During germline regeneration, FOXC2+ SSCs were activated and able to completely restore the process. Germ cell-specific Foxc2 knockout resulted in an accelerated exhaustion of SSCs and eventually led to male infertility. Furthermore, FOXC2 prompts the expressions of negative regulators of cell cycle thereby ensures the SSCs reside in quiescence. Thus, this work proposes that the quiescent FOXC2+ SSCs are essential for maintaining the homeostasis and regeneration of spermatogenesis in adult mammals.In adult mammals, spermatogenesis embodies the complex developmental process from spermatogonial stem cells (SSCs) to spermatozoa. At the top of this developmental hierarchy lie a series of SSC subpopulations. Their individual identities as well as the relationships with each other, however, remain largely elusive. Using single-cell analysis and lineage tracing, we discovered both in mice and humans the quiescent adult SSC subpopulation marked specifically by forkhead box protein C2 (FOXC2). All spermatogenic progenies can be derived from FOXC2 Gfra1, ID4, Ret, Eomes, Pax7, Nanos2, Shisa6, T, Pdx1, Lhx1, Egr2, and Plvap . It is critical for this process to be continuous and successful that SSCs are maintained in a homeostatic balance between self-renewal and differentiation . The SSCnd Plvap . ParticuAdult stem cells, as the undifferentiated primitive cells that can be found in nearly all types of tissues in mammals, are characteristic for a unique quiescent status reflected by both reversible cell cycle arrest and specific metabolic alterations . The rols and Apr spermatogonia of mouse testes and is required for SSC maintenance , a member of the forkhead/winged helix transcription factor family, plays essential roles in the development of various tissues in mice . Recentlntenance . HoweverMcam (Gfra1 (Tcl1 and Egr2 (+ uSPG in mice (s (59.9%) than other subtypes including Apr (5.2%), Apr-1 (4.1%), Aal4-1 (1.83%), Aal8-1 (1.5%), and Aal16-1 (1.67%)  of the uSPG from adult mice testes marked by THY1, a widely recognized surface marker for uSPG with self-renewing and transplantable state , to dissMcam , Gfra1  . There w (1.67%) , suggest tracing , we foun tracing , indicat subsets .-  . As expe- . Pseudote MKI67- . Immunofe MKI67- , represee MKI67- . These riCreERT2/+;Rosa26LSL-T/G/LSL-T/GFoxc2 mice in which FOXC2+ cells were specifically labeled with GFP to enable the progeny tracing after tamoxifen treatment (+) was tracked at d3 (day3), w1 (week1), w2, w4, w6, m4 (month 4), m7, and m12, respectively . The testes of these mice were examined at d3, d7, and d14 post tamoxifen induction , FOXC2+ cells constituted the majority of uSPGs , especially at the advanced state5  sequencing to explore the underlying mechanism , for whiquencing . Specifiquencing  showed fquencing . By overby FOXC2 . Furtherby FOXC2 . More spby FOXC2 . Meanwhiby FOXC2  via the by FOXC2 , which wby FOXC2 . Overall+ SSCs as a quiescent SSC subpopulation in adult mice. Further investigation through functionality analysis confirmed FOXC2 is essential for SSC self-renewal and stemness, thereby is required for maintaining the SSC population that is critical for continuous spermatogenesis. Importantly, our data demonstrated that the colonies formed by FOXC2+ cells constituted nearly the total length of the seminiferous tubules (99.31%), implying that the FOXC2+ SSCs can support the complete spermatogenesis in adult mice.In this work, a comprehensive analysis of uSPG populations with scRNA-seq and the following lineage tracing study by whole-mount immunofluorescence assay led to the identification of FOXC2+ Apr and Aal cells are found to break randomly and a portion of them can return to the stem cell state , and further revealed that FOXC2 functioned in the negative regulation of cell cycle progression, thus confirming that FOXC2-expressing SSCs are quiescent SSC population in adult mice. The finding that FOXC2 inhibited cell cycle and differentiation of SSCs in testis is consistent with that reported in other tissues  at 120 days, implying that the quiescent FOXC2+ cells are able to transform into the proliferative FOXC2+ cells to replenish the SSC pool to maintain the SSC homeostasis and normal spermatogenesis. We further confirmed by lineage tracing analysis that FOXC2-expressing cells were the only remaining SSC population and were responsible for germline regeneration after the busulfan treatment, indicating that FOXC2+ SSCs represent a functionally important stem cell population with regenerative ability. In the future, more insights into the unique regulation of SSCs can be drawn from studying and comparing the transition between the quiescent and proliferative states in FOXC2+ and other SSC subpopulations.We observed that the FOXC2 tissues . In gene tissues . In our + SSC subpopulation  constitute the SSC pool, of which only a small proportion (~5.1%) cells are proliferative while the majority remains quiescent  appeared more active in proliferation than the adult counterpart , Cre/+Ddx4 mice (stock no. 000692) and EGFPTg/+ mice (stock no. 021930) were bought from the Jackson Laboratory. The iCreERt2Foxc2 mice and the flox/floxFoxc2 (f/fFoxc2) mice were constructed and bought from the Biocytogen. The LSL-DTA/+Rosa26 mice were bought from GemPharmatech. All mice were housed and bred under specific pathogen-free conditions  in the animal facility at the Institute of Basic Medical Sciences. DNA was isolated from the tails, and the genotypes of the mice were checked using PCR with specific primers  has been provided in supplementary file. The 8-week-old C57BL/6J wild-type mice were used for magnetic-activated cell sorting (MACS). The  primers . All micf/+Foxc2 and f/-;Ddx4Cre/+Foxc2 mice (n=4) were minced and digested in the collagenase type IV  and DNase I  at 37°C for 15 min. The cell suspension was pipetted up and down once every 5 min and the digestion process was stopped with DMEM (containing 10% FBS). The cell suspension was filtered through a 40 μm nylon mesh, and after centrifugation, the cells were resuspended in 8 mL PBS. The 15 mL conical centrifuge tubes were slowly overlayed with 2 mL of 70% Percoll solution, 2 mL of 30% Percoll solution, and then 2 mL of testicular cell suspension and centrifuge at 600×g for 10 min at 4°C without using the centrifuge brake. After centrifugation, the cells at the interface between the 70% and the 30% Percoll solution were carefully removed into the new conical centrifuge tubes, washed with PBS, and then centrifuge at 600×g for 10 min at 4°C. After centrifugation, the cells were resuspended in 360 μL MACS buffer, added with 40 μL of magnetic microbeads conjugated with anti-Thy-1 antibody , and mixed well. Incubate the cell suspension containing Thy-1 microbeads for 20 min at 4°C. Mix gently by tapping every 10 min. Add 20 mL of MACS buffer to the tube to dilute Thy-1 microbeads and centrifuge at 300×g for 10 min at 4°C. Remove the supernatant completely and resuspend in 2 mL of MACS buffer. Place the separation columns  in the magnetic field of the mini MACS Separation Unit (Miltenyi Biotec 130-142-102) and rinse with 0.5 mL of MACS buffer. Apply the cell suspension to the columns (500 μL/column). After the cell suspension has passed through the column and the column reservoir is empty, wash the column with 0.5 mL of MACS buffer three times. Remove the column from the MACS Separation Unit and elute the magnetically retained cells slowly into a 50 mL conical centrifuge tube with 1 mL of MACS buffer using the plunger supplied with the column. Centrifuge the tube containing the cells at 600×g for 10 min at 4°C and resuspend the cell pellet with 10 mL of MACS buffer for rinsing. Repeat this step once. After the final rinsing step, resuspend cells in 0.04% BSA and count the cell number.The testes from 8-week-old C57BL/6J wild-type mice or 4-month-old + cells were used for loading onto the Chromium Single Cell 3′ Chip kit v2  according to the instructions. Cell capturing and library preparation was performed following the kit instructions of the Chromium Single Cell 3’ v2 Library and Gel Bead Kit . In brief, 5000 cells were targeted for capture, and after cDNA synthesis, 10–12 cycles were used for library amplification. The libraries were then size-selected, pooled, and sequenced on a Novaseq 6000 (Illumina). Shallow sequencing was performed to access the library quality and to adjust the subsequent sequencing depth based on the capture rate and the detected unique molecular indices (UMI).The MACS-sorted THY1Raw sequencing reads were processed using the Cell Ranger v.3.0.1 pipeline of the 10x Genomics platform. In brief, reads from each sample were demultiplexed and aligned to the mouse mm10 genome, and UMI counts were quantified for each gene per cell to generate a gene-barcode matrix. Default parameters were used. The UMI counts were analyzed using the Seurat R Package  v.3.0.1. We used2 in Dig-Hisalt Buffer) and incubated at 37°C for 1 hr. The tagmentation was terminated by adding 2.25 mL of 0.5 M EDTA, 2.75 mL of 10% SDS, and 0.5 mL of 20 mg/mL proteinase K at 55°C for 1 hr. The DNA fragments were extracted by phenol chloroform and used for sequencing on an Illumina HiSeq instrument (Illumina NovaSeq 6000) to generate 2×150 bp paired-end reads following the manufacturer’s instructions.CUT&Tag assay was performed using CUT&Tag 2.0 High-Sensitivity Kit . The detailed procedures were described in Raw reads were analyzed by removing low-quality or adaptor sequences using Trim_galore (v0.5.0) and cleaned reads were mapped to the reference genome mm10 using Bowtie2 (v2.2.5). We used MACS2 (v2.1.2) to call peaks found in different groups. Homer (v4.11.1) de novo motif discovery tool was used for finding the binding motifs of Foxc2 with the findMotifsGenome.pl command. The binding potential of candidate target genes at the binding motif was predicated using the JASPAR Scan function . The peaks filtered by fold change more than 5 and transcription start site less than 3000 bp were annotated by R package Chip Seeker for gene category analysis. R package Cluster profiler was used for gene function annotation such as KEGG and GO analysis.GO and KEGG pathway enrichment analyses