Multimodal label-free microscopy

This paper reviews the different multimodal applications based on a large ext ent of label-freeinaging modalities,ranging from linear to nonlinear optics,while also inchuding spectroscopicmeasurements.We put specific emphasis on multimodal measurements going across the usual boundaries between imaging modalities,whereas most multimodal platforms combine techniquesbased on similar light interactions or similar hardware implementations.In this review,we limitthe scope to focus on applications for biology such as live cells or tissues,since by their nat ure ofbeing alive or fragile,we are often not free to take liberties with the image acquisition times andare forced to gather the maximum amount of information possible at one time.For such samples,imaging by a given label-free method usually presents a challenge in obt aining suficient opticalsignal or is limited in terms of the types of observable targets.Multimodal imaging is thenparticularly attractive for these samples in order to maximize the amount of measured infor-mation.While multimodal imaging is always useful in the sense of acquiring additional infor-mation from additional modes,at times it is possible to attain information that could not bediscovered using any single mode alone,which is the essence of the progress that is possible usinga multimodal approach.

Dark-vacuole Bodies Studying by High-resolution Label-free Microscopy Assisted with Fluorescence Technology

The intracellular logistics system,consisting of vesicles,plays a crucial role in cellular transport.However,there is a lack of research on the types and functions of intracellular vesicles,and new technologies are needed for further investigation.Recently,researchers have discovered a new subcellular structure known as Dark-vacuole bodies.The composition,function,and potential synergy with other organelles of these Dark-vacuole bodies remain unclear.In this study,we utilized the high-resolution label-free Fourier ptychographic microscopy,developed by our research group,along with fluorescence confocal technology,to study and analyze Dark-vacuole bodies.Our findings provide evidence of the influence of Dark-vacuole bodies on the morphology,distribution,movement,and cell cycle of living cells.Specifically,we analyzed the effects of drug induced stimulation of lipid drops and endosomes,promotion of cell endocytosis,and induction of cellular senescence on Dark-vacuole bodies.Our results indicate that Dark-vacuole bodies show little correlation with lipid drops and endocytosis vesicles,but are significantly associated with late endosomes.Furthermore,cellular senescence leads to a significant increase in the number and size of Dark-vacuole bodies.This study serves as a foundation for further confirming the nature of Dark-vacuole bodies as new organelles.

Tb^(3+)-nucleic acid probe-based label-free and rapid detection of mercury pollution in food

Mercury is a threatening pollutant in food,herein,we developed a Tb^(3+)-nucleic acid probe-based label-free assay for mix-and-read,rapid detection of mercury pollution.The assay utilized the feature of light-up fluorescence of terbium ions(Tb^(3+))via binding with single-strand DNA.Mercury ion,Hg^(2+)induced thymine(T)-rich DNA strand to form a double-strand structure(T-Hg^(2+)-T),thus leading to fluorescence reduction.Based on the principle,Hg^(2+)can be quantified based on the fluorescence of Tb^(3+),the limit of detection was 0.0689μmol/L and the linear range was 0.1-6.0μmol/L.Due to the specificity of T-Hg^(2+)-T artificial base pair,the assay could distinguish Hg^(2+)from other metal ions.The recovery rate was ranged in 98.71%-101.34%for detecting mercury pollution in three food samples.The assay is low-cost,separation-free and mix-to-read,thus was a competitive tool for detection of mercury pollution to ensure food safety.

Label-free in-vivo classi-cation and tracking of red blood cells and platelets using Dynamic-YOLOv4 network

In-vivo flow cytometry is a noninvasive real-time diagnostic technique that facilitates continuous monitoring of cells without perturbing their natural biological environment,which renders it a valuable tool for both scientific research and clinical applications.However,the conventional approach for improving classification accuracy often involves labeling cells with fluorescence,which can lead to potential phototoxicity.This study proposes a label-free in-vivo flow cytometry technique,called dynamic YOLOv4(D-YOLOv4),which improves classification accuracy by integrating absorption intensity fluctuation modulation(AIFM)into YOLOv4 to demodulate the temporal features of moving red blood cells(RBCs)and platelets.Using zebrafish as an experimental model,the D-YOLOv4 method achieved average precisions(APs)of 0.90 for RBCs and 0.64 for thrombocytes(similar to platelets in mammals),resulting in an overall AP of 0.77.These scores notably surpass those attained by alternative network models,thereby demonstrating that the combination of physical models with neural networks provides an innovative approach toward developing label-free in-vivoflow cytometry,which holds promise for diverse in-vivo cell classification applications.

