NIR-II fluorescence imaging in liver tumor surgery: A narrative review

In liver tumor surgery,the recognition of tumor margin and radical resection of microcancer focis have always been the crucial points to reduce postoperative recurrence of tumor.However,naked-eye inspection and palpation have limited effectiveness in identifying tumor boundaries,and traditional imaging techniques cannot consistently locate tumors in real time.As an intraoperative real-time navigation imaging method,NIRfluorescence imaging has been extensively studied for its simplicity,reliable safety,and superior sensitivity,and is expected to improve the accuracy of liver tumor surgery.In recent years,the research focus of NIRfluorescence has gradually shifted from the-rst near-infrared window(NIR-I,700–900 nm)to the second near-infrared window(NIR-II,1000–1700 nm).Fluorescence imaging in NIR-II reduces the scattering effect of deep tissue,providing a preferable detection depth and spatial resolution while signi-cantly eliminating liver autofluorescence background to clarify tumor margin.Developingfluorophores combined with tumor antibodies will further improve the precision offluorescence-guided surgical navigation.With the development of a bunch offluorophores with phototherapy ability,NIR-II can integrate tumor detection and treatment to explore a new therapeutic strategy for liver cancer.Here,we review the recent progress of NIR-IIfluorescence technology in liver tumor surgery and discuss its challenges and potential development direction.

Self-confocal NIR-II fluorescence microscopy for multifunctional in vivo imaging

Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.

Pay attention to the application of indocyanine green fluorescence imaging technology in laparoscopic liver cancer resection

Traditional laparoscopic liver cancer resection faces challenges,such as difficultiesin tumor localization and accurate marking of liver segments,as well as theinability to provide real-time intraoperative navigation.This approach falls shortof meeting the demands for precise and anatomical liver resection.The introductionof fluorescence imaging technology,particularly indocyanine green,hasdemonstrated significant advantages in visualizing bile ducts,tumor localization,segment staining,microscopic lesion display,margin examination,and lymphnode visualization.This technology addresses the inherent limitations oftraditional laparoscopy,which lacks direct tactile feedback,and is increasinglybecoming the standard in laparoscopic procedures.Guided by fluorescenceimaging technology,laparoscopic liver cancer resection is poised to become thepredominant technique for liver tumor removal,enhancing the accuracy,safetyand efficiency of the procedure.

Application value of indocyanine green fluorescence imaging in guiding sentinel lymph node biopsy diagnosis of gastric cancer: Meta-analysis