were conducted using the ClusterProfiler package (v3.12.0)  and the 3 cells/testis), and 2 months after transplantation, the testes were harvested and observed under a fluorescence microscope.The 8-week-old C57BL/6J WT mice were treated with busulfan (40 mg/kg) and used as recipient mice 1 month later. SSCs were transplanted into the testis of recipient mice  to isolate the GFPMouse testes were fixed in 4% paraformaldehyde (PFA) at 4°C overnight, dehydrated, embedded in paraffin, and cut into 5 μm thick sections. The rehydrated mouse or human testis sections were subjected to antigen retrieval, blocked in 5% BSA with 0.1% Triton X-100, and incubated with primary antibody  at 4°C oTotal RNA was extracted from the testes or cultured cells using the RNeasy kit (QIAGEN), reverse-transcribed using RevertAid First Strand cDNA Synthesis kit (Thermo), and processed for qRT-PCR using PowerUp SYBR Green Master Mix (Applied Biosystems) and a LightCycler 480 system (Roche) with gene-specific primers . ReactioAccording to a previous report for activation of iCre , the mic+ cells.The cyst length was obtained according to the previous report . Briefly+, GFRA1+, LIN28A+, or FOXC2+ cells were measured on the seminiferous tubules with whole-mount staining, the numbers of which per 1000 Sertoli cells were determined.The cell density was counted according to a previous report . Briefly2 for 30 min to allow the sperm to swim out. Then, the sperm were diluted in water and counted using a hemocytometer.Total sperm counts were obtained according to the previous report . Brieflyf/-;Ddx4Cre/+Foxc2 mice, a few tubule cross-sections lacked one or more out of the four germ cell layers, which was defined as ‘degenerative tubules’ in this study.Testes of WT and mutant mice were fixed with PFA fixative and processed for paraffin-embedded section preparation (5 μm thick) and hematoxylin and eosin staining, according to standard procedures. The percentage of degenerating seminiferous tubules was calculated based on the cross-sections of seminiferous tubules (n>200) that appeared on one transverse section for each testis. In normal (WT) mouse testes, four generations of germ cells, each synchronously progressing through spermatogenesis, form cellular associations of fixed composition . In the testes of All statistical analyses were performed using GraphPad Prism (v7.0). All experiments were repeated at least three times, and data for evaluated parameters are reported as mean ± s.e.m. The p-values were obtained using two-tailed unpaired Student’s t-tests or one-way ANOVA followed by Tukey test . important study reports that Foxc2+ cells in the testis represent the quiescent spermatogonial stem cells (SSCs). The data supporting this claim are solid. The finding is of great significance to reproductive and stem-cell biology as male fertility depends on the fine balance between self-renewal and differentiation activities of the male germline stem cells, i.e., SSCs.This  The expression and localization of Foxc2 strongly suggest that its role is mainly confined to As undifferentiated spermatogonia (uSPGs). Lineage tracing demonstrated that all germ cells were derived from the FOXC2+ uSPGs. Specific ablation of the FOXC2+ uSPGs led to the depletion of all uSPG populations. Full spermatogenesis can be achieved through the transplantation of Foxc2+ uSPGs. Male germ cell-specific ablation of Foxc2 caused Sertoli-only testes in mice. CUT&Tag sequencing revealed that FOXC2 regulates the factors that inhibit the mitotic cell cycle, consistent with its potential role in maintaining a quiescent state in As spermatogonia. These data made the authors conclude that the FOXC2+ uSPG may be the true SSCs, essential for maintaining spermatogenesis. The conclusion is supported by the data presented. The authors found FOXC2 is mainly expressed in As of mouse undifferentiated spermatogonia (uSPG). About 60% of As uSPG were FOXC2+ MKI67-, indicating that FOXC2 uSPG were quiescent. Similar spermatogonia (ZBTB16+ FOXC2+ MKI67-) were also found in human testis.The lineage tracing experiment using Foxc2CRE/+;R26T/Gf/f mice demonstrated that all germ cells were derived from the FOXC2+ uSPG. Furthermore, specific ablation of the FOXC2+ uSPGs using Foxc2Cre/+;R26DTA/+ mice resulted in the depletion of all uSPG population. In the regenerative condition created by busulfan injection, all FOXC2+ uSPG survived and began to proliferate at around 30 days after busulfan injection. The survived FOXC2+ uSPGs generated all germ cells eventually. To examine the role of FOXC2 in the adult testis, spermatogenesis of Foxc2f/-;Ddx4-cre mice was analyzed. From a 2-month-old, the degenerative seminiferous tubules were increased and became Sertoli cell-only seminiferous tubules, indicating FOXC2 is required to maintain normal spermatogenesis in adult testes. To get insight into the role of FOXC2 in the uSPG, CUT&Tag sequencing was performed in sorted FOXC2+ uSPG from Foxc2CRE/+;R26T/Gf/f mice 3 days after TAM diet feeding. The results showed some unique biological processes, including negative regulation of the mitotic cell cycle, were enriched, suggesting the FOXC2 maintains a quiescent state in spermatogonia.Lineage tracing experiments using transgenic mice of the TAM-inducing system was well-designed and demonstrated interesting results. Based on all data presented, the authors concluded that the FOXC2+ uSPG are primitive SSCs, an indispensable subpopulation to maintain adult spermatogenesis. The conclusion of the mouse study is supported by the data presented. By popular single-cell RNA-seq, the authors identified FOXC2 as an undifferentiated spermatogonia-specific expressed gene. The FOXC2+-SSCs can sufficiently initiate and sustain spermatogenesis, the ablation of this subgroup results in the depletion of the uSPG pool. The authors provide further evidence to show that this gene is essential for SSCs maintenance by negatively regulating the cell cycle in adult mice, thus well-established FOXC2 as a key regulator of SSCs quiescent state.The experiments are well-designed and conducted, the overall conclusions are convincing. This work will be of interest to stem cell and reproductive biologists. The following is the authors’ response to the original reviews.Reviewer #1 (Public Review):The expression and localization of Foxc2 strongly suggest that its role is mainly confined to As undifferentiated spermatogonia (uSPGs). Lineage tracing demonstrated that all germ cells were derived from the FOXC2+ uSPGs. Specific ablation of the FOXC2+ uSPGs led to the depletion of all uSPG populations. Full spermatogenesis can be achieved through the transplantation of Foxc2+ uSPGs. Male germ cell-specific ablation of Foxc2 caused Sertoli-only testes in mice. CUT&Tag sequencing revealed that FOXC2 regulates the factors that inhibit the mitotic cell cycle, consistent with its potential role in maintaining a quiescent state in As spermatogonia. These data made the authors conclude that the FOXC2+ uSPG may be the true SSCs, essential for maintaining spermatogenesis. The conclusion is largely supported by the data presented, but two concerns should be addressed: (1) terminology used is confusing: primitive SSCs, primitive uSPGs, transit amplifying SSCs... (2) the GFP+ cells used for germ cell transplantation should be better controlled using THY1+ cells.Thanks for your good comments. According to your suggestions, we have addressed your two concerns as follows:1> Overall our work suggest that FOXC2+ SSCs are a subpopulation of SSCs in a quiescent state, thus we have replaced the term ‘primitive’ with ‘quiescent’ in the revised manuscript. In general, ‘transient amplifying SSCs’ is considered to be ‘progenitors’, thus we have replaced ‘transient amplifying SSCs’ with ‘progenitors’ in the revised manuscript.2> The transplantation experiment was conducted using MACS-sorted THY1+, FACS sorted THY1+, and FACS-sorted GFP+ (FOXC2+) uSPGs simultaneously. To be consistent with the single-cell RNA-seq using the MACS-sorted THY1+ uSPGs, we only presented the results from MACS-sorted THY1+ and FACS-sorted GFP+ (FOXC2+) uSPGs in the previous manuscript. Following the reviewer’s suggestion, we have included the results derived from FACS sorted THY1+ uSPGs as the control. The overall conclusion is still fully supported by the more comprehensive dataset, i.e. FOXC2+ cells generated significant higher numbers of colonies than THY1+ cells after transplantation .Reviewer #2 (Public Review):The authors found FOXC2 is mainly expressed in As of mouse undifferentiated spermatogonia (uSPG). About 60% of As uSPG were FOXC2+ MKI67-, indicating that FOXC2 uSPG were quiescent. Similar spermatogonia (ZBTB16+ FOXC2+ MKI67-) were also found in human testis.The lineage tracing experiment using Foxc2iCreERT2/+;Rosa26LSL-T/G/LSL-T/G mice demonstrated that all germ cells were derived from the FOXC2+ uSPG. Furthermore, specific ablation of the FOXC2+ uSPGs using Foxc2iCreERT2/+;Rosa26LSL-DTA/+ mice resulted in the depletion of all uSPG population. In the regenerative condition created by busulfan injection, all FOXC2+ uSPG survived and began to proliferate at around 30 days after busulfan injection. The survived FOXC2+ uSPGs generated all germ cells eventually. To examine the role of FOXC2 in the adult testis, spermatogenesis of Foxc2f/-;Ddx4Cre/+ mice was analyzed. From a 2-month-old, the degenerative seminiferous tubules were increased and became Sertoli cell-only seminiferous tubules, indicating FOXC2 is required to maintain normal spermatogenesis in adult testes. To get insight into the role of FOXC2 in the uSPG, CUT&Tag sequencing was performed in sorted FOXC2+ uSPG from Foxc2iCreERT2/+;Rosa26LSL-T/G/LSL-T/G mice 3 days after TAM diet feeding. The results showed some unique biological processes, including negative regulation of the mitotic cell cycle, were enriched, suggesting the FOXC2 maintains a quiescent state in spermatogonia.Lineage tracing experiments using transgenic mice of the TAM-inducing system was well-designed and demonstrated interesting results. Based on all data presented, the authors concluded that the FOXC2+ uSPG are primitive SSCs, an indispensable subpopulation to maintain