Ionically Imprinting-Based Copper(Ⅱ)Label-Free Detection for Preventing Hearing Loss

Copper is a microelement with important physiological functions in the body.However,the excess copper ion(Cu^(2+))may cause severe health problems,such as hair cell apoptosis and the resultant hearing loss.Therefore,the assay of Cu^(2+)is important.We integrate ionic imprinting technology(IIT)and structurally colored hydrogel beads to prepare chitosan-based ionically imprinted hydrogel beads(IIHBs)as a low-cost and high-specificity platform for Cu^(2+)detection.The IIHBs have a macroporous microstructure,uniform size,vivid structural color,and magnetic responsiveness.When incubated in solution,IIHBs recognize Cu^(2+)and exhibit a reflective peak change,thereby achieving label-free detection.In addition,benefiting from the IIT,the IIHBs display good specificity and selectivity and have an imprinting factor of 19.14 at 100μmol·L^(-1).These features indicated that the developed IIHBs are promising candidates for Cu^(2+)detection,particularly for the prevention of hearing loss.

Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept:a quantitative analysis based on 4D label-free technique

AIM:To identify different metabolites,proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy(PDR)and resistance to anti-vascular endothelial growth factor(VEGF)drugs,and to provide biomarkers for the diagnosis and treatment of PDR.METHODS:Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry(LC-MS/MS)analyses based on 4D label-free technology.Statistically differentially expressed proteins(DEPs),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway representation and protein interactions were analyzed.RESULTS:A total of 12 samples were analyzed.The proteomics results showed that a total of 58 proteins were identified as DEPs,of which 47 proteins were up-regulated and 11 proteins were down-regulated.We found that C1q and tumor necrosis factor related protein 5(C1QTNF5),Clusterin(CLU),tissue inhibitor of metal protease 1(TIMP1)and signal regulatory protein alpha(SIRPα)can all be specifically regulated after aflibercept treatment.GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR.In addition,protein-protein interaction(PPI)network evaluation revealed that TIMP1 plays a central role in neural regulation.In addition,CD47/SIRPαmay become a key target to resolve anti-VEGF drug resistance in PDR.CONCLUSION:Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR.Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept,among which C1QTNF5,CLU,TIMP1 and SIRPαmay become targets for future treatment of PDR and resolution of anti-VEGF resistance.

Label-free visualization of cholesteatoma in the mastoid and tympanic membrane using CARS microscopy

Objective:The present study aimed to evaluate the possibility of using coherent anti-Stokes Raman spectroscopy（CARS） microscopy to determine the specific molecular morphology of cholesteatoma by detecting the natural vibrational contrast of the chemical bonds without any staining.Materials and methods:Specimens from the mastoid and tympanic membrane with and without cholesteatoma were analyzed using CARS microscopy,two-photon excited fluorescence（TPEF） microscopy,and the second harmonic generation（SHG） microscopy.Results:In cholesteatoma tissues from the mastoid,a strong resonant signal at 2845 cm;was observed by CARS,which indicated the detection of the CH;hydro-carbon lipid bonds that do not generate visible signals at 2940 cm;suggestive of CH;bonds in amino acids.A strong resonant signal at 2940 cm;appeared in an area of the same specimen,which also generated abundant signals by TPEF and SHG microscopy at 817 nm,which was suggestive of collagen.In the tympanic membrane specimen with cholesteatoma,a strong resonant signal with corrugated morphology was detected,which indicated the presence of lipids.A strong signal was detected in the tympanic membrane with chronic otitis media using TPEF/SHG at 817 nm,which indicated collagen enrichment.The CARS and TPEF/SHG images were in accordance with the histology results.Conclusion:These results suggest the need to develop a novel CARS microendoscope that can be used in combination with TPEF/SHG to distinguish cholesteatoma from inflammatory tissues.