BACKGROUND Gastric cancer is a common malignant tumor of the digestive system worldwide,and its early diagnosis is crucial to improve the survival rate of patients.Indocyanine green fluorescence imaging(ICG-FI),as a new imaging technology,has shown potential application prospects in oncology surgery.The meta-analysis to study the application value of ICG-FI in the diagnosis of gastric cancer sentinel lymph node biopsy is helpful to comprehensively evaluate the clinical effect of this technology and provide more reliable guidance for clinical practice.AIM To assess the diagnostic efficacy of optical imaging in conjunction with indocya-nine green(ICG)-guided sentinel lymph node(SLN)biopsy for gastric cancer.METHODS Electronic databases such as PubMed,Embase,Medline,Web of Science,and the Cochrane Library were searched for prospective diagnostic tests of optical imaging combined with ICG-guided SLN biopsy.Stata 12.0 software was used for analysis by combining the"bivariable mixed effect model"with the"midas"command.The true positive value,false positive value,false negative value,true negative value,and other information from the included literature were extracted.A literature quality assessment map was drawn to describe the overall quality of the included literature.A forest plot was used for heterogeneity analysis,and P<0.01 was considered to indicate statistical significance.A funnel plot was used to assess publication bias,and P<0.1 was considered to indicate statistical significance.The summary receiver operating characteristic(SROC)curve was used to calculate the area under the curve(AUC)to determine the diagnostic accuracy.If there was interstudy heterogeneity(I2>50%),meta-regression analysis and subgroup analysis were performed.analysis were performed.RESULTS Optical imaging involves two methods:Near-infrared(NIR)imaging and fluorescence imaging.A combination of optical imaging and ICG-guided SLN biopsy was useful for diagnosis.The positive likelihood ratio was 30.39(95%CI:0.92-1.00),the sensitivity was 0.95(95%CI:0.82-0.99),and the specificity was 1.00(95%CI:0.92-1.00).The negative likelihood ratio was 0.05(95%CI:0.01-0.20),the diagnostic odds ratio was 225.54(95%CI:88.81-572.77),and the SROC AUC was 1.00(95%CI:The crucial values were sensitivity=0.95(95%CI:0.82-0.99)and specificity=1.00(95%CI:0.92-1.00).The Deeks method revealed that the"diagnostic odds ratio"funnel plot of SLN biopsy for gastric cancer was significantly asymmetrical(P=0.01),suggesting significant publication bias.Further meta-subgroup analysis revealed that,compared with fluorescence imaging,NIR imaging had greater sensitivity(0.98 vs 0.73).Compared with optical imaging immediately after ICG injection,optical imaging after 20 minutes obtained greater sensitivity(0.98 vs 0.70).Compared with that of patients with an average SLN detection number<4,the sensitivity of patients with a SLN detection number≥4 was greater(0.96 vs 0.68).Compared with hematoxylin-eosin(HE)staining,immunohistochemical(+HE)staining showed greater sensitivity(0.99 vs 0.84).Compared with subserous injection of ICG,submucosal injection achieved greater sensitivity(0.98 vs 0.40).Compared with 5 g/L ICG,0.5 and 0.05 g/L ICG had greater sensitivity(0.98 vs 0.83),and cT1 stage had greater sensitivity(0.96 vs 0.72)than cT2 to cT3 clinical stage.Compared with that of patients≤26,the sensitivity of patients>26 was greater(0.96 vs 0.65).Compared with the literature published before 2010,the sensitivity of the literature published after 2010 was greater(0.97 vs 0.81),and the differences were statistically significant(all P<0.05).CONCLUSION For the diagnosis of stomach cancer,optical imaging in conjunction with ICG-guided SLN biopsy is a therapeut-ically viable approach,especially for early gastric cancer.The concentration of ICG used in the SLN biopsy of gastric cancer may be too high.Moreover,NIR imaging is better than fluorescence imaging and may obtain higher sensitivity.

Near-Infrared Fluorescence Imaging Contrast Agents for Clinical Research: Limitations and Alternatives

Introduction: Near-infrared fluorescence imaging is a technique that will establish itself in the short term at the international level because it is recognized for its potential to improve the performance of surgical interventions, its moderate investment and operating costs and its portability. Although the technology is now mature, there is currently the problem of the availability of contrast agents to be injected IV. The aim of this methodology article is to propose an alternative solution to the need for contrast agents for clinical research, particularly in oncology. Methodology: They consist of coupling a fluorescent marker in the form of an NHS derivative, such as IR DYE manufactured in compliance with GMP, with therapeutic monoclonal antibodies having marketing authorization for molecular imaging. For a given antibody, the marking procedure must be the subject of a validation file on the final preparation filtered on a sterilizing membrane at 0.22 μm. Once the procedure has been validated, it would be unnecessary to repeat the tests before each clinical research examination. A check of the marking by thin-layer chromatography (TLC) and place it in a sample bank at +4˚C for 1 month of each injected formulation would be sufficient for additional tests if necessary. Conclusion: Molecular near-infrared fluorescence imaging is experiencing development, the process of which could be accelerated by greater availability of clinical contrast agents. Alternative solutions are therefore necessary to promote clinical research in this area. These methods must be shared to make it easier for researchers.