adult spermatogenesis.The conclusion of the mouse study is mostly supported by the data presented, but to accept some of the authors' claims needs additional information and explanation. Several terminologies define cell populations used in the paper may mislead readers.1. "primitive spermatogonial stem cell (SSC)" is confusing. SSCs are considered the most immature subpopulation of uSPG. Thus, primitive uSPGs are likely SSCs. The naming, primitive SSCs, and transit-amplifying SSCs  are weird. In general, the transit-amplifying cell is progenitor, not stem cell. In human and even mouse, there are several models for the classification of uSPG and SSCs, such as reserved stem cells and active stem cells. The area is highly controversial. The authors' definition of stem cells and progenitor cells should be clarified rigorously and should compare to existing models.Thanks for your good comments. Considering that our results showed that FOXC2+ SSCs are in a quiescent state and that Mechanistically FOXC2 maintained the quiescent state of SSCs by promoting the expression of negative regulators of cell cycle, we have replaced ‘primitive SSCs’ with ‘quiescent SSCs’ in the revised manuscript. We agree with the reviewer that ‘transient amplifying SSCs’ is considered to be ‘progenitors’, thus we have replaced ‘transient amplifying SSCs’ with ‘progenitors’ in the revised manuscript. Further，from our point of view, the FOXC2+Ki67+ SSCs could be regarded as active stem cells, and the FOXC2+Ki67- SSCs could be regarded as reserved stem cells, although further research evidence is still needed to confirm this.1. scRNA seq data analysis and an image of FOXC2+ ZBTB16+ MKI67- cells by fluorescent immunohistochemistry are not sufficient to conclude that they are human primitive SSCs as described in the Abstract. The identity of human SSCs is controversial. Although Adark spermatogonia are a candidate population of human SSCs, the molecular profile of the Adark spermatogonia seems to be heterogeneous. None of the molecular profiles was defined by a specific cell cycle phase. Thus, more rigorous analysis is required to demonstrate the identity of FOXC2+ ZBTB16+ MKI67- cells and Adark spermatogonia.We agree with the reviewer that the identity of human SSCs remain elusive even though Adark population demonstrates certain characteristics of SSCs. To acknowledge this notion, we have revised our conclusion as such that only suggests FOXC2+ZBTB16+MKI67- represents a quiescent state of human SSCs.1. FACS-sorted GFP+ cells and MACS-THY1 cells were used for functional transplantation assay to evaluate SSC activity. In general, the purity of MACS is significantly lower than that of FACS. Therefore, FACS-sorted THY1 cells must be used for the comparative analysis. As uSPGs in adult testes express THY1, the percentage of GFP+ cells in THY1+ cells determined by flow cytometry is important information to support the transplantation data.Thanks for your good comments. According to your suggestions, we have addressed your concerns as follows:1> The transplantation experiment was conducted using MACS-sorted THY1+, FACS sorted THY1+, and FACS-sorted GFP+ (FOXC2+) uSPGs simultaneously. To be consistent with the single-cell RNA-seq using the MACS-sorted THY1+ uSPGs, we only presented the results from MACS-sorted THY1+ and FACS-sorted GFP+ (FOXC2+) uSPGs in the previous manuscript. Following the reviewer’s suggestion, we have included the results derived from FACS sorted THY1+ uSPGs as the control. The overall conclusion is still fully supported by the more comprehensive dataset, i.e. FOXC2+ cells generated significant higher numbers of colonies than THY1+ cells after transplantation .2> We performed FACS analysis to determine the proportion of GFP+ cells in FACS-sorted THY1+ cells from Rosa26LSL-T/G/LSL-T/G or Foxc2iCreERT2/+;Rosa26LSL-T/G/LSL-T/G mice at day 3 post TAM induction, and the result showed that GFP+ cells account for approximately 20.9±0.21% of THY1+ cells, See Author response image 1.1. The lineage tracing experiments of FOXC2+-SSCs in Foxc2iCreERT2/+;Rosa26LSL-T/G/LSL-T/G showed ~95% of spermatogenic cells and 100% progeny were derived from the FOXC2+ (GFP+) spermatogonia  at month 4 post-TAM induction, although FOXC2+ uSPG were quiescent and a very small subpopulation . This means that 40% of As spermatogonia and most of Apr/Aal spermatogonia, which were FOXC2 negative, did not contribute to spermatogenesis at all eventually. This is a striking result. There is a possibility that FOXC2CRE expresses more widely in the uSPG population although immunohistochemistry could not detect them.Thanks for your good comments. From our lineage tracing results, over 95% of the spermatogenic cells are derived from the FOXC2+ SSCs in the testes of 4-month-old mice, which means that FOXC2+ SSCs maintain a long-term stable spermatogenesis. In addition, previous studies have shown that only a portion of As spermatogonia belong to SSCs with complete self-renewal ability , which is consistent with our findings. Therefore, we speculate that 40% of As spermatogonia and most of Apr/Aal spermatogonia, which were FOXC2 negative, did contribute to spermatogenesis but cannot maintain a long-term spermatogenesis due to limited self-renewal ability.1. The CUT&Tag_FOXC2 analysis on the FACS-sorted FOXC2+ showed functional enrichment in biological processes such as DNA repair and mitotic cell cycle regulation . The cells sorted were induced Cre recombinase expression by TAM diet and cut the tdTomato cassette out. DNA repair process and negative regulation of the mitotic cell cycle could be induced by the Cre/lox recombination process. The cells analyzed were not FOXC2+ uSPG in a normal physiological state.We do appreciate the reviewer’s concern on the possibility of the functions enriched in the analysis as referred might be derived from Cre/lox recombination. However, we think it is unlikely that the Cre/lox recombination process, supposed to be rather local and specific, can trigger such a systemic and robust response by the DNA damage and cell cycle regulatory pathways. The reasons are as follows: First, as far as we are aware, there has been sufficient data to support this suggested scenario. Second, we did not observe any alteration in either the SSC behaviors or spermatogenesis in general upon the TAM-induced genomic changes, suggesting the impact from the Cre/lox recombination on DNA damage or cell cycle was not significant. Third, no factors associated with the DNA repair process were revealed in the differential analysis of single-cell transcriptomes of FOXC2-WT and FOXC2-KO.1. Wei et al  have published that FOXC2 is expressed predominately in As and Apr spermatogonia and requires self-renewal of mouse SSCs; however, the authors did not mention this study in Introduction, but referred shortly this at the end of Discussion. Their finding should be referred to and evaluated in advance in the Introduction.Thanks for your good comments. According to your suggestion, we have revised the introduction to refer this latest parallel work on FOXC2. We are happy to see that our discoveries are converged to the important role of FOXC2 in regulating SSCs in adult mammals.Reviewer #3 (Public Review):By popular single-cell RNA-seq, the authors identified FOXC2 as an undifferentiated spermatogonia-specific expressed gene. The FOXC2+-SSCs can sufficiently initiate and sustain spermatogenesis, the ablation of this subgroup results in the depletion of the uSPG pool. The authors provide further evidence to show that this gene is essential for SSCs maintenance by negatively regulating the cell cycle in adult mice, thus well-established FOXC2 as a key regulator of SSCs quiescent state.The experiments are well-designed and conducted, the overall conclusions are convincing. This work will be of interest to stem cell and reproductive biologists.Thanks for the positive feedback.Reviewer #1 (Recommendations for the Authors):The authors should address the following concerns:1. The most primitive uSPGs should be the true SSCs. The term "primitive SSCs" is very confusing.1. In addition to FACS-sorted GFP+ cells, FACS-sorted THY1+ cells should also be used for transplantation.Thanks for your good comments. According to your suggestions, we have addressed your two concerns as follows:1. Overall our work suggest that FOXC2+ SSCs are a subpopulation of SSCs in a quiescent state, thus we have replaced the term ‘primitive’ with ‘quiescent’ in the revised manuscript.2. The transplantation experiment was conducted using MACS-sorted THY1+, FACS sorted THY1+, and FACS-sorted GFP+ (FOXC2+) uSPGs simultaneously. To be consistent with the single-cell RNA-seq using the MACS-sorted THY1+ uSPGs, we only presented the results from MACS-sorted THY1+ and FACS-sorted GFP+ (FOXC2+) uSPGs in the previous manuscript. Following the reviewer’s suggestion, we have included the results derived from FACS sorted THY1+ uSPGs as the control. The overall conclusion is still fully supported by the more comprehensive dataset, i.e. FOXC2+ cells generated significant higher numbers of colonies than THY1+ cells after transplantation .Reviewer #3 (Recommendations for the Authors):The experiments are well-designed and conducted, the overall conclusions are convincing. The only concerns are the writing, especially the introduction which was not well-rationalized. Sounds the three subtypes and three models for SSCs' self-renew are irrelevant to the major points of this manuscript. I don't think you need to talk too much about the markers of SSCs. Instead, I suggest you provide more background about the quiescent or activation states of the SSCs. In addition to that, as a nuclear-localized protein, it cannot be used to flow cytometric sorting, I don't think it should be emphasized as a marker. You identified a key transcription factor for maintaining the quiescent state of the primitive SSCs, that's quite important!Appreciate the positive feedback and constructive suggestions on the writing. We have substantially revised our manuscript to include the relevant advances and understanding from the field as well as highlight the importance of FOXC2 in regulating the quiescent state of SSCs.


