion, which are generally detrimental for the fabrication of photonic devices. In additional to glass processing, laser-written waveguides were also inscribed in crystalline material by taking advantage of the stress induced between several linear type III modifications13.DLW consists of focusing femtosecond laser pulses inside a transparent glass substrate resulting in a permanent change in the refractive index. This new glass processing scheme based on nonlinear absorption was originally reported by type I modifications due to the smooth nature of the index change associated with this type. In general, type I refractive index change is caused by the formation of color centers16, and/or the change of the glass density, which is due to the local heating and subsequent glass restructuration during the cooling stage following laser passage18.For the direct inscription of photonic components such as waveguides, one is usually interested in 20. In this paper, we present a novel type of positive refractive index modification (∆n) following irradiation with femtosecond laser pulses which highly differs from the standard type I modification and allows the inscription of a “new type” of waveguides. First, we characterized the fluorescent photo-induced structures using a confocal microscope, then we investigated the dependence of ∆n as a function of the irradiation conditions. Following this, 7 mm long waveguides were written inside the glass where the waveguiding aspects such as morphology, ∆n profile, near field mode profile, simulated mode profile and propagation losses were investigated. For the sake of brevity, only one single mode waveguide is presented in detail in this paper. Indeed, all modification traces exhibit rather similar waveguiding aspects. However, it should be noted that increasing ∆n results in multimode waveguides which will not be addressed in this paper. Next, a 50–50 beam splitter was successfully fabricated to illustrate the potential of this new writing process. Finally, the waveguiding properties were transposed from bulk samples to ribbon-shaped fibers both made of the same photosensitive silver-containing zinc phosphate glasses.Recently, our group has developed a silver containing zinc phosphate glass in which DLW induces the creation of fluorescent silver clusters distributed around the laser-glass interaction voxel2O) at a depth of 160 µm below the surface. The energy deposited via the nonlinear four-photon absorption processes inside the interaction voxel20 leads to the formation of photoinduced silver clusters Agmx+. This is based on a pulse-to-pulse process by heat accumulation, which results in the aggregation of silver atomic and ionic species. The process of ion migration has been proposed to occur radially from the center of the beam to the edge. Therefore, a combination of electrons production, silver reduction, and silver migration leads to the formation of fluorescent silver clusters Agmx+ at the periphery of the interaction voxel22. These clusters are characterized by two main absorption bands, typically centered at 290 nm and 345 nm and extending in the visible, and by a broad fluorescence emission covering the whole visible range20. The glass sample was translated perpendicularly to the laser beam to form linear fluorescent structures . Results are presented in Figs. mx+ features being photo-induced by a Gaussian profile laser beam21  that the corresponding ranges of irradiance and number of pulses do not affect the glass matrix itself and (ii) that the laser-matter energy deposition occurs through multi-photon ionization of the silver ions. Therefore, the formation of silver clusters occurs for pulse energies lower than the onset of the type I modification in this glass. The involved DLW process corresponds to an extrinsic laser structuring since it fully preserves the glass matrix while acting on the incorporated silver ions. In order to illustrate the potential of this new DLW process for practical applications, we have inscribed waveguiding structures in the bulk of our photosensitive glass. A series of 7 mm long straight waveguides were written 160 µm below the surface, while the laser parameters were varied. To get a single mode waveguide, the laser irradiance as well as the number of pulses were both optimized to get the appropriate size and ∆n.To further support the hypothesis that this ∆n is related to the creation of silver clusters, a non-silver containing zinc phosphate glass was irradiated using identical irradiation conditions and even higher irradiance. Neither fluorescence nor refractive index change (∆n) was observed in this case, which indicates that the formation of silver clusters is the main and direct cause for refractive index change in silver containing zinc phosphate glass, in the low pulse