A SIMULTANEOUS MULTI-PROBE DETECTION LABEL-FREE OPTICAL-RESOLUTION PHOTOACOUSTIC MICROSCOPY TECHNIQUE BASED ON MICROCAVITY TRANSDUCER

We demonstrate the feasibility of simultancous multi-probe detection for an optcal-resolution photoacoustic microscopy(OR-PAM)system.OR-P AM has elicited the attention of biomedical imaging researchers because of its optical absorption contrast and high spatial resolution with great imaging depth.OR-PAM allows label-free and noninvasive imaging by maximizing the optical absorption of endogenous biomolecules.However,given the inadequate absoption of some biomolcules,detection sensitivity at the same incident intensity requires improvement.In this study,a modulated continuous wave with power density less than 3mW/cm^(2)(1/4 of the ANSI safety limit)excited the weak photoacoustic(PA)signals of biological cells.A microcavity traneducer is developed based on the bulk modulus of gas five orders of magnitude lower than that of solid;air pressure variation is inversely proportional to cavity volume at the same temperature increase.Considering that a PA wave expands in various directions,detecting PA signals from different positions and adding them together can increase detection sensitivity and signal-to-noise ratio.Therefore,we employ four detectors to acquire tiny PA signals simul-taneously.Experimental results show that the developed OR-PAM system allows the label-free imaging of cells with weak optical absorption.

基于label-free定量蛋白质组学方法筛选沉默CHAF1B基因后心肌细胞差异表达蛋白及调控网络分析

目的分析沉默染色质装配因子1亚基B(chromatin assembly factor 1 subunit B,CHAF1B)基因后心肌细胞中差异表达蛋白,预测CHAF1B基因调控网络,为寻找促进心肌细胞修复的潜在治疗靶点提供参考。方法采用转染和蛋白质印迹法筛选沉默CHAF1B基因的有效小干扰RNA(small interfering RNA,siRNA)。应用有效siRNA沉默人源心肌AC16细胞CHAF1B基因后,采用细胞活力检测方法检测细胞活力;提取总蛋白质进行定量、还原、烷基化和胰蛋白酶裂解成肽段,利用高效液相串联质谱法鉴定肽段;搜索UniProt蛋白库筛选差异表达的蛋白质进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集和蛋白质互作网络(protein-protein interaction networks,PPI)分析。结果siRNA有效沉默CHAF1B基因后,心肌细胞存活明显受到抑制;label-free定量蛋白质组学方法鉴定结果显示,共有69个差异表达蛋白质,其中50个表达显著上调(差异倍数≥2,P<0.05),19个表达显著下调(差异倍数≤0.5,P<0.05)。GO分析显示,差异表达蛋白质主要参与大分子复合亚基体、细胞组分生物合成和组装等生物学过程,分布在细胞质和囊泡等区域,发挥蛋白质结合等分子功能。KEGG通路富集和PPI分析显示,差异表达蛋白质参与的信号通路包括蛋白酶体、氨酰tRNA生物合成、胞吞、嘧啶代谢和氨基酸生物合成等10条信号途径;表达显著上调的蛋白质如蛋白酶体亚单位A2和B7、26 S蛋白酶体调节亚单位6B和10B参与蛋白酶体途径,丝氨酸、甘氨酸、谷氨酰胺和赖氨酸tRNA合成酶介导氨酰tRNA生物合成;表达显著下调的蛋白质包括骨架相关蛋白2/3复合体亚单位3和热休克70蛋白1样参与胞吞作用,核糖核苷-二磷酸还原酶大亚基介导嘧啶代谢等通路。实时荧光定量聚合酶链式反应结果证实,转染CHAF1B siRNA后心肌细胞中合成骨架相关蛋白2/3复合体亚单位3的基因ARPC3和氨酰tRNA生物合成关键基因QARS1的mRNA水平均显著降低。结论CHAF1B为心肌细胞存活的关键蛋白质,参与调控心肌细胞的胞吞和氨基酸生物合成等多种生物学过程,参考其调控网络可帮助寻找促进心肌细胞修复的干预环节。

Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

Atherosclerosis has been recognized as a chronic inflammation disease,in which many types of cells participate in this process,including lymphocytes,macrophages,dendritic cells(DCs),mast cells,vascular smooth muscle cells(SMCs).Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions.The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques.Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation(SHG)were developed to image mast cells,SMCs and collagen in plaque ex vivo using endogenous optical signals.Mast cells were imaged with two-photon tryptophan autofluorescence,SMCs were imaged with two-photon NADH auto fluorescence,and collagen were imaged with SHG.This development paves the way for further study of mast cell degranulation,and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases(MMPs)which participate in the degradation of collagen.