We modified the multifrequency atomic force microscope platform to combine photo-induced force microscope with electrostatic/Kelvin probe force microscope constituting a novel hybrid nanoscale optical-electrical force imaging system. This enables to observe a correlation between the IR response, doping level, and topographic information of the graphene layers. Through the nanoscale spectroscopic image measurements, we demonstrate that the charge imbalance at the graphene interface can be controlled by chemical (doping effect via Redox mechanism) and mechanical (triboelectric effect by the doped cantilever) approaches. Moreover, we can also diagnosis the subsurface cracks on the stacked few-layer graphene at nanoscale, by monitoring the strain-induced IR phonon shift. Our approach provides new insights into the development of graphene-based electronic and photonic devices and their potential applications.We, for the first time, report the nanoscopic imaging study of anomalous infrared (IR) phonon enhancement of bilayer graphene, originated from the charge imbalance between the top and bottom layers, resulting in the enhancement of E Fdip, Fes and FvdW are simultaneously applied and demodulated at the fundamental, second and third eigenmodes of a cantilever for PiFM, KPFM/EFM and topography images, respectively.Optical-electrical force nanoscopy.  Recently bilayer graphene has been focused due to its controllable band structure with respect to the gate voltage and twisted angle, providing a wider range of electronic and photonic properties. Moreover, bilayer graphene has higher mechanical strength and flexibility as well as higher thermal conductivity compared to monolayer graphene, making it suitable for designing sophisticated nanoscale devices. In particular, one such novel property comes from optical phonons which are collective vibrations of the carbon atoms that can be excited by the EM field of light.In recent two decades, the study of graphene has been a rapidly growing field, with the discovery of its exceptional properties and potential applications in various areas, including electronics and photonics6. Specifically, in bilayer graphene (2L), there exists an in-plane optical phonon modes with a frequency of approximately 0.2 eV9, known as the E2g and E1u modes, which are symmetric and antisymmetric modes between the layers as shown in Fig. 1u mode can only be detected through IR spectroscopy, while E2g mode can only be detected through Raman spectroscopy. Note that, when we consider that graphene is a monoatomic compound, the presence of a finite IR phonon intensity in pristine undoped 2 L is quite puzzling. Because the IR active vibration requires the change of dipole moment, if all carbon atoms are equivalent, no IR intensity would be expected. This phenomenon is explained by the charged-phonon theory, which describes how the optical phonon mode borrows effective charge from the electronic transitions to which it becomes coupled11. The charged-phonon theory has been successfully applied to 2 L to explain the significant dependence of phonon intensity and Fano asymmetry on gating12. This effect can be utilized to determine the doping level of graphene devices since the electron doping leads to changes in the electron-phonon coupling strength13. In Fig. 15.Raman scattering and infrared (IR) absorption spectroscopies have been the predominant approaches for optical phonon studies in graphene; two technologies provide complementary information due to their different selection rulesSince the IR optical phonon modes of 2 L highly depend on the spatial distribution of local doping level as well as the local strain, the comprehensive understanding between optical, electrical and mechanical information with a high spatial resolution is required to engineer the graphene-based nanoscale devices. However, the conventional optical techniques has been limited in their ability to probe individual nanostructures including the correlative information. This is because the spatial resolution of these techniques is typically on the order of a few hundred nanometers to microns, which is much larger than the size of individual nanostructures. In this purpose, the near-field imaging techniques such as tip-enhanced Raman spectroscopy (TERS) and scanning near-field optical microscopy (SNOM) are implemented to visualize the optical phonon modes in accordance with its topography at nanoscale. However, these techniques are still not able to probe the correlative optical-electrical information at nanoscale.VCPD) as sketched in Fig. CPD between the sharp metallic probe and the sample provides a means to monitor the local electrical doping level of the sample, by retrieving its work function (W). Here, we present a novel approach to characterize and control the IR phonon anomaly of a bilayer graphene by combining photo-induced force microscope (PiFM) with KPFM to develop the optical-electrical hybrid nanoscope through the force detection methods. By simultaneously measuring the nano-IR response and surface potential of 2 L, we figured out that there is an anomalous IR phonon enhancement of 2 L and it is originated from the charge imbalance between layers. We also demonstrate that this anomalous response can be controlled by the chemical (doping effect via Redox mechanism) and mechanical (triboelectric effect by a highly doped cantilever) approaches. Furthermore, we extend our study to more complex example such as the extrinsically stacked few-layer graphene (FLG) by diagnosing its crystallinity such as lattice mismatch or incomplete recombination, and the subsurface cracks by monitoring the strain-induced IR phonon shift. We believe that this technique has the potential to revolutionize the fields of materials science by providing new insights into the optimization of 2-D material-based electronic and photonic devices.The local electrical property can be probed by using Kelvin probe force microscope (KPFM) with a few tens of nm spatial resolution. When in contact, two dissimilar metals or semi-conductor materials generate a contact potential difference  and auto gain control in the feedback loop, so that it is easy to implement in the conventional AFM system. The first three eigenmodes of a gold-coated Si tip  is plotted in Fig. FvdW) to generate the gap control signal. Applied to the second eigenmode  is exerted on the fundamental eigenmode of the cantilever with the heterodyne PiFM mode. Each of the three driving forces with respect to the eigenmodes can be written as below:F0, A3, VAC, VCPD and VDC, are the mechanical driving force, the amplitude of the carrier motion (third eigenmode), the AC voltage for electrostatic force between tip and sample, the contact potential difference between tip and sample, the DC voltage for KPFM feedback, respectively. The VCPD is measured by turning on the KPFM feedback which nullifies the electrostatic motion at the second resonance by using the VDC20. The entire measurements in this study have been performed in the small oscillation limit so that one can regard the cantilever as the superposition of independent simple harmonic oscillators, which prevents any coupled motion between eigenmodes24. Then each force signal is demodulated at each eigenmode to obtain the amplitude and phase of each motion. The detailed calculations lie in the Section An optical-electrical hybrid nanoscope is developed to observe a correlation between the IR response, doping level and topographic information. We modified the amplitude modulated (AM) multifrequency atomic force microscopee18 Fig.  with ele 20 Fig.  through Fdip is proportional to the real part of the electrostatic reflection factor, β, which is given as below26:z is the tip-sample distance, ε is relative permittivity of substrate, σ is conductivity of graphene, ω is frequency of incident light, q is in-plane photon momentum. The data presented in Fig. 26, which is the theoretically calculated real part of graphene’s conductivity around 0.2 eV for both Bernal and Rhombohedral structures, ranging from a monolayer (1 L) to tetralayers (4 L). It was calculated using the tight-binding Hamiltonian and Kubo formula, where only electronic contribution is considered without taking into account the effect of IR phonon modes27. Thus, under the vibrational off-resonance condition, we expect that the PiFM response is proportional to the number of layers n because, when the conductivity is small enough, the real part of nσ028, where σ0 and n are the conductivity of 1 L and number of graphene layers, respectively. In this work, we have confirmed that it is really the case, and found that the PiFM signal around a vibrational phonon resonance shows an interesting anomalous behavior of 2 L, which is not simply proportional to the number of layers n.The photo-induced dipole force can be related to the conductivity of graphene sheet, as 2 substrate as the optical image in Fig. 32. Figure −1 and 1590 cm−1 are also conducted to confirm the number of layers. As shown in Fig. 2)), it is deduced that the dominant interaction force in PiFM is the photo-induced dipole force (PiDF) rather than the photo-induced thermal force (PiTF)34. By normalizing the PiFM responses on each layer with that of the SiO2 substrate, a proportional increase at the off-resonance (1570 cm−1 and at 1590 cm−1) is well represented in Fig. −1 IR phonon resonance, the PiFM image in Fig. We prepared exfoliated FLG on a SiO35. The IR signal on the bilayer at 1584 cm−1 is more significantly enhanced than that on the other layers. Given that the PiFM signal is proportional to the IR dipole moment, this effect may be explained by a charge imbalance between the top and bottom layer of 2 L, which originates from the charge transfer between the bottom layer and the SiO2 surface via Redox mechanism, resulting in the spontaneous P-doping on the bottom layer38. Since the doping effect at the graphene interface is getting screened for more than bilayer, it can be presumed that the IR response on the phonon resonance of E1u mode is strongest in the bilayer. To support the suggested scenario, we measured the The PiFM spectra on four different layers of the sample in Fig. 35, there can be a threshold potential difference between layers to exhibit the anomalous IR phonon enhancement. Building on this concept, we endeavored to reduce the charge imbalance between layers by positively doping the top layer with ethanol/water, resulting in a comparable electrostatic potential in both layers. As plotted in Fig. VCPD between layers is reduced, and the entire work function curve shifts upward compared to the Fig. −1 IR resonance in Fig. −1 and 1595 cm−1. These findings provide compelling evidence that the anomalous IR phonon response of 2 L can be effectively controlled through chemical doping.According to Ref. 2 substrate, where the p-doping on the graphene-SiO2 interface is reduced due to the decreased water layer on the substrate. As depicted in Fig. −1 IR resonance (red line-dots) in Fig. −1 and 1595 cm−1), displaying a proportional increase. Based on the results in Fig. Furthermore, to minimize the charge imbalance between the layers, we prepared FLG on a plasma-cleaned SiO39, which commonly suffer from lattice mismatch or incomplete recombination, may show different IR phonon response compared to the exfoliated layers. We prepared a 1 L on a SiO2 substrate via exfoliation, followed by the transfer of an additional exfoliated FLG onto the 1 L, using PMMA as the transfer agent. The overlapping region is depicted by the orange dashed line in the optical image displayed in Fig. The in-plane optical phonon anomaly of 2 L can be influenced by the crystallinity of the sample, as well as defects and disorder. The extrinsically stacked graphene layers with the transfer method−1, when the measurement is conducted at off-resonance, the PiFM signal is influenced only by the conductivity of graphene, enabling clear differentiation of the number of stacked layers. The trilayers in overlapping region, denoted as 3(o), exhibit incomplete lattice recombination compared to the trilayers in non-overlapping region, 3 L, by showing a slightly lower PiFM signal. At the IR phonon resonance of 1582 cm−1 in Fig. The PiFM images in Fig. −1 and slightly negative contrast at 1588 cm−1. This can be explained by the strain-induced phonon shifts, as previously reported in the literature41. If the tensile (compress) strain is applied to the layered graphene, the IR phonon resonance redshifts (blueshifts), as sketched in Fig. Additionally, a subsurface crack shown as a bright narrow straight line in the PiFM images Fig.  can be iPPP-NCHR probe from Nanosensors, with the soft contact force of 25 nN to avoid the sample damage. The aggregation is the PMMA residue which is demonstrated by the PiFM spectrum on it in the Fig. 24.Since the anomalous IR phonon of 2 L is enhanced by the charge imbalance between the top and bottom layers, this may be locally controlled by friction charging with high spatial resolution, which occurs when the p-doped graphene layer is frictionally brought into contact with the n-doped cantilever. We rubbed the extrinsically stacked graphene layers, indicated by the white dashed rectangular areas in topography of Fig. VCPD map shows that the transferred top layer (green colored