energy regime. Indeed, we claim that our index change is not based on a standard index modification of the glass matrix itself, due to laser-induced modifications of the local density as it is the case for The structures/waveguides were imaged using a confocal fluorescence microscope  and a relatively low ∆n (~2.5 × 10−3), the horizontal intensity mode profile . The overall propagation losses of 1.2 dB/cm are the upper bound, estimated considering the simulated mismatch, Fresnel losses of 0.43 dB (n = 1.59) and the measured insertion losses of the 7-mm waveguide (see methods). Also, in our case using the traditional methods for characterizing propagation losses is not highly efficient due to the large mismatch between the fiber and the waveguide geometry, and the short length of the waveguide. Further efforts are required to get an accurate evaluation of the propagation losses of such waveguides.2O), by sequentially writing two partially overlapping S-bended waveguides. The optical component divides the input light power equally through each of the two output branches . The fluorescent structures composed of silver clusters exhibit the same morphology as those of the structures written in bulk glasses, which also demonstrates the equivalence of the index modification profiles in bulk and fiber-shaped samples. A 630 nm laser was injected inside the 1.4 cm waveguide and the near field-mode profile was imaged.Finally, DLW was also conducted on silver containing zinc phosphate ribbon fibers. Our group previously reported the fabrication of photosensitive silver-containing zinc phosphate flat substrate fibers and the capabilities of performing DLW on such fibersLight is guided over the full length of the waveguide and the near-field intensity profile is similar to the profile of waveguides inscribed in bulk sample. The normalized horizontal and vertical intensity profiles in the ribbon fiber are much the same to the one observed in bulk glasses which are not shown in this paper for the sake of similarity.29. Furthermore, such laser-induced modifications had already led to original second-order effective nonlinearities by means of direct laser poling, allowing for a localized second-harmonic generation that showed a significant efficiency with an effective coefficient close to the pm/V level30. In addition, a post laser irradiation thermal treatment (Tg + 20 °C) in silver-containing zinc phosphate glass had allowed the growth of silver metallic nanoparticles exhibiting an effective dielectric/metal medium bearing a plasmonic resonance response in the structured glass matrix31. To the best of our knowledge, combining optical and plasmonic waveguiding in a same glass sample is not achievable otherwise, which highlights the novelty and tremendous potential of these new type waveguides. Finally, one must emphasize that the creation of silver clusters is not only limited to the silver-containing zinc phosphate glasses but can also be generated in other silver containing glass matrixes such as sodocalcique and borosilicate glasses as previously reported by our group22. This proves the universality in terms of processes when considering tailored materials as silver-containing photosensitive glasses32, and the novelty of this technique in making hybrid integrated circuits in glasses.Therefore, we demonstrate the capability of creating waveguides in ribbon fibers, thus paving the way for fiber applications in spectroscopy sensing, filtering and/or fully integrated photonics. DLW in silver-containing zinc phosphate bulk glasses and ribbon fibers allows the formation of a perennial new type of waveguides which take advantage of the thermal stability and bleaching-free fluorescence of the photo-induced silver clusters up to the glass transition temperature type I modification as it takes origin in the creation of the photo-induced silver clusters around the interaction voxel, involving such new chemical bonds with more polarizable electron clouds than those of the initial ions Ag+, and this leading to a local refractive index increase independently from any modification of the glass matrix itself. A 3 × 6 µm2 single-mode waveguide was written inside bulk glass, exhibiting a ∆n of 2.5 × 10−3, a single mode profile supported by each of the two silver clusters traces and showing an upper propagation losses of 1.2 dB/cm at λ = 630 nm. Moreover, a 50–50 beam splitter was written paving the way for 3D integrated optical circuits in such glasses. Finally, a 1.4 cm waveguide was inscribed inside ribbon fibers of these silver-containing zinc phosphate glass, demonstrating the waveguiding properties of such fibers once they are structured. These findings provide unique opportunities for applications such as spectroscopy fiber sensing, filtering and/or fully integrated photonics.As