Label-free,background-free detection of nucleic acid with immobilization-free heterogeneous biosensor and one-pot hybridization chain reaction amplification

Although immobilization-free and label-free electrochemical DNA(E-DNA)biosensors have engaged tremendous interest due to their superior properties,such as easy operation,time-saving and cost-saving,most of them are fabricated in homogeneous modes and usually produce high background current.In the present work,we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction(HCR)for one-pot nucleic acid detection with zero background.The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins,resulting in the generation of HCR products with multibranched arms,which can be captured onto the electrode viaπ-πstacking interactions between multibranched arms and reduced graphene oxide(rGO).Prior to the incubation process with an electrode,two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes.Thus,an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized.This strategy also presents additional merits of simplicity and cheap cost,since probe immobilization,signal tag labeling,and multiple incubation processes are avoided.Therefore,the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.

Applications of nanostructures in wide-field, label-free super resolution microscopy

Super resolution imaging capable of resolving details beyond the diffraction limit is highly desired in many scientific and application fields, including bio-medicine, nanomaterial science, and opto-electronic integration. Up to now, many different methods have been proposed, among which wide-field, label-free super resolution microscopy is indispensable due to its good applicability to diverse sample types, large field of view（FOV）, and high imaging speed. In recent years,nanostructures have made a crucial contribution to the wide-field, label-free subdiffraction microscopy, with various working mechanisms and configuration designs. This review summarizes the recent applications of the nanostructures in the wide-field, label-free super resolution microscopy, with the emphasis on the designs of hyperlens with hyperbolic dispersion, microsphere with ＂nano-jets＂, and nanowire ring illumination microscopy based on spatial frequency shift effect. The bottlenecks of the current techniques and possible solutions are also discussed.

A promising approach for quantifying focal stroke modeling and assessing stroke progression:optical resolution photoacoustic microscopy photothrombosis

To investigate the mechanisms underlying the onset and progression of ischemic stroke,some methods have been proposed that can simultaneously monitor and create embolisms in the animal cerebral cortex.However,these methods often require complex systems and the effect of age on cerebral embolism has not been adequately studied,although ischemic stroke is strongly age-related.In this study,we propose an optical-resolution photoacoustic microscopy-based visualized photothrombosis methodology to create and monitor ischemic stroke in mice simultaneously using a 532 nm pulsed laser.We observed the molding process in mice of different ages and presented age-dependent vascular embolism differentiation.Moreover,we integrated optical coherence tomography angiography to investigate age-associated trends in cerebrovascular variability following a stroke.Our imaging data and quantitative analyses underscore the differential cerebrovascular responses to stroke in mice of different ages,thereby highlighting the technique's potential for evaluating cerebrovascular health and unraveling age-related mechanisms involved in ischemic strokes.

Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

The large use of nonlinear laser scanning microscopy in the past decade paved the way forpot ential clinical application of this imaging technique.Modern nonlinear microscopy techniquesoffer promising label-free solutions to improve diagnostic performances on tissues.In particular,the combination of multiple nonlinear imaging techniques in the same microscope allows inte-grating_morphological with functional information in a morpho-functional scheme.Suchapproach provides a high-resolution label-free alternative to both histological and immunohis-tochemicai examination of tissues and is becoming increasingly popular among the clinicalcommumity.Nevertheless,several technical improvements,including automatic scanning andimage analysis,are required before the technique represents a standard diagnostic method.In thisreview paper,we highlight the capabilit ies of multimodal nonlinear microscopy for tissue inaging,by providing various examples on colon,arterial and skin tissues.The comparison between images acquired using multimodal nonlinear microscopy and histology shows a good agreement bet ween the two methods.The results demonstrate that multimodal nonlinear microscopy is apowerful label-free alternative to standard histopathological methods and has the potential tofind a stable place in the clinical setting in the near future.