area) is already p-doped due to its contact with the PMMA surface, and its work function is approximately 4.74 eV. This value is slightly lower than the p-doped bottom 1 L on the SiO2 surface (light blue colored area) whose work function is around 4.81 eV. On the other hand, the VCPD in the rubbed areas (red colored) are increased by around 50–150 meV, which corresponds to the decreased work function by that amount. In Fig. Dirac point in a graphene) of our sample, which is around 4.6 eV (black dashed line).In Fig. The block diagram of each domain in Fig. −1 off resonance, the PiFM image of the rubbed areas reveals a heightened signal level compared to the pristine areas. This phenomenon arises because the PiFM signal is proportional to the photo-induced dipole, which is more sensitive to electron density than hole density. At the IR phonon resonance of 1582 cm−1 in Fig. In Fig. −1 off resonance, the PiFM signals on the rubbed areas, 1 L’, 2 L’ and 3 L’ (black dashed line-squares) shifts upwards by approximately 10% from that on the pristine areas, 1 L, 2 L and 3 L (black solid line-squares), maintaining similar layer dependence. At the 1582 cm−1 IR resonance, the PiFM signal on pristine 2 L (red solid line-squares) surpasses that on pristine 3 L by 6%, while the signal on rubbed 2 L’ (red dashed line-squares) is 3% lower than that on rubbed 3 L’.The line graphs in Fig. VCPD experiences a 150 mV increase from 1 L to 1 L’, whereas 2 L’, 3 L’, 3(o)’, and 4(o)’ exhibit a smaller shift of approximately 50 mV from their respective pristine areas. These trends are delineated in two ellipsoidal areas of data, with the pristine layers represented by the blue shaded ellipsoidal region and the rubbed layers by the red shaded ellipsoidal region.The PiFM-KPFM correlation graphs offer valuable insights into understanding the comprehensive interplay between the IR response and friction charging with respect to the stacking structures. The correlation graphs presented in Fig. −1 off resonance is compressed at 1582 cm−1 on the IR phonon resonance. The distinctive bilayer enhancement is perceptible exclusively in the pristine 2 L, showcasing a 2 L (red) surpass that of 3 L (blue) in PiFM responses. In contrast, the on-resonance anomalous IR enhancement in the rubbed area displays a relatively reduced effect while preserving the same layer sequence. Furthermore, given that the trilayer and tetralayer in overlapping region, 3(o) and 4(o), show akin behavior to pristine 2 L and 3 L, the PiFM signal is slightly higher in 3(o) than 4(o) on resonance, while the response in rubbed 3(o)’ is lower than rubbed 4(o)’. This PiFM-KPFM correlation graph serves as a clear representation of the intuitive relationship between the sample crystallinity and the friction charging effect, even in the complex systems.The data distribution observed at the 1574 cm42. In particular, since the IR vibration requires the change of dipole moment, the IR phonon response can be further amplified under the charge imbalance between the graphene layers. Our results show that the remarkable strength of the anomalous IR enhancement in the bilayer, compared to other layers in FLG, can be attributed to the charge imbalance between the top and bottom layers in 2L, which can be controlled by chemical (doping effect via Redox mechanism) and mechanical (triboelectric effect by the doped cantilever) approaches. Furthermore, we confirm the threshold potential for triggering the anomalous IR enhancement, which is originated from the electronic band transition11. These information can be very useful in the development of graphene-based electronic and photonic devices such as chemical sensors to monitor the change in environmental parameters, including humidity, temperature, and gas concentration, which highly affect on the doping level of the surface of the devices45.We develop a novel hybrid nanoscale optical-electrical force imaging system and, for the first time, report the nanoimaging study of the IR phonon anomaly of bilayer graphene by changing its local doping level at the nanoscale. This can be explained by the charged phonon theory which suggests that the intensity of the optical phonon can be strongly enhanced when graphene is doped46, which measures the thermal expansion of sample near its IR absorption resonance with its surface potential. Recently, the Xiaoji group [46] successfully implemented this system to monitor the degradation of perovskite by analyzing the correlative response of charge doping/ion migration. The correlation plot between VCPD and PFIR intensity well distinguishes the two different sites (pristine and degraded) of the perovskite, and shows good feasibility of the quality check for the perovskite. However, it is worth noting that this system requires the tip to contact the sample in order to measure its thermal expansion, which may have a limitation to highly dopant-sensitive sample which can be easily affected by the doping level of tip. Moreover, it is limited to access some thin inorganic samples such as few layered 2-D materials which have a very small thermal expansion. For instance, the thermal expansion of 2 L on the resonance of E1u phonon mode is around sub pm/(mW/cm2) which closes to the noise level of PFIR. Thus, the PFIR-KPFM is expected to be very challenge to characterize the optical-electrical correlative response in 2 L. Furthermore, since the photo-induced dipole force (PiDF) depends on the near-field reflection factor (β) of a sample, the PiFM can reveal the subsurface structures of the devices as its refractive index contrast at even the out-of-resonance, where the thermal expansion of the 2-D materials is almost suppressed. In that case, our PiFM-KPFM system can be a good complement to this measurement, since the system can operate via photo-induced dipole force in non-contact manner.The optical-electrical hybrid nanoscope can be configured by the other manner, the peak-force IR (PFIR)-KPFM50. Recently, the photo-induced force response of highly conductive polymer is purely extracted in the visible region by implementing the frequency modulated (FM) PiFM-KPFM system under the ultrahigh vacuum-low temperature21. The conductive polymer shows the electrostatic artifact in PiFM image without the KPFM feedback, while the (FM) PiFM-KPFM clearly removes the artifact and shows pure photo-induced response of the conductive polymer. We expect that the pure IR response of the chemical reactions and functional groups of the conductive polymers can be extracted by implementing our ambient IR-PiFM-KPFM technique. Moreover, since our configuration is the amplitude modulated (AM) PiFM-KPFM system which slightly modifies the conventional AFM setup, it does not require the massive vacuum system and the complex frequency modulation electronics such as phase lock loop (PLL) and auto gain control in the feedback loop. Thus, the (AM) PiFM-KPFM can be very common platform for commercialization.The strong electrostatic potential can create additional tip-sample distance change, which may lead an artifact in the IR response. Since the KPFM feedback nullifies the contact potential difference, it is very useful to extract pure IR response of the conductive samples. This is very similar idea to the magnetic (or piezoelectric) force microscopy with KPFM feedback to extract its pure magnetic (or piezoelectric) responses2 substrate anomalously enhances the IR response of 2 L. We demonstrate that the charge imbalance at the graphene interface can be controlled by chemical (doping effect via Redox mechanism) and mechanical (triboelectric effect by the doped cantilever) approaches. Finally, we extend our study to more complex and practical example, the extrinsically stacked FLG, by diagnosing its crystallinity such as lattice mismatch or incomplete recombination, and the subsurface cracks by monitoring the strain-induced IR phonon shift. By using the highly doped cantilever, we can control its local doping level and anomalous IR response at nanoscale, suggesting the feasibility to engineer the graphene devices. Our findings will have significant implications for a range of applications across various fields, such as graphene-based electronics, environmental monitoring including humidity and gas concentration, and material science of heterogeneous interface.In summary, we have presented a novel approach to observe a correlation between the IR response, doping level, and topographic information in FLG by developing the optical-electrical hybrid nanoscope through the force detection methods. Our results show that the spontaneous P-doping effect at the interface of graphene-SiO2 (thickness of 285 nm)/Si substrate. Confocal Raman spectroscopic measurements (an excitation wavelength of 532 nm) are carried out on the sample to determine the number of layers (n) in section 39. One can find the optical image of the bottom and stacked top layer in the Section The intrinsically stacked FLG samples are prepared by mechanically exfoliating graphite and transferring to a SiO−1 and a tuning range of 765 to 1900 cm−1. The laser beam is side-illuminated to the sample with an angle of 40 degree by a parabolic mirror whose numerical aperture (NA) is around 0.4. The average illumination power was 5 mW with around 2λ μm diameter focal spot, and had a pulse duration of 40 ns. The gold-coated silicon cantilever, PPP-NCLAu from Nanosensors which is the 20 nm gold coated tip with the tip radius of 30 nm, is used for the measurements in ambient condition. The AC and DC voltages were simultaneously applied to the tip from the VistaScope controller to obtain electrostatic force response and contact potential difference. By implementing amplitude modulated (AM) multifrequency atomic force microscope technique, the photo-induced force microscope and Kelvin probe force microscope/electrostatic force microscope are combined. Typically the resonance frequency of PPP-NCLAu for the fundamental, second and third eigenmodes are around 150 kHz, 941 kHz and 2633 kHz. The cantilever responses to the photo-induced, the electrostatic and the van der Waals forces are demodulated at fundamental, second and third eigenmodes. The more details lie in the section The hybrid nanoscope is developed by modifying the VistaScope system from Molecular Vista in the United States. The QCL laser system from Block Engineering was used with a wavenumber resolution of 1 cmSupplementary Information


Lenacapavir (LEN) is a highly potent, long-acting, first-in-class inhibitor of HIV-1 capsid protein for the treatment of HIV-1 infection in adults with multidrug resistance (MDR) in combination with other antiretrovirals.CAPELLA is an ongoing, phase 2/3 study in heavily treatment-experienced people with HIV-1 with multidrug-resistance. Participants received oral LEN for loading followed by subcutaneous LEN Q6M in addition to an investigator-selected optimized background regimen (OBR). After reporting the efficacy and safety data through 52 weeks (W), the protocol was amended to allow longer follow up; we report W104 results.Of 72 participants enrolled (36 in each cohort), 25% were female, 38% were Black, median age was 52 years, 64% had CD4 <200 cells/µL, and 46% had HIV-1 resistant to all 4 major classes . One participant (of 72) decided not to continue in the post-W52 extension. At W104, 62% (44/71) had HIV-1 RNA <50 c/mL via FDA Snapshot algorithm, and 14% (10/71) had HIV-1 RNA >50 c/mL; 24% discontinued for reasons other than lack of efficacy (13/71) or had missing data but were on study drug (4/71). When analyzed as missing=excluded, 81% (44/54) had HIV-1 RNA <50 c/mL. CD4 count increased by a median 97 cells/µL (Q1 to Q3: 18 to 224) and the proportion of participants with CD4 count ≥200 cells/µL increased from 36% (26/72) at baseline to 71% (39/55) at W104. Fourteen participants had emergent LEN resistance (9 previously reported), 6 of whom subsequently suppressed while continuing LEN. The median (Q1–Q3) duration of follow-up on LEN was 125 (111–140) weeks. One participant discontinued due to injection site nodule at W52 ; no participant discontinued due to an AE after W52. Most common AEs (excluding injection site reactions [ISRs] and COVID-19) were diarrhea and nausea . LEN-related ISRs were mostly mild-to-moderate.In people with MDR HIV-1 and limited treatment options, LEN in combination with an OBR was well tolerated and maintained virologic suppression at W104. These data support the use of LEN for treatment of people with MDR HIV-1.Onyema Ogbuagu, MD, Gilead Sciences, Inc: Advisor/Consultant|Gilead Sciences, Inc: Honoraria|Janssen: Advisor/Consultant|Viiv: Advisor/Consultant Edwin DeJesus, MD, Gilead Sciences, Inc: Advisor/Consultant|Theratechnology: Advisor/Consultant|Theratechnology: Honoraria Mezgebe Berhe, MD, MPH, Gilead Sciences, Inc: Clinical Trial Investigator Gary J Richmond, MD, Gilead Sciences, Inc: Grant/Research Support|Glaxo Smith Kline: Grant/Research Support|Insmed: Grant/Research Support|TaiMed: Grant/Research Support Peter J Ruane, MD, Gilead Sciences, Inc: Advisor/Consultant|Viiv: Advisor/Consultant|Viiv: Honoraria Gary I. Sinclair, MD, Abbvie: Grant/Research Support|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Janssen: Honoraria|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Honoraria|Thera: Advisor/Consultant|Thera: Grant/Research Support|Thera: Honoraria|ViiV: Advisor/Consultant|ViiV: Grant/Research Support|ViiV: Honoraria Moti N Ramgopal, MD, Abbvie: Honoraria|Gilead Sciences, Inc: Advisor/Consultant|Gilead Sciences, Inc: Honoraria|Janssen: Advisor/Consultant|Janssen: Honoraria|Merck: Advisor/Consultant|Viiv: Advisor/Consultant|Viiv: Honoraria William Sanchez, MD, Gilead Sciences, Inc: Grant/Research Support|GSK: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support Gordon E Crofoot, MD, Gilead Sciences, Inc: Clinical Trial Investigator Nicolas A Margot, MA, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Hui Wang, PhD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Hadas Dvory-Sobol, PhD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Martin Rhee, MD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Jared Baeten, MD, PhD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Sorana Segal-Maurer, MD, Gilead Sciences, Inc: Advisor/Consultant|Gilead Sciences, Inc: Honoraria|Janssen: Advisor/Consultant|Janssen: Honoraria|Theratechnologies: Advisor/Consultant|Theratechnologies: Honoraria|Viiv: Advisor/Consultant|Viiv: Honoraria


