Background:Pathological scars are a disorder that can lead to various cosmetic,psychological,and functional problems,and no effective assessment methods are currently available.Assessment and treatment of pathological...Background:Pathological scars are a disorder that can lead to various cosmetic,psychological,and functional problems,and no effective assessment methods are currently available.Assessment and treatment of pathological scars are based on cutaneous manifestations.A two-photon microscope(TPM)with the potential for real-time non-invasive assessment may help determine the under-surface pathophysiological conditions in vivo.This study used a portable handheld TPM to image epidermal cells and dermal collagen structures in pathological scars and normal skin in vivo to evaluate the effectiveness of treatment in scar patients.Methods:Fifteen patients with pathological scars and three healthy controls were recruited.Imaging was performed using a portable handheld TPM.Five indexes were extracted from two dimensional(2D)and three dimensional(3D)perspectives,including collagen depth,dermo-epidermal junction(DEJ)contour ratio,thickness,orientation,and occupation(proportion of collagen fibers in the field of view)of collagen.Two depth-dependent indexes were computed through the 3D second harmonic generation image and three morphology-related indexes from the 2D images.We assessed index differences between scar and normal skin and changes before and after treatment.Results:Pathological scars and normal skin differed markedly regarding the epidermal morphological structure and the spectral characteristics of collagen fibers.Five indexes were employed to distinguish between normal skin and scar tissue.Statistically significant differences were found in average depth(t=9.917,P<0.001),thickness(t=4.037,P<0.001),occupation(t=2.169,P<0.050),orientation of collagen(t=3.669,P<0.001),and the DEJ contour ratio(t=5.105,P<0.001).Conclusions:Use of portable handheld TPM can distinguish collagen from skin tissues;thus,it is more suitable for scar imaging than reflectance confocal microscopy.Thus,a TPM may be an auxiliary tool for scar treatment selection and assessing treatment efficacy.展开更多
The low signal-to-noise ratio(S/N)and single functionality of fluorescence imaging agents have limited their practical applications.Bright two-photon excitation(2PE)imaging probes are highly desirable in vivo with lar...The low signal-to-noise ratio(S/N)and single functionality of fluorescence imaging agents have limited their practical applications.Bright two-photon excitation(2PE)imaging probes are highly desirable in vivo with larger imaging depth,minor autofluorescence background,and less photodamage.Herein,we developed responsive aggregated gold nanoparticles(Au NPs)as high contrast 2PE imaging agents,capable of emitting red fluorescence upon excitation of near-infrared(NIR)laser and possessing a high signal-to-noise ratio(S/N of 2,475).By forming aggregates in situ inside tumor tissue,doxorubicin hydrochloride(DOX)-loaded mix-charged gold nanoparticles(DOX-MC-Au NPs)were utilized to act as selective fluorescence imaging probes and precise therapy agents.These high-contrast theranostic agents offer a promising potential for precise cancer imaging and therapy,which might open a new venue to multifunctional and noninvasive theranostics.展开更多
To overcome the conflict between the long-wavelength excitation and high singlet oxygen quantum yield of photosensitizers,we conjugated a two-photon fluorophore,tetrahydroquinoxaline coumarin(TQ),and an efficient phot...To overcome the conflict between the long-wavelength excitation and high singlet oxygen quantum yield of photosensitizers,we conjugated a two-photon fluorophore,tetrahydroquinoxaline coumarin(TQ),and an efficient photodynamic therapeutic agent,benzo[a]phenothiazinium(NBS-NH_(2)),through a hexamethylene linker to build a two-photon photosensitizer,TQ-NBS.In TQ-NBS,TQ served as an energy donor and NBS-NH_(2) acted as an energy acceptor;and TQ-NBS was a F?rster resonance energy transfer(FRET)cassette with a 92.8%efficiency.The large two-photon absorption cross-section of TQ allowed photosensitizer TQ-NBS to work in a 900 nm two-photon excitation(TPE)mode,which greatly benefited the deep tissue penetration in PDT treatment.Meanwhile,the excellent phototoxicity and near-infrared fluorescence of NBS-NH2was kept in TQ-NBS under a TPE mode via a FRET process.Photosensitizer TQ-NBS exhibited a high phototoxic efficacy in living cells and tumor-bearing mice.展开更多
With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in tradition...With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in traditional fluorescence microscopes have limited their imaging applications mostly to superficial tissues. To overcome these disadvantages, researchers have developed the graded-index （GRIN） probes with small diameters for imaging internal organs of small animals in a minimally invasive fashion. However, dynamic imaging based on GRIN lens has not been studied extensively. Here, this paper presented a fluorescence endoscopic imaging system based on GRiN lenses using one-photon and two-photon excitation. GRIN lenses with 1.15 mm diameter and 7.65 mm length were used in the system. The images were acquired by a compact laser scanning imaging system with a resonant galvo-mirror system to scan the laser beam and a photomultiplier tube （PMT） to detect fluorescence signals. Experimental results showed that this system using two-photon excitation could implement dynamic fluorescence microendoscopic imaging and monitor the movement of blood flow beneath the skin in anesthetized mice while producing images with higher contrast and signal to noise ratio （SNR） than those using one photon excitation. It would be a useful tool for studying biological processes of small animals or plants in vivo.展开更多