Activatable fluorescent probes for imaging and diagnosis of rheumatoid arthritis

Rheumatoid arthritis(RA)is a systemic autoimmune disease that is primarily manifested as synovitis and polyarticular opacity and typically leads to serious joint damage and irreversible disability,thus adversely affecting locomotion ability and life quality.Consequently,good prognosis heavily relies on the early diagnosis and effective therapeutic monitoring of RA.Activatable fluorescent probes play vital roles in the detection and imaging of biomarkers for disease diagnosis and in vivo imaging.Herein,we review the fluorescent probes developed for the detection and imaging of RA biomarkers,namely reactive oxygen/nitrogen species(hypochlorous acid,peroxynitrite,hydroxyl radical,nitroxyl),pH,and cysteine,and address the related challenges and prospects to inspire the design of novel fluorescent probes and the improvement of their performance in RA studies.

In vivo and in situ real-time fluorescence imaging of peripheral nerves in the NIR-II window

The peripheral nervous system(PNS)is essential for performing and maintaining various motor and sensory functions.Abnormalities can lead to a series of peripheral neurological conditions,such as paraesthesia,pain,or spasms,which are debilitating and lowering the quality of life.Thecurrent guidelines for diagnosis rely predominantly on clinical symptoms resulting from PNS dysfunction,which occur already at an advancedstage.There are currently no effective methods that visually reflect the extent of peripheral neuropathy.In our study,we present a novel in vivoand in situ real-time imaging of peripheral nerves based on the second near-infrared window(NIR-II)fluoresce nee.In NIR-II system,lead sulfidequa ntum dots(PbS Qds)with NIR-II fluoresce nee specifically bound to motor neuro rvspecific protein agrin,acting as image con trast.In micemodel,peripheral nerves were visible as soon as after 2 h post injection.We provide evidenee for the efficacy of this approach,which allows todirectly dem on strate peripheral nerves,their structure,and pote ntial damagesites and degree.Furthermore,our products were of goodbiocompatibility,while the n eural fluoresce nee signal was solid,bright and stable for 4 h in vivo.Thus,overall,our results suggest that NIR-II isan effective new method for direct imaging of peripheral nerves in vivo,opening new horizons on early,improved and more precise,targeteddiag no sis.A resulti ng more rapid installatio n of perso nalized therapy facilitates a better prognosis of clinical peripheral neuropathy.

Automated apoptosis identification in fluorescence imaging of nucleus based on histogram of oriented gradients of high-frequency wavelet coefficients

The automatic and accurate identification of apoptosis facilitates large-scale cell analysis.Most identification approaches using nucleus fluorescence imaging are based on specific morphological parameters.However,these parameters cannot completely describe nuclear morphology,thus limiting the identification accuracy of models.This paper proposes a new feature extraction method to improve the performance of the model for apoptosis identification.The proposed method uses a histogram of oriented gradient(HOG)of high-frequency wavelet coefficients to extract internal and edge texture information.The HOG vectors are classified using support vector machine.The experimental results demonstrate that the proposed feature extraction method well performs apoptosis identification,attaining 95:7% accuracy with low cost in terms of time.We confirmed that our method has potential applications to cell biology research.

Real-time and high-transmission middle-infrared optical imaging system based on a pixel-wise metasurface micro-polarization array

Real-time polarization medium-wave infrared(MIR)optical imaging systems enable the acquisition of infrared and polarization information for a target.At present,real-time polarization MIR devices face the following problems:poor real-time performance,low transmission and high requirements for fabrication and integration.Herein,we aim to improve the performance of real-time polarization imaging systems in the MIR waveband and solve the above-mentioned defects.Therefore,we propose a MIR polarization imaging system to achieve real-time polarization-modulated imaging with high transmission as well as improved performance based on a pixel-wise metasurface micro-polarization array(PMMPA).The PMMPA element comprises several linear polarization(LP)filters with different polarization angles.The optimization results demonstrate that the transmittance of the center field of view for the LP filters is up to 77%at a wavelength of4.0μm and an extinction ratio of 88 d B.In addition,a near-diffraction-limited real-time MIR imaging optical system is designed with a field of view of 5°and an F-number of 2.The simulation results show that an MIR polarization imaging system with excellent real-time performance and high transmission is achieved by using the optimized PMMPA element.Therefore,the method is compatible with the available optical system design technologies and provides a way to realize real-time polarization imaging in MIR wavebands.