基于label-free技术的梅花鹿(Cervus nippon)茸角蛋白组分比较

旨在探究不同生长时期梅花鹿鹿茸的蛋白质组分信息,为鹿茸有效活性物质的挖掘提供理论依据。本研究利用label-free蛋白质组学技术及生物信息学方法对不同生长时期(10、20、40、60、130与360天)梅花鹿茸角蛋白质组分进行比较研究。结果显示,梅花鹿茸角含有丰富的蛋白质,10、20、40、60、130与360天蛋白质含量依次为65.35、70.90、74.00、82.25、56.00、28.02mg·g^-1。应用label-free蛋白质组学技术共鉴定出636种梅花鹿茸角蛋白质,其中218种蛋白质为显著差异表达,主要参与了蛋白质合成、发育、细胞骨架、转运等生物学过程。不同生长阶段茸角的蛋白质表达有各自的特点,为梅花鹿茸角药理活性成分的筛选及茸角相关产品的开发奠定理论基础。

活动性肺结核患者外周血CD4+T淋巴细胞的label-free蛋白组学初步分析

目的应用非标记label-free蛋白质组学方法筛选出活动性肺结核患者外周血CD4+ T淋巴细胞差异表达蛋白质,为阐明结核病的发病机制和早期诊断提供理论依据.方法收集分离9例活动性肺结核患者和9例健康人外周血CD4 T淋巴细胞,应用label-free非标记蛋白组学方法检测外周血CD4+ T淋巴细胞蛋白质谱,筛选出差异表达蛋白质。并用基因本体论(GO)及京都基因与基因组百科全书(KEGG)生物信息学分析软件对差异表达蛋白质进行分析.结果活动性肺结核与健康人外周血CD4+T淋巴细胞中的蛋白质组分布的基本框架很相似,但可发现二者之间有38个明显差异表达的蛋白点,其中有26种蛋白质在肺结核组中表达上调(差异倍数>1.5, P<0.05), 12种蛋白质在肺结核组中表达下调(差异倍数<0.67, P<0.05 ).GO分析结果表明,大部分的差异蛋白质主要定位于胞内区域,结核感染相关差异蛋白质功能体现在结合、调控以及代谢过程中.KEGG分析表明,磷酸戊糖途径、cGMP/PKG信号通路、磷脂酰肌醇信号系统等与结核分枝杆菌感染密切相关.结论对活动性肺结核患者外周血CD4+T淋巴的比较蛋白质组学研究一共鉴定岀38种差异蛋白质,他们可能在肺结核发病过程中起着重要作用,也可能可做为诊断结核病的潜在生物学标志物。

Label-free与iTRAQ蛋白质组学方法在IgA肾病中的应用

目的:探讨蛋白质非标记(label-free)定量技术与同位素标记相对和绝对定量(iTRAQ)技术两种尿液蛋白组学技术在IgA肾病(IgA-N)中的应用价值。方法:选取正常人(健康对照组)和IgA-N患者(IgA-N组)各12例,均留取新鲜晨尿,组内混合,预实验判断样品质量,初步确定蛋白质鉴定数量,FASP酶解后分两部分进行:一部分iTRAQ标记分级后与另一部分进行label-free的LC-MS/MS质谱、数据分析。比较两种实验方法蛋白覆盖情况、实验重复性及差异蛋白数量。结果:经过质谱鉴定,label-free经质谱鉴定到的肽段长度7~26,高峰10~18,各肽段长度范围内的数目最多达800种。iTRAQ实验经质谱鉴定到的肽段长度6~26,高峰分布6~16,各肽段长度范围内的数目最多达1400种。iTRAQ计算组内变异系数(CV)为5.7%~11.2%,组间CV为7.9%~22.0%(CV<0.3)。Label-free实验计算组内CV为3.7%~13.2%,组间CV为5.9%~19.0%(CV<0.3)。Label-free鉴定到624种蛋白,iTRAQ鉴定到621种蛋白。通过火山图分析两种实验方法均可以很好筛选出差异蛋白,且聚类效果好;经GO分析两种实验方案均显示出相似度较高的生物分类功能。结论:iTRAQ标记可检测到更多的蛋白,且在低分子量检测中占优势,而label-free实验得到的CV相对较小,需要依据实验目的优化实验方案。