a conclusion, we have demonstrated that direct laser writing (DLW) in silver-containing zinc phosphate glasses produces a novel type of positive refractive index change (∆n) that is compatible for waveguiding applications. This ∆n differs from the standard 2O5 – 0.55ZnO – 0.01Ga2O3 – 0.04 Ag2O glass was elaborated from a mix of powders Zn(PO3)2, ZnO, Ga2O3, and Ag(NO3). Glass samples were made using a melt-quenching technique: a platinum crucible containing the powders was heated at 1200 °C during 12 h. The samples were quenched, ground and melted twice with the intention of improving the chemical homogeneity of the glass and then limit the local refraction index variations in order to get the best optical quality. Afterwards, the samples were annealed at 40 °C below the glass transition for 4 h to relax the accumulated mechanical constraints. Then samples were cut and polished for DLW. Moreover, after DLW, the facets were cut and polished again to reveal the waveguides to the surface, to allow laser injection and the visualization of the guided mode profile. Preforms for fiber drawing were produced by casting the glass in a specifically designed brass mold, pre-heated at ~Tg-10 °C and annealed at Tg-40 °C for 12 hours.A 0.4 P−1). The preform-holder motion and capstan rotation velocity were controlled in real-time to produce the targeted fiber diameter. Following this procedure, meters of fibers were drawn with dimensions ranging from 600 µm × 300 µm, down to 200 µm × 100 µm.Thermal drawing was performed using a dedicated 3-meter-high optical fiber draw tower composed of an annular electrical furnace with a sharp temperature profile, a diameter monitor, a tension dancer and a collecting drum. The rectangular preform was slowly fed into the furnace and the temperature was gradually increased up to ~700 °C under continuous oxygen gas flow . Direct laser writing was performed using a microscope objective  and all the structures were written 160 µm below the surface except for the ribbon fibers.The glass samples were irradiated using a Yb:KGW femtosecond oscillator  and emitting at 1030 nm). The laser irradiance was controlled with an acousto-optic modulator enabling the accumulation of N = 102 to 10 TW/cm2 while the writing speed varied between 10 µm/s and 50 µm/s corresponding to 1.7 × 106 deposited pulses and 3.3 × 105 deposited pulses respectively.The cartographies of square linear structures were written using irradiances going from 6 TW/cm2 laser irradiance and 60 µm/s writing speed. The beam splitter consisted of two partially-overlapping opposite s-bend waveguides.The waveguide and the beam splitter were written using 9 TW/cm2 and a writing speed of 25 µm/s. Following the waveguide inscription, the fiber facets were carefully polished to reveal the waveguide to the surface allowing laser injection and the mode profile imaging.The waveguide was written 50 µm below the surface using an irradiance of 10 TW/cmNear field mode profiles were observed after injecting light of a red diode laser emitting at 630 nm using a single mode HP-460 fiber (core diameter 2.5 µm - NA = 0.13) into the waveguides and beam splitter. Then, a 100x – 0.55 NA Mitutoyo objective was focused on the output facet to visualize the guided mode profile and conjugate it on a CCD camera.The losses were estimated by measuring the power of injected light in the waveguide and the power transmitted. Moreover, the mode mismatch between the fiber and the waveguide was taken into consideration as well as Fresnel losses. The mode mismatch was simulated using Lumerical software. A power coupling coefficient between the HP-460 fiber and the waveguide was simulated to be ~30% i.e. 5.22 dB of loss in ideal conditions. Fresnel losses were calculated to 0.43 dB corresponding to the two glass facets. Finally, taking into consideration the simulated mismatch, Fresnel losses and the measured insertion losses, the higher bond of the propagation losses was estimated to be 1.2 dB/cm.−3. The propagation of a 630 nm beam was simulated inside the above-mentioned structure.Simulations of the guided mode were conducted using Lumerical’s Mode solutions software. The waveguide geometry was simulated based on the confocal fluorescence images and the refractive index change measurement. The simulations consisted of two parallel ellipsoids of 550 nm thickness, spaced by 1.85 µm, with a depth of 6 µm (z axis) and a step index refractive index change of 2.5 × 10Were obtained using an Olympus FLUOVIEW FV1200 confocal microscope. An oil immersion objective of 60x was used to resolve the structures. Excitation wavelength was at 480 nm while the emission wavelength was broad band between 500 nm and 600 nm.Refractive index variation (∆n) between the unmodified glass and the silver clusters was measured using a phase-contrast microscopy method with commercially SID4Bio available equipment SID4Bio from PHASICS Inc. A 100x – 1.3 NA oil immersion objective was used to image the structures using white light, typically leading to 800 nm transverse spatial resolution (FWHM).The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