Adaptive Design of Fluorescence Imaging Systems for Custom Resolution, Fields of View, and Geometries

Objective and Impact Statement:We developed a generalized computational approach to design uniform,high-intensity excitation light for low-cost,quantitative fluorescence imaging of in vitro,ex vivo,and in vivo samples with a single device.Introduction:Fluorescence imaging is a ubiquitous tool for biomedical applications.Researchers extensively modify existing systems for tissue imaging,increasing the time and effort needed for translational research and thick tissue imaging.These modifications are applicationspecific,requiring new designs to scale across sample types.Methods:We implemented a computational model to simulate light propagation from multiple sources.Using a global optimization algorithm and a custom cost function,we determined the spatial positioning of optical fibers to generate 2 illumination profiles.These results were implemented to image core needle biopsies,preclinical mammary tumors,or tumor-derived organoids.Samples were stained with molecular probes and imaged with uniform and nonuniform illumination.Results:Simulation results were faithfully translated to benchtop systems.We demonstrated that uniform illumination increased the reliability of intraimage analysis compared to nonuniform illumination and was concordant with traditional histological findings.The computational approach was used to optimize the illumination geometry for the purposes of imaging 3 different fluorophores through a mammary window chamber model.Illumination specifically designed for intravital tumor imaging generated higher image contrast compared to the case in which illumination originally optimized for biopsy images was used.Conclusion:We demonstrate the significance of using a computationally designed illumination for in vitro,ex vivo,and in vivo fluorescence imaging.Applicationspecific illumination increased the reliability of intraimage analysis and enhanced the local contrast of biological features.This approach is generalizable across light sources,biological applications,and detectors.

Compact and robust dual-color linearly polarized illumination source for three-photon fluorescence imaging

The miniaturized femtosecond laser in near infrared-Ⅱregion is the core equipment of threephoton microscopy.In this paper,we design a compact and robust illumination source that emits dual-color linearly polarized light for three-photon microscopy.Based on an all-polarizationmaintaining passive mode-locked fiber laser,we shift the center wavelength of the pulses to the 1.7m band utilizing cascade Raman effect,thereby generate dual-wavelength pulses.To enhance clarity,the two wavelengths are separated through the graded-index multimode fiber.Then we obtain the dual-pulse sequences with 1639.4 nm and 1683.7 nm wavelengths,920 fs pulse duration,and 23.75 MHz pulse repetition rate.The average power of the signal is 53.64mW,corresponding to a single pulse energy of 2.25 nJ.This illumination source can be further amplified and compressed for three-photon fluorescence imaging,especially dual-color three-photon fluorescence imaging,making it an ideal option for biomedical applications.

Fluorescence molecular imaging system and fusion algorithm based on 2CCD camera

Infrared and visible light images can be obtained simultaneously by building fluorescence imaging system,which includes fluorescence excitation,images acquisition,mechanical part,image transmission and processing section.This system studied the 2charge-coupled device(CCD)camera(AD-080CL)of the JAI company.Fusion algorithm of visible light and near infrared images was designed for the fluorescence imaging system with wavelet transform image fusion algorithm.In order to enhance the fluorescent moiety of the fusion image,the luminance value of the green component of the color image was changed.And using microsoft foundation classes(MFC)application architecture,the supporting software system was bulit in VS2010 environment.