Thermopower and the Lorentz number for an edge-free (Corbino) graphene disk in the quantum Hall regime is calculated within the Landauer–Büttiker formalism. By varying the electrochemical potential, we find that amplitude of the Seebeck coefficient follows a modified Goldsmid–Sharp relation, with the energy gap defined by the interval between the zero and the first Landau levels in bulk graphene. An analogous relation for the Lorentz number is also determined. Thus, these thermoelectric properties are solely defined by the magnetic field, the temperature, the Fermi velocity in graphene, and fundamental constants including the electron charge, the Planck and Boltzmann constants, being independent of the geometric dimensions of the system. This suggests that the Corbino disk in graphene may operate as a thermoelectric thermometer, allowing to measure small temperature differences between two reservoirs, if the mean temperature magnetic field are known. Following the discovery of graphene ,2,3, a tT, the electron charge In addition to conductivity measurements, thermoelectric phenomena including Seebeck and Nerst effects in graphene ,33,34,35rp value ,41(1)|Sons, see ).The thermoelectric properties of graphene disks at zero (or low) magnetic fields have also been considered ,43. In tT and field B) can still be truncated by a closed-form function of the quantity In this paper, we present numerical results on the Seebeck coefficient and Lorentz number  for the ballistic disk in graphene see . The resoperties ,49.The remaining parts of the paper are organized as follows. In E and uniform magnetic field B, which can be written as Our analysis starts with the wave equation for massless Dirac fermions in graphene at energy e height ,19.j, can be written asBecause of the symmetry, the wave function can be written in the formhe leads . . Fulj s. To reduce round-off errors that occur in finite-precision pure mathematics due to exponentially growing (or decaying) spinor components, we have divided the interval M equally wide parts, bounded by For the disk area, l-th interval has the formThe resulting wave function for the The matching conditions, namelyj. The scattering matrix,Analogously, assuming the scattering from j s are decoupled, numerous software packages can be used to find their solutions up to machine precision. We use the double precision LAPACK routine zgesv, see [Since linear systems for different esv, see . The traFor heavily doped leads  and (9) was performed for each e method  was alsoWithin the Landauer–Büttiker formalism, the linear-response conductance ,54 and oFor zero temperature, Equation (23) reduces toS and d-wave systems [For some specific r metals . For gap of μ/kBT, with a  systems , before  systems ,60,61.u and fundamental constants.In the presence of a transport gap, one can consider a simplified model for ented in ,42 to thIn a case where more Before discussing the thermoelectric properties, we present zero-temperature conductance spectra, related to the transmission-energy dependence via Equation (26), thus representing the input data for the Seebeck coefficient and Lorentz number calculations. For the rectangular barrier of infinite height, n is an integer  and (25) for In the quantum Hall regime, i.e., for EnLL see . In suchS, surrounding an odd zero of S  and (25) results in the following features visible in In These results support our conjecture that the model ∞, see For the sake of completeness, in this section we revisit the effects of smooth potential barriers, considered earlier for the zero magnetic field . The elem, the Fermi wavelength For a finite barrier height, the particle-hole symmetry of the conductance spectrum F/L (see ). The vaFor m). Due to this asymmetry, the global conductance minimum In both cases, i.e., for The consequences of the above-mentioned features of m (specified for each panel) are chosen. For sufficiently large In m is varied from T is chosen to keep the constant The effect of B values reveal common behaviours upon proper rescaling  is outside the scope of this work. Although the transmission resonances with FQHE states have been observed in ultra-clean graphene samples , existin


Resistive random access memory is very well known for its potential application in in-memory and neural computing. However, they often have different types of device-to-device and cycle-to-cycle variability. This makes it harder to build highly accurate crossbar arrays. Traditional RRAM designs make use of various filament-based oxide materials for creating a channel that is sandwiched between two electrodes to form a two-terminal structure. They are often subjected to mechanical and electrical stress over repeated read-and-write cycles. The behavior of these devices often varies in practice across wafer arrays over these stresses when fabricated. The use of emerging 2D materials is explored to improve electrical endurance, long retention time, high switching speed, and fewer power losses. This study provides an in-depth exploration of neuro-memristive computing and its potential applications, focusing specifically on the utilization of graphene and 2D materials in RRAM for neural computing. The study presents a comprehensive analysis of the structural and design aspects of graphene-based RRAM, along with a thorough examination of commercially available RRAM models and their fabrication techniques. Furthermore, the study investigates the diverse range of applications that can benefit from graphene-based RRAM devices. Graphene-based resistive random access memory (RRAM) devices have gained significant attention in recent years for their potential applications in neural computing. Graphene, a two-dimensional carbon material, has exceptional electrical and mechanical properties, making it an attractive candidate for RRAM devices. RRAM is considered one of the most promising emerging non-volatile memory, a potentially universal memory device that comes under the broad category of memristive systems  compatibility with RRAM are also discussed.Memristive devices have been studied for their potential to create artificial neural networks that can learn and adapt in a manner similar to biological neural networks  device. This new computing system proposed by Sano et al.  can storThese devices offer several advantages over conventional memory technologies such as flash, dynamic random access memory (DRAM), and static random access memory (SRAM), including high density, low power consumption, and fast switching speeds  memristor array typically refers to a configuration where memristors are organized in a regular grid pattern. The purpose of a single memristor array is to enable the simultaneous operation and interconnection of multiple memristors  and N bit lines (BLs). The transistor switch enables or disables the column-wise memristor nodes. In v1, v2, … vn from the inputs, conductance gi, j of memristors as weights and columns current i1, i2, … im as outputs, where i, j are the coordinates of the crossbar node. The output currents indicate the weighted summation of input voltages. The bias is included as an additional input line.The DNN is implemented using memristor crossbar arrays. Each DNN layer is implemented using one transistor/memristor (1T 1M) configuration as in There are several analog memristive crossbar implementations of CNN architecture  to its neighbors is defined by cloning templates, A and B, for feedback and feedforward connections  through the feedforward weights B. The output of the cell yk, l is fed to C through the feedback weights A. The state equation can be mathematically expressed as Chua and Yang  to get the gate values. ft is the output value of forget gate, i(t) is the output of input/update gate, o(t) denotes the output from the output gate, and c(t) denotes the cell state.The recurrent neural networks-based methods demonstrated outstanding ability in prediction tasks using time-series data by combining large dynamical memory and adaptable computational capabilities. Long short-term memory (LSTM), the special configuration of RNN, is aimed at overcoming the vanishing gradient problems in conventional RNN , using a scaling factor a and not trained. The weights of the reservoir are sampled from u. Hence, the ESNs are conceptually simple and practically easy to implement.The calculation time in LSTM is very heavy and time-consuming. Echo state network (ESN), a reservoir computing architecture, has emerged as an alternative to the gradient descent training method for RNN . STDP is based on presynaptic and postsynaptic impulses. The implementation of SNN architectures with STDP using memristive crossbar arrays is presented in As discussed in the section, the field of neuromorphic computing using memristor crossbar arrays is advancing and the exploration of novel materials and devices for in-memory computing is required to improve efficiency and scalability. The RRAM devices are promising candidates for synapses and neurons in neuromorphic circuits. The analog tunable capability of RRAM devices enables novel computing functions for the realization of neuromorphic computing. The material class for RRAM devices is from magnetic alloys, metal oxides, 2D materials, and organic materials. Existing studies in the literature report that 2D material-based RRAM devices have better properties compared to conventional electrode materials which enhances the characteristics of RRAM in such a way to improve its application in neural computing. The coming section reviews the mechanism of the working principle of RRAM and the use of 2D materials for enhancing the properties are discussed in detail.Graphene and other 2D materials have the potential to revolutionize neural computing due to their unique electrical, mechanical, and optical properties. Graphene is a single layer of carbon atoms arranged in a hexagonal lattice, and it is a highly conductive and transparent material. Other 2D materials, such as transition metal dichalcogenides (TMDs) and hexagonal boron nitride (h-BN), also exhibit interesting properties that make them promising for use in neural computing  and chalcogen elements (such as sulfur or selenium). TMDs can be used to create synaptic devices, which are fundamental building blocks of artificial neural networks  - The copper or nickel substrate is heated in a reactor chamber while introducing a hydrocarbon gas (such as methane) to the chamber. These hydrocarbons react with the substrate to form graphene.Epitaxial growth - Substrates similar to crystal structure of graphene  can be used to grow graphene for obtaining epitaxial growth via CVD process.Mechanical exfoliation - The bulk crystal graphite consists of multiple layers of graphene. These layers are peeled off using tape or a sharp object.Electrochemical exfoliation - The electrolyte solution is used to exfoliate graphene from graphite.Solvothermal synthesis - The exfoliation of graphene from a bulk crystal of graphite is done in an autoclave having high pressure and temperature.Thermal reduction of graphene oxide - The repeated reduction of graphene oxide by heating in a hydrogen gas environment can result in graphene formation.The discovery of graphene was through the mechanical exfoliation  can be used for the fabrication of RRAM, where the graphene is transferred to polydimethylsiloxane (PDMS) from polyimide (PI)   resistance can be changed by the application of electrical current to the RRAM. The change in resistance to a high or low resistance is mapped to binary states of “0” and “1”, thereby allowing digital storage. By applying voltage pulses to the RRAM electrode resistance of the TiO2 film can be changed. The change in resistance is dependent on the frequency as well as the amplitude of the pulses applied. The RRAM can be read by applying a small voltage pulse and reading the output currents without disturbing the resistance state.The popular resistive switching material such as titanium dioxide .The insulating and conducting mechanisms in the RRAM occur from the breakdown and growth of the filament on the application of an external voltage. Depending on the resistive mechanism, RRAM can be classified into (i) metal ion-based RRAM and (ii) oxygen vacancies filament-based RRAM. In metal ion-based RRAM, the switching mechanism happens by the migration of metal ions in the filament and the oxidation and reduction mechanism. The steps followed in the process of transitioning of conducting state to the insulating state are depicted in This type of mechanism happens in the case of metal electrodes such as Au, Ni, or Cu at the top-level electrode. The migration of metal ions occurs through the dielectric layer, and the subsequent reduction or oxidation happens at the bottom. This will create a metal filament between the two metal electrodes through the dielectric barrier. This metal filament formation possesses the LRS state, and the disappearance of the same enables the HRS state. In In the case of oxygen vacancies-based RRAM, the resistance-switching mechanism occurs with the creation of oxygen vacancies. The reaction of oxygen ions with the anode material will create the conducting filament. The properties of RRAM will depend on the type of materials present in the top electrode, bottom electrode, and middle layer. Different substitutions of the top and bottom electrodes and middle layers with different materials can enhance the properties of RRAM. The use of 2D materials has shown an enhancement in endurance, switching speed, threshold voltage, retention time, etc. The graphene-based RRAM shows promising results in the modification of RRAM toward better performance and for making the system a multilevel cell storage device for the application of MAC computing.Different parameters will affect the performance of the RRAM device. This study mainly focuses on the variability-averse multi-level cell storage in the graphene-based RRAM system. The RRAM devices have shown a large variability due to the stochastic nature of the switching process.Improving the reliability, scalability, and cost-effectiveness of the RRAM device is an essential requirement for practically realizing in-memory and neural computing applications. Graphene-based RRAMs (GRRAM) have different characteristics: low power consumption, higher density, transparency, and homogeneity. GRRAM can be divided into two sub-parts: graphene RRAM and graphene oxide (GO) /reduced graphene oxide (rGO) RRAM. In graphene RRAM, graphene is used as an electrode, whereas in graphene oxide/reduced graphene oxide RRAM, GO or rGO can either be used as a dielectric layer or electrode to enhance the device's performance.The main property of RRAM is the resistive switching mechanism which has various difficulties related to the selection of electrodes and the dielectric layer. The high conductivity and high surface area-to-volume ratio of graphene makes it suitable for electrodes. The power consumption is significantly less in graphene-based electrodes in RRAM compared to conventional metal electrodes in RRAM memory devices. Graphene as an electrode offers various advantages over traditional metal electrodes. The greater mechanical scalability, higher conductivity, and ultrathin nature of graphene help to design non-volatile RRAM memory devices. The mechanical properties of graphene, including exceptional strength, flexibility, and elasticity, make it an ideal candidate for use in RRAM devices. These properties enable the fabrication of ultrathin memory cells and provide the potential for integrating RRAM into complex, multi-layered device architectures  characteristics. The bending cycle is 10 orders larger  is considered an active layer for resistive switches where the memory device has been fabricated with a minimum process cost and less complexity. The fabricated device exhibits a switching ratio of 102, an endurance of 80 voltage sweeps, and a unipolar switching mechanism independent of the supply voltage. The formation of a memristor on a graphene electrode is shown in Park et al.  demonstrx active layer, indium tin oxide, and graphene as bottom and top electrodes with the glass substrate. Studies on the various device sizes are pursued to enhance the reliability of non-volatile memory. Their study revealed that the conduction filament generated in SiO2 active layer maintains the constant current as the device size increases or decreases. The switching ratio (105) and electrical endurance (300 voltage sweeps) have improved compared to Park et al. (x-indium tin oxide (ITO) device.As transparent electronics devices are in high demand for the electronics industry. Yao et al.  have conk et al. . They ha3) . With its vertical two-terminal configuration, the device has good resistive switching performance and a high on-off ratio (switching ratio)  has been proposed by Qian et al.  and preso et al. , they hax/Al2O3/TiO2 dielectric layer is sandwiched between the top and bottom electrodes. The device exhibits forming-free switching characteristics where the device transitions between different resistance states (HRS/LRS) without requiring a separate “forming” process. The forming-free behavior reduces the device complexity and faster the switching process. The proposed device has increases the non-linearity of the current-voltage characteristic with a reduced value of current compliance. When the device exhibits increased non-linearity, the relationship between voltage and current is not linear and more complex and may involve various mechanisms, such as threshold effects, hysteresis, or other non-linear behaviors. This non-linearity can be influenced by factors such as the material properties of the dielectric layer and the electrodes as well as the specific design and operating conditions of the device. A stable retention time of 104s, a switching ratio of 104, and a greater endurance value (>200 cycles) have been obtained for the graphene-insulator-graphene (G-I-G) based RRAM configuration. Sohn et al. reported a graphene-based 3D RRAM structure where the oxygen ions originating from HfOx migrate toward the graphene layer, where they aggregate to create a conductive filament . He et al.  based RRAM device flexible non-volatile memory. For the purpose of this study, aluminum (Al) has been chosen as the top and bottom electrodes, while GO functions as the active layer. When a negative voltage is applied, it induces an electric field that prompts the migration of oxygen ions within the GO layer. This migration leads to the formation of localized conductive filaments (CFs), consequently causing the device to switch to a LRS. Notably, at the LRS, ohmic conduction is not observed due to the transformation of the GO film into a sp3-bonded state in the absence of CFs . Subsequently, when a negative voltage bias is applied to the TE Al layer, the reverse diffusion of oxygen ions occurs, resulting in the formation of CFs that lead to the low-resistance state (LRS) near the contact interface, driven by the influence of a negative electric field . Graphene oxide offers a large hydrophilic surface area with oxygen-containing functional groups, facilitating microbial attachment and tailored electrochemical reactions on its electrode surface  can be used for non-volatile and bistable memory devices for its high optical transparency and flexibility. Vasu et al.  studied 2 and a retention time of 105. Ho et al.  and tungsten diselenide (WSe2) etc.), which offer a diverse range of electronic properties as discussed in section 3. TMDS based RRAM is an emerging technology in the field of non-volatile memory and nanoelectronics  (molybdenum disulfide (MoS2) and titanium dioxide (TiO2) provide unique electronic properties suitable for different device applications  and phase change memory. RRAM shows excellent scalability beyond the 10 nm technology node. The resistive switching mechanism in RRAM helps to attain different intermediate levels by varying the programming current. The size of the conducting filament in an RRAM device depends directly on the applied current. Thus, by adjusting the value of the current, different resistance states can be attained in the system.Multilevel cell storage in RRAM helps to increase the storage density of the memory cell without reducing its size of. In the normal method, the cell size needs to be reduced to increase the density, which requires complex patterning techniques. In the case of multilevel cell storage, the number of bits stored per cell can be increased to n (any integer above 2), increasing the density to n times with 22/Pt is studied by Lei et al.  varying compliance current, (ii) adjusting reset voltage, and (iii) changing the pulse width of program/erase operation  gradual set and reset, where the filament formation process in RRAM is not required for achieving the resistance states and which is particularly advantageous for linear weight updating in neuromorphic computing. However, some renowned company has developed various kind of RRAM devices. Adesto Technologies has recently launched a new chip family called Moneta, which utilizes CBRAM (Conductive Bridging Random Access Memory) technology. The Moneta family offers ultra-low power memory solutions that are designed to significantly reduce the overall energy consumption of connected devices. The chips demonstrate read and write operations at 50-100 times lower power compared to competitive solutions. The company has already begun shipping samples of the Moneta family in four different densities, including 32 Kbit, 64 Kbit, 128 Kbit, and 256 Kbit. Fujitsu recently developed RRAM product which offers 1.5 times higher memory density compared to the existing 8 Mbit RRAM. Other renowned foundries such as Intel, Panasonic, and Samsung have been developing RRAM technology. These companies have been investing heavily in RRAM research and development to improve the performance, reliability, and scalability of this promising memory technology.For several years, researchers have demonstrated the potential of memristive devices in laboratory experiments. As a result, there have been successful demonstrations of these devices in commercial applications, with RRAM devices being particularly noteworthy in solid-state drives (SSDs) and Internet of Things (IoT) devices. Li et al.  proposedThe researchers are investigating using graphene or graphene oxide (GO) as electrodes or switching material of RRAM targeting in-memory computing for neuromorphic behavior  in analog device applications (Banerjee et al., 5, and endurance value of 105 Hz. AlN has high thermal stability, allowing it to withstand the elevated temperatures used in CMOS processes. This makes it possible to integrate AlN-based RRAM fabrication steps into standard CMOS processes without causing significant damage to the underlying circuitry (Jackson et al., Nowadays, graphene with Si CMOS circuits can also be constructed together for making heterogeneous devices. The demonstration of graphene and Si CMOS hybrid circuits has reduced barriers to entry of graphene in electronics. Huang et al.  construcDue to its unique and interesting features, graphene has surpassed all other nanomaterials in terms of its use in electronic devices. Additionally, it was shown that graphene's greater mobility, less light absorption, and excellent mechanical qualities enhance the functionality of transparent flexible electronic devices. The difficulty is that the cost of manufacturing graphene will increase the overall price of the device. The transfer of graphene from one substrate to another without causing any damage is a tedious process, which requires the need of sophisticated instruments. Efficient methods need to be implemented to overcome these drawbacks.The past several years have seen a substantial increase in research into new memory technologies, and numerous prototype RRAM products have been created to show the potential for high-speed and low-power applications. The CMOS compatibility and ability to fabricate in smaller dimensions make the RRAM a suitable candidate for device applications. A high endurance is reported in graphene-based RRAM devices. To date, in a single RRAM device, no technology has reported fast switching, low power, and stable operation simultaneously. In a graphene-based RRAM device, the properties need to be enhanced for better performance of the device.This review article offers an insightful look into the topic of developing graphene-based RRAM devices in terms of neural computing by giving a concise overview of the development of memory architecture, the current trends, and the constraints. The importance of graphene based RRAM, as well as its structure, operation, and classification, have all been highlighted in a thorough discussion. The methodology and a detailed investigation on the MLC capabilities of RRAM have been presented. It is proposed that the graphene-based RRAM can be used for multilevel cell storage. This modified memory device, with 2D material can be used as a synapse. Along with this, the implementation of graphene based RRAM for various important applications such as hardware security and neuromorphic computing have been highlighted.RTR, RD, and CR: Conducted literature review, prepared a part of the draft copy of the manuscript, and revision of the manuscript. AJ: Contributed to the theoretical framework development, provided critical insights during data analysis and interpretation, manuscript correction and writing, funding acquisition and supervision.