The highly scalable and low cost process to fabricate flexible thermoelectric materials and devices demonstrated here opens up many opportunities to transform thermoelectric energy harvesting and cooling applications.Screen printing allows for direct conversion of thermoelectric nanocrystals into flexible energy harvesters and coolers. However, obtaining flexible thermoelectric materials with high figure of merit ZT through printing is an exacting challenge due to the difficulties to synthesize high-performance thermoelectric inks and the poor density and electrical conductivity of the printed films. Here, we demonstrate high-performance flexible films and devices by screen printing bismuth telluride based nanocrystal inks synthesized using a microwave-stimulated wet-chemical method. Thermoelectric films of several tens of microns thickness were screen printed onto a flexible polyimide substrate followed by cold compaction and sintering. The n-type films demonstrate a peak ZT of 0.43 along with superior flexibility, which is among the highest reported ZT values in flexible thermoelectric materials. A flexible thermoelectric device fabricated using the printed films produces a high power density of 4.1 mW/cm Thermoelectric conversion is a solid-state and environmentally friendly energy conversion technology with broad applications including solid-state cooling, energy harvesting, and waste heat recovery2σT/κ, where α, σ, κ and T are the Seebeck coefficient, electrical conductivity, thermal conductivity, and absolute temperature respectively35689The efficiency of thermoelectric materials is determined by the figure of merit ZT defined as ZT = αAmong all the methods to fabricate thermoelectric materials, wet deposition of nanocrystal-based colloidal inks using screen printing, inkjet printing, direct writing, or other layer-by-layer methods hold many advantages due to the ability to directly convert nanocrystal inks into micro/macroscale functional materials and devices with great scalability, flexibility, and cost effectiveness1214161718Here, we report a study of flexible thermoelectric films by screen printing colloidal inks composed of bismuth telluride based nanoplates fabricated using a scalable microwave-stimulated wet chemical approach2Te2.8Se0.2 were synthesized using a microwave stimulated wet-chemical synthesis method based on inexpensive organic solvents and metal salts described earlierOur doped and functionalized pnictogen chalcogenide nanocrystals of Bi2Se2.8Se0.2 nanopowders, 39 wt.% Solvent , 2 wt.% Binder , 1 wt.% Glass Frits . The ink is thoroughly mixed using a planetary centrifugal mixer for 20 minutes followed by a vortex mixer for 10 minutes to get a uniform mixture.The dried nanocrystals with the TGA capping agent were mixed with solvent and binder to produce viscous and thixotropic inks for screen printing. The optimized ink contains 58 wt.% BiAs-prepared ink is screen printed on flexible polyimide substrates. The thermoelectric films of various thicknesses in the range of 10–100 μm were obtained by controlling the screen mesh size and the number of repeated print passes. The printed films were first dried in air at 200 °C on a hot plate to remove the solvent and binder, followed by a cold compaction using a hydraulic press to consolidate the films. The film was finally sintered at 430 °C for 45 minutes in vacuum in order to remove the TGA surfactant and further improve the film density. The sintering temperatures are kept below the melting point of the polyimide substrate, though better thermoelectric properties could be obtained at higher sintering temperatures.The temperature-dependent in-plane electrical conductivity and Seebeck coefficient of the film sample were measured simultaneously using a commercial Linseis Seebeck and resistivity instrument. The above two properties of the same sample were also measured using a home-built testing system, and the two sets of measurement results are within 2%. In order to measure the thermal conductivity of the sample, a freestanding film of about 100 μm thickness was prepared under the same conditions as those for preparing thinner films on substrate. The temperature-dependent cross-plane thermal diffusivity of the freestanding film was measured using a laser flash instrument. The cross-plane thermal conductivity was then determined using the sample density measured using Archimedes method and the specific heat measured using a DSC instrument. The in-plane thermal conductivity