Response of Ficus microcarpa L.Foliage to Water Stress Determined by Chlorophyll Fluorescence Imaging Technique

[Objective] This study was to determine the response of Ficus microcarpa L. foliage to polyethylene glycol （PEG） simulated water stress using chlorophyll fluo- rescence imaging technique. [Method] The responses of detached leaves from Ficus microcarpa, Ficus benjamina and Nerium oleander to PEG-6000 simulated water stress were detected, and the chlorophyll fluorescence imaging technique was used to detect and analyze the stress at different spots of a single leaf simultaneously. [Result] The responses of Ficus microcarpa, Ficus benjamina and Nerium oleander to dehydration showed that： ~1~） the maximal photochemical efficiency （Fv/Fm） and non- photo-chemical quenching （NPQ） values were small in the reaction center among different detected spots of leaves, and there were great differences between relative electron transport rate （ETR）, photochemical quenching （qP） and quantum efficiency of PSII photochemistry （（φPSII）; （2） the differences of these parameters were more ob- vious among different spots of water-stressed leaves; （3） the discrete degrees of the species with strong resitances decreased significantly. [Conclusion] This study lays the foundation for the further research on the response of plants to drought stress using chlorophyll fluorescence imaging technique.

Optical molecular imaging in cancer research:current impact and future prospect

Cancer has long been amajor threat to human health.Recent advancements inmolecular imaging have revolutionized cancer research by enabling early and precise disease localization,essential for effective management.In particular,optical molecular imaging is an invaluable cancer detection tool in preoperative planning,intraoperative guidance,and postoperative monitoring owing to its noninvasive nature,rapid turnover,safety,and ease of use.The tumor microenvironment and cells within it express distinct biomarkers.Optical imaging technology leverages these markers to differentiate tumor tissues from surrounding tissues and capture real-time images with high resolution.Nevertheless,a robust understanding of these cancer-relatedmolecules and their dynamic changes is crucial for effectivelymanaging cancer.Recent advancements in opticalmolecular imaging technologies offer novel approaches for cancer investigation in research and practice.This review investigates themodern opticalmolecular imaging techniques employed in both preclinical and clinical research,including bioluminescence,fluorescence,chemiluminescence,photoacoustic imaging,and Raman spectroscopy.We explore the current paradigm of optical molecular imaging modalities,their current status in preclinical cancer research and clinical applications,and future perspectives in the fields of cancer research and treatment.

indocyanine green-based fluorescence imaging in visceral and hepatobiliary and pancreatic surgery:State of the art and future directions

In recent years, the use of fluorescence-guided surgery(FGS) to treat benign and malignant visceral, hepatobiliary and pancreatic neoplasms has significantly increased. FGS relies on the fluorescence signal emitted by injected substances(fluorophores) after being illuminated by ad hoc laser sources to help guide the surgical procedure and provide the surgeon with real-time visualization of the fluorescent structures of interest that would be otherwise invisible. This review surveys and discusses the most common and emerging clinical applications of indocyanine green(ICG)-based fluorescence in visceral, hepatobiliary and pancreatic surgery. The analysis, findings, and discussion presented here rely on the authors' significant experience with this technique in their medical institutions, an up-to-date review of the most relevant articles published on this topic between 2014 and 2018, and lengthy discussions with key opinion leaders in the field during recent conferences and congresses. For each application, the benefits and limitations of this technique, as well as applicable future directions, are described. The imaging of fluorescence emitted by ICG is a simple, fast,relatively inexpensive, and harmless tool with numerous different applications in surgery for both neoplasms and benign pathologies of the visceral and hepatobiliary systems. The ever-increasing availability of visual systems that can utilize this tool will transform some of these applications into the standard of care in the near future. Further studies are needed to evaluate the strengths and weaknesses of each application of ICG-based fluorescence imaging in abdominal surgery.