基于Label-free技术分析牛卵泡蛋白质组分及关键调控蛋白

旨在研究牛卵泡发育过程中蛋白质组表达变化并筛选卵泡发育关键调控蛋白,利用非标记(label-free)定量蛋白质组学技术对牛卵泡颗粒细胞(granulesa cells, GCs)蛋白质组分进行比较分析。采集牛发情周期优势卵泡(dominant follicles, DF)和从属卵泡(subordinate follicles, SF),分别分离GCs,并提取总蛋白,胰蛋白酶酶解,液相色谱-串联质谱(LC-MS/MS)进行蛋白质组分分析,数据库检索分析DF和SF中蛋白质表达情况,并应用生物信息学方法筛选牛卵泡发育关键调控蛋白。结果表明:本试验从30 321个肽段中共成功鉴定出3 409种蛋白质(FDR≤0.01),其中,DF中表达2 895种蛋白质,SF中表达3 102种蛋白质,获得差异表达蛋白质(差异倍数>2,P<0.05) 259种,17种差异表达蛋白质可能与牛卵泡优势化过程相关,SERPINB2可能调控牛卵泡闭锁。该研究筛选获得的牛卵泡差异表达蛋白质和特异表达蛋白质,丰富了牛卵泡发育调控理论,为进一步研究卵泡闭锁及优势化奠定基础。

基于Label-free组学研究南极磷虾油对脂代谢的影响

为了研究南极磷虾油对脂代谢的调节作用,利用非标记(Label-free)蛋白质定量技术,比较分析灌胃南极磷虾油对高脂模型小鼠肝脏蛋白的表达影响。结果表明,与模型组相比,灌胃磷虾油后,试验组和正常组中差异蛋白表达上调的分别有125个和109个,下调表达的分别有99个和95个。进一步分析脂代谢差异蛋白发现,在试验组和正常组中表达上调的分别是棕榈酰蛋白硫酯酶1 (PPT1)、载脂蛋白B100(APOB100)、短支链酰基辅酶A脱氢酶(ACADSB)、3-羟基酰基-CoA脱水酶3(HACD3)和磺基转移酶1A1(SULT1A1);表达下调的分别为酰基辅酶A合成酶中链家族成员3(ACSM3)和酰基辅酶A合成酶家族成员2(线粒体)(ACSF2)。结合脂代谢通路分析和蛋白质相互作用网络图进一步推测,ACADSB、ACSM3和ACSF2等蛋白质在南极磷虾油调节脂代谢中起着重要的调控作用。本研究结果为深入解析南极磷虾油的作用机理和调节脂代谢的分子机制提供了依据。

基于label-free技术的青杨3个叶位叶片比较蛋白质组学分析

为了研究青杨不同叶位叶片蛋白质组的表达变化规律和青杨叶片衰老的蛋白质组学机制,采用非标记蛋白质组学(label-free)方法对杂交青杨子代3个叶位叶片蛋白质组变化进行了分析。结果表明:3个不同叶位叶片共检测到111个差异表达蛋白质,其中55%的差异蛋白质仅在25叶位与5叶位的比较中差异显著;差异表达蛋白质功能分析表明,青杨叶片成熟到衰老过程中与光合作用、生长发育相关蛋白在10和25叶位多呈现下调表达,逆境反应、营养分解转运相关蛋白在10和25叶位多上调表达,而一些转录翻译与修饰调控相关蛋白质则主要呈现波动趋势;青杨3个叶位中,10叶位光合作用和物质合成比5叶位活跃,25叶位叶片由于衰老营养物质分解转运加强,物质合成减弱。蛋白质是细胞功能执行者,可见,青杨叶片成熟到衰老是受蛋白质表达变化有序调控的过程。