of the freestanding film was measured directly using a steady-state method in vacuum, which is within 5% of the cross-plane thermal conductivity, indicating the sample is isotropic. Details about the thermal conductivity measurement are included in the 2Te2.8Se0.2 elements were printed onto a flexible polyimide substrate with 4 mm spacing. Thin copper foils were soldered to the five elements in order to connect them electrically in series. A custom test bed was built using two commercial Peltier modules, one operating as a heater and the other as a cooler. The hot side and cold side of the TE device were thermally grounded to the two Peltier modules to create a temperature gradient. Two 75 μm diameter k-type thermocouples were mounted to the hot and cold sides of the device to measure the temperature difference. The device was connected electrically in series with a shunt resistor and a variable resistor for impedance matching at each measurement temperature. The open circuit voltage, load voltage, current, internal resistance, and power output from the device were measured at each hot-side temperature while the cold-side temperature is maintained constant.Five 10 mm × 2 mm × 0.01 mm n-type Bi2Te2.8Se0.2 nanocrystals, indicating the plate-like structures of several tens of nanometers thickness. 2Te2.8Se0.2 film of about 10 μm thickness fabricated by screen printing. The films have about 85% relative density, and contain nanoscale pores primarily due to incomplete sintering of the nanocrystals and the removal of the additives in the ink.19 cm−3 versus 1.42 × 1019  cm−3), whereas the film mobility is about 56% lower than the pellet mobility (127 cm2V−1s−1 versus 290 cm2V−1s−1) due to increased electron scattering by impurities and porosity present in the printed films.Temperature-dependent thermoelectric properties were obtained on a flexible film of 10 μm thickness printed using the nanocrystal ink and a reference pellet sample of 500 μm thickness made by the pure nanocrystal powders using cold compaction and sintering under the same conditions. The relative densities of the film and the pellet are 85% and 90% respectively. As shown in L of the film and the pellet is estimated to be 0.41 Wm−1K−1 and 0.66 Wm−1K−1, respectively, using the equation κL = κ − σTL, where κ is the total thermal conductivity, σ is the electrical conductivity, T is the absolute temperature, and L is the Lorenz number determined from our previous workL of these samples is significantly lower than their bulk counterpart attributed to the nanoscale grains and porosities originated from the nanocrystalsL of the film is lower than the pellet mainly due to additional phonon defects scattering caused by the addition of glass particles as well as a small contribution from slightly higher porosities.The room-temperature lattice thermal conductivity κAs shown in In order to test the flexibility, the room-temperature electrical resistance of the printed films was tested using Van der Pauw method as a function of bending cycles on two cylinders of 7 mm radius and 5 mm radius respectively. Electrical resistance is chosen here to evaluate film flexibility because it is very sensitive to any cracks that may develop during bending test. After 150 bending cycles, the electrical resistances of the film show 1.4% increase for the 7 mm bending radius and 4.5% increase for the 5 mm bending radius respectively, indicating superior bending flexibility shown in .2 with 60 °C ΔT. The power density was determined based on the total cross-sectional area (2 mm × 0.01 mm × 5) of the five thermoelectric elements. The experimental results are within 10% of the finite element simulation results based on the thermoelectric properties shown in A thermoelectric generator device consisting of 5 n-type elements shown in  inset wa2 with a small temperature difference of 60 °C, opening up lots of applications for low-temperature energy harvesting. The performance of the printed thermoelectric films and devices can be further improved by increasing the electrical conductivity through optimization of the ink formulation and refinement of the sintering process.Flexible thermoelectric films were screen printed at ambient conditions using nanocrystals synthesized by a highly scalable microwave assisted wet chemical method. The films show an unprecedented peak ZT of 0.43 at 175 °C and superior flexibility with negligible changes of electrical conductivity after 150 bending cycles. The flexible thermoelectric device fabricated using the printed n-type thermoelectric elements produces a high power density of 4.1 mW/cmHow to cite this article: Varghese, T. et al. High-performance and flexible thermoelectric films by screen printing solution-processed nanoplate crystals. Sci. Rep.6, 33135; doi: 10.1038/srep33135 (2016).