Evaluation of COC183B2 antibody targeting ovarian cancer by near-infrared fluorescence imaging

Objective: To evaluate the imaging potential of a novel near-infrared(NIR) probe conjugated to COC183 B2 monoclonal antibodies(MAb) in ovarian cancer(OC).Methods: The expression of OC183 B2 antigen in OC was determined by immunohistochemical(IHC) staining using tissue microarrays with the H-score system and immunofluorescence(IF) staining of tumor cell lines.Imaging probes with the NIR fluorescent dye cyanine 7(Cy7) conjugated to COC183 B2 Mab were chemically engineered. OC183 B2-positive human OC cells(SKOV3-Luc) were injected subcutaneously into BALB/c nude mice. Bioluminescent imaging(BLI) was performed to detect tumor location and growth. COC183 B2-Cy7 at 1.1,3.3, 10, or 30 μg were used for in vivo fluorescence imaging, and phosphate-buffered saline(PBS), free Cy7 dye and mouse isotype immunoglobulin G(IgG)-Cy7(delivered at the same doses as COC183 B2-Cy7) were used as controls.Results: The expression of OC183 B2 with a high H-score was more prevalent in OC tissue than fallopian tube(FT) tissue. Among 417 OC patients, the expression of OC183 B2 was significantly correlated with the histological subtype, histological grade, residual tumor size, relapse state and survival status. IF staining demonstrated that COC183 B2 specifically expressed in SKOV3 cells but not HeLa cells. In vivo NIR fluorescence imaging indicated that COC183 B2-Cy7 was mainly distributed in the xenograft and liver with optimal tumor-to-background(T/B)ratios in the xenograft at 30 μg dose. The highest fluorescent signals in the tumor were observed at 96 h postinjection(hpi). Ex vivo fluorescence imaging revealed the fluorescent signals mainly from the tumor and liver. IHC analysis confirmed that xenografts were OC183 B2 positive.Conclusions: COC183 B2 is a good candidate for NIR fluorescence imaging and imaging-guided surgery in OC.

A fast and adaptive method for automatic weld defect detection in various real-time X-ray imaging systems

A first and effective method is proposed to detect weld deject adaptively in various Dypes of real-time X-ray images obtained in different conditions. After weld extraction and noise reduction, a proper template of median filter is used to estimate the weld background. After the weld background is subtracted from the original image, an adaptite threshold segmentation algorithm is proposed to obtain the binary image, and then the morphological close and open operation, labeling algorithm and fids＇e alarm eliminating algorithm are applied to pracess the binary image to obtain the defect, ct detection result. At last, a fast realization procedure jbr proposed method is developed. The proposed method is tested in real-time X-ray image,s obtairted in different X-ray imaging sutems. Experiment results show that the proposed method is effective to detect low contrast weld dejects with few .false alarms and is adaptive to various types of real-time X-ray imaging systems.

Enzymatic activity and chlorophyll fluorescence imaging of maize seedlings (Zea mays L.) after exposure to low doses of chlorsulfuron and cadmium

The aim of this research was to study the influence of chlorsulfuron residue and cadmium on the enzymatic activity and photosynthetic apparatus of maize(Zea mays L.) plants. Chlorsulfuron and cadmium at 0.001 and 5.0 mg kg–1, respectively, were mixed and applied to soil prior to planting. The levels of chlorsulfuron-and cadmium-induced stress to plants were estimated by growth, chlorophyll content, lipid peroxide content, enzyme activities, and major fluorescence parameters of chlorophyll(revealed by the fluorescence imaging system Fluor Cam). Chlorsulfuron negatively affected the chlorophyll content, photochemical efficiency of photosystem II in the dark-adapted state, the maximum efficiency of photosystem II, photochemical quenching coefficient, and steady-state fluorescence decline ratio in the leaves of maize seedlings. However, cadmium did not produce noticeable changes. Plants that were exposed to both chlorsulfuron and cadmium showed an obvious increase in the steady-state fluorescence decline ratio. These results implied that the seedlings possessed more resistance to cadmium than to chlorsulfuron and their resistance to chlorsulfuron toxicity was enhanced by the presence of cadmium. The results also suggested that chlorophyll fluorescence imaging reveals overall alterations within the leaves but may not reflect small-scale effects on tissues, as numeric values of specific parameters are averages of the data collected from the whole leaf.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.