This work investigates the behavior of commercial and custom Quartz tuning forkss (QTF) under humidity variations. The QTFs were placed inside a humidity chamber and the parameters were studied with a setup to record the resonance frequency and quality factor by resonance tracking. The variations of these parameters that led to a 1% theoretical error on the Quartz Enhanced Photoacoustic Spectroscopy (QEPAS) signal were defined. At a controlled level of humidity, the commercial and custom QTFs present similar results. Therefore, commercial QTFs appear to be a very good candidates for QEPAS as they are also affordable and small. When the humidity increases from 30 to 90 %RH, the variations in the custom QTFs’ parameters remain suitable, while commercial QTFs show unpredictable behavior. First introduced in 2002 by Kosterev et al. , Quartz Subsequently, the geometry was adapted, targeting different frequencies . The inff0) and/or decreasing the quality factor (Q) [f0 and Q evolution in real time and operate a feedback correction on the optical and electrical setup, i.e., on the modulation of the laser and the LIA parameters.Follow the Reduce their influence by maintaining their variations in a given controlled range.While it is almost immune to acoustic noises, QEPAS is sensitive to environmental parameter variations. The temperature, pressure and humidity can modify the QTF properties, generating a shift in the resonance frequency (ctor (Q) . The varIn this paper, we propose to evaluate the stability of commercial and custom QTF parameters under humidity variations. As part of a collaboration with Polysense, we were able to build this study between a standard QTF and four of their designed QTFs.f0 and Q to not exceed this 1% error.Most industrial gas sensors assure an accuracy of a few percent on the displayed value . We have decided to follow this criterion to define an acceptable relative error of 1% on the QEPAS signal (Equation (1)). Therefore, the first step is to calculate the required accuracy for the f0 as the resonant frequency, describing the frequency response of a typical QTF. It corresponds to the well-known Butterworth-Von Dyke (BVD) electrical model of the component. The squared QEPAS signal is proportional to the squared admittance of the QTF, representing a Lorentzian function [0f and Q as well. This 1% amplitude error can be converted in an error on the measured frequency We consider here a QEPAS signal S modelized by a Lorentzian lineshape, centered on function . This shIsolating f0 = 32 kHz and typical Q of 8000:Using (3), and noting that:f = f0, the QEPAS signal has a linear relationship with Q, thus the error is easily obtained:This variation in the frequency corresponds to the green curve in In this study, we will consider ten QTFs: two under the “AV-08” reference, two under the “T1-08” reference and six under the “commercial QTF” reference. “AV-08” and “T1-08” are custom QTFs realized by Polysense. The commercial QTFs come from Fox Electronics (NC38LF).f0 and ΔQ in relation to a 1% signal variation is calculated. For each tested QTF, an estimation of Δfexc and amplitude Vexc was generated using a waveform generator (Tektronix AFG 1022).When an external perturbation is brought to an oscillating device, it oscillates according to the frequency imposed by the perturbation. When this perturbation is stopped, the oscillation returns to the inner frequency of the device: it represents the relaxation phase. In the presented experimental setup, a sinewave of frequency exc).The sinewave was used to electrically excite the QTF . The signal recorded from the QTF was amplified with a homemade transimpedance amplifier and demodulated at fexc by the LIA. The collected signal has an exponential decay shape. Its period and envelop are, respectively, described by Equations (8) and (9). While the former gives information about the instantaneous frequency of the QTF, the latter gives access to its quality factor.The frequency excf must be chosen to be close enough to f0 to obtain a good accuracy on the demodulated signal, but far enough to avoid f0= fexc, leading to huge values of T. In parallel, Vexc must be chosen to be big enough to avoid uncertainty on the demodulated signal, but small enough to avoid the saturation of the LIA.For instance, fexc and Vexc) on the QTF parameters (f0 and Q) was measured.To determine the best experimental conditions, the effect of the sinewave parameters . When the excitation amplitude increases, the dispersion decreases, and the mean values quickly converge. This preliminary study was useful for quantifying the errors in the characterization setup.On one hand, for variations of |f0 − fexc|was chosen to be equal to 20 Hz, 2 Hz and 3 Hz for the commercial QTF, AV-08 group and T1-08 group, respectively. Vexc was chosen to be equal to 100 mVpp in all of the experiments.The same study was conducted for each QTF described in this paper. Finally, according to the results, |f0 and Q under the humidity variation.Once these parameters are fixed, one can move on to the measurement and analysis of the shifts in The behavior of the QTF parameters under humidity variation was assessed with a benchtop humidity chamber (ESPEC SH-242). The temperature in the chamber was set to 22 °C. The humidity was changed from 30% to 90% RH (percentage of relative humidity), with an increasing step of 10% occurring every 20 min. To confirm the observations and ensure their repeatability, a step to 50% and 30 %RH was added after the 90 %RH step. The humidity steps were long due to the response time of the humidity chamber, which was in the range of a few minutes.The resonance frequency of each QTF, its quality factor, as well as the temperature and the different humidity steps were recorded and are presented in f0 and Q during a stabilized value of humiditydeviation of  (arbitrary measured for 70 %RH) as it could be the case in a QEPAS experiment with controlled humidity.Investigating the f0 and Q between 30% and 90 %RHThe difference of the mean values of  is investigated considering the case of a real-life measurement, where the humidity would increase rapidly.The study of the variation in the QTF parameters was made in two steps:f0 variations were very small: ~0.025 Hz. The same investigations were realized for the custom QTFs, and the results are summarized in f0 occurred at 90 %RH. The variation in f0 when moving from 80 to 90 %RH was approximately equal to the shift that occurred when the humidity was increased from 30 to 80 %RH. It is also at 90 %RH that the largest error in the temperature was observed. This error, approximately equal to ±0.2 °C, occurred due to the difficulties of keeping this humidity value stable.In Usually, before any QEPAS measurement, a frequency sweep can be realized to estimate the exact resonance frequency of the QTF. Simultaneously, the measured frequency is used as the laser modulation frequency and demodulation frequency in the LIA. This frequency is also used as a reference in the demodulation of the collected signal. In controlled experimental conditions, there is no reason to observe significative changes between the measured frequency and the one used as a reference by the LIA. However, in real-life applications, the humidity, temperature and gas flow do not always remain constant. For example, in environmental gas monitoring, the humidity depends on the weather conditions. In breath analysis, the concentration of water is considered very high: close to 5%, i.e., 90 %RH, and can vary from one subject to another. The effect of the temperature on the QTF is well-known and specified by manufacturers. The frequency evolves in a parabolic shape when the temperature increases . The efff0 leading this 1% error were equal to 0.28, 0.036 and 0.06 Hz, respectively, for the commercial, AV-08 and T1-08 groups.In QEPAS measurements, humidity can be useful to enhance the photoacoustic effect, but at the same time, can be a source of interferences and error. In this paper, the presented results are interpreted on the basis of an acceptable error of 1% on the QEPAS signal. The calculated variation of f0 and the calculated values leading to 1% of error for each group of QTFs.At a constant value of humidity, i.e., by keeping the humidity level stable during the experiment, both the commercial and custom QTFs presented non-significative variations in their resonance frequency, showing that the characterization setup is stable and accurate. In fact, there was a factor of approximately 0.1 between the measured variations of f0, ranging between 0.5 and 3 Hz, was above the acceptable defined values. In this comparison, the AV-08 group presented more stable results than the T1-08. The measured values for AV-08  were approximately 0.0055  and were five times  smaller than the calculated values.The comparison of the frequency variations between 30 and 90 %RH showed a difference in behavior between the custom QTFs and the commercial ones. Several commercial QTFs were tested; the dispersion of the data shows unpredictable variations in high humidity conditions (80–90 %RH), whereas both groups of custom QTF presented sustainable variation in the same conditions. For the commercial QTFs, the variation of Even if they were not especially designed for photoacoustic uses, the commercial QTFs presented very comparable QEPAS performances to the custom QTFs, as long as the humidity was stabilized during the measurement. Therefore, commercial QTFs remain a cheaper, smaller and efficient option for gas sensing. Nevertheless, in some applications, such as breath analysis, the relative humidity can reach 90% at the end of the exhalation, implying a large increase in the humidity in the sensor that usually operates at room humidity. The results obtained through these measurements showed more that the custom QTFs gave more reliable performances in this regard, while the commercial QTFs’ behavior can be unpredictable.f0 leads us to focus our concern of this crucial parameter, which was taken into consideration at different stages in the experiments. However, the inconsistent variations in the quality factor are more complex to describe. For the commercial QTFs, the variation of Q from 30 to 90 %RH might be a cause of the instability in the system, while they are acceptable at a constant humidity value. For the custom QTFs, no trends can be extracted in the measurement of Q.Most field sensors do not monitor only one type of information, such as the gas concentration, but often integrate temperature and humidity probes. The data of these environmental parameters can be displayed in real-time and eventually be used for post-process, data analysis and correlations, as well as for the real-time compensation of these parameters onto the output signal. The irregular variation of f0 and Q variations:f0 − fexc| = 20 Hz and Vexc = 100 mVpp) is less significant than that reported during the measurement performed with humidity variations.During the measurements presented in The QTFs were exposed to high levels of humidity. The experiments presented in this paper were conducted at least two times. The surface of the QTFs was observed with a microscope. However, with a magnification down to 100x, no physical degradations of the QTFs were noticed.In the investigation of the QTF as a humidity sensor , the preDifferent hypotheses can be proposed to explain the f0 and Q variations under temperature and humidity changes. In real-life applications, such as breath analysis, the regulation of these parameters’ effect on the measurements becomes crucial. The consistency of the QTFs especially designed for gas sensing was proven during the experiment, while the commercial QTFs displayed more instability at high values of humidity. While we are still uncertain of the origin of the high dispersion observed in the commercial QTF, one must consider the necessity to perform additional measurements on each group of QTF in order to build a statistic and to potentially observe a trend. Further measurements for a better understanding of the humidity effect may imply the use of several coatings on the commercial QTFs or the development of custom QTFs with dimensions that are closer to the commercial ones.In this work, the evaluation of custom and commercial QTFs’ parameters, along with humidity variations, was presented. Our attention was focused on the variation in the resonance frequency of the transducer as the QEPAS signal strongly relies on that parameter. The performed measurement has shed light on the importance of controlling the humidity during spectroscopy and sensing experiments as it has a non-neglectable impact on the stability of this frequency. By the mean of the developed experimental setup, we were able to monitor the