Combined with the fluorescence labeling technique, two-photon microscopy excited with femtosecond pulse laser has become an important tool for neuroscience research. In this research, the calcium signals from neurons ...Combined with the fluorescence labeling technique, two-photon microscopy excited with femtosecond pulse laser has become an important tool for neuroscience research. In this research, the calcium signals from neurons in rat cortex slice were monitored by a custom-built two-photon microscopy, and the spontaneous calcium signals and the pharmacological responses as well as the responses to femtosecond pulse laser stimulation were recorded. The results showed that the amplitude of the cal-cium signals increased in direct proportion to the corresponding electrical activities. Glutamate induced a calcium transient, but continuous application resulted in smaller response. Simultaneous monitoring of neuronal populations distinguished the neurons of different microcircuits. The femtosecond pulse laser induced local or global calcium signals in the pyramidal neurons. The approach of interrogation and control of neural activities using femtosecond pulse laser is non-contact, nondestructive, repeatable, and without any additional substrates, which will contribute to the development of neuroscience.展开更多
We report a newly synthesized polymer of a star-shaped porphyrin compound(TPA-FxP) with four oligofluorene arms at its meso positions with the pronounced enhancement of the two-photon properties and the generation of ...We report a newly synthesized polymer of a star-shaped porphyrin compound(TPA-FxP) with four oligofluorene arms at its meso positions with the pronounced enhancement of the two-photon properties and the generation of singlet oxygen by utilizing the two-photon excited fluorescence resonance energy transfer.The steady-state spectra and transient triplet-triplet absorption spectra give evidence that the enhanced two-photon absorption cross section results from not only the through-space energy transfer(Frster) but also the through-bond energy transfer between conjugated peripheral oligofluorene arms and the porphyrin core.The two-photon absorption cross section at 780 nm up to 3360 GM(1 GM = 10-50 cm4·s/photon) of TPA-FxP was obtained,which is comparable to the highest values reported from other similar chemically modified porphyrin core compounds.Furthermore,the enhanced production of singlet oxygen under two-photon absorption conditions is also reported.展开更多
Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuores...Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuorescence lifetime imaging microscopy(TP-FLIM)allows non-invasive,label free,three-dimensional,high-resolution imaging of living tisues with not only histological but also biochemical characterization ability in both qualitative and quantitative way.Benefiting from these advantages,this technology is protmising for clinical diagnosis of digestive tract tumors.In recent years,many efforts have'been made in this field and some remarkable progress has been achieved.In this paper,we overview the recent progress of TP-FLIM-based researches on digestive tract tumor detection.Among them,our latest results on the gastric cancer and esophageal cancer are elaborately depicted.Finally,we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.展开更多
Direct,in situ selective detection of intracellular formaldehyde(FA)is of great significance for understanding its function in FA-related diseases.Herein,red carbon dots(RCD)are reported as label-free two-photon fluor...Direct,in situ selective detection of intracellular formaldehyde(FA)is of great significance for understanding its function in FA-related diseases.Herein,red carbon dots(RCD)are reported as label-free two-photon fluorescent nanoprobes for detecting and imaging of FA.Upon addition of FA,the-NH2 groups of RCD could quickly and specially react with aldehydes to form Schiff base and then the strong fluorescence of RCD with blue-shift emission is recovery due to the destruction of the hydrogen bond interaction between RCD and water.In addition,the nanoprobes exhibit outsta nding photo stability,rapid response(<1 min),high sensitivity(~9.9μmol/L)and excellent selectivity toward FA over other aldehyde group compounds.Notably,owing to the good cell-membrane permeability and biocompatibility,as well as the large two-photon absorption cross-section,the as-prepared RCD can be used as label-free nanoprobes for selectively detecting and imaging FA in living cells and zebrafishes through one-photon and two-photon excitation.Moreover,RCD could stain the tissue of zebrafishes at depths interval of up to 240μm under two-photon excitation.This research implied that RCD are promising tools for directly and in situ imaging FA in vivo,thus providing critical insights into FA-related pathophysiological processes.展开更多
基金supported by grants from Beijing Municipal Science and Technology Commission Medicine Collaborative Science and Technology Innovation Research Project(No.Z191100007719001)To Establish a Database and Study the Imaging Features of Common Skin Diseases based on Two-photon Imaging Technology(No.SK2021090379-1)
文摘Background:Pathological scars are a disorder that can lead to various cosmetic,psychological,and functional problems,and no effective assessment methods are currently available.Assessment and treatment of pathological scars are based on cutaneous manifestations.A two-photon microscope(TPM)with the potential for real-time non-invasive assessment may help determine the under-surface pathophysiological conditions in vivo.This study used a portable handheld TPM to image epidermal cells and dermal collagen structures in pathological scars and normal skin in vivo to evaluate the effectiveness of treatment in scar patients.Methods:Fifteen patients with pathological scars and three healthy controls were recruited.Imaging was performed using a portable handheld TPM.Five indexes were extracted from two dimensional(2D)and three dimensional(3D)perspectives,including collagen depth,dermo-epidermal junction(DEJ)contour ratio,thickness,orientation,and occupation(proportion of collagen fibers in the field of view)of collagen.Two depth-dependent indexes were computed through the 3D second harmonic generation image and three morphology-related indexes from the 2D images.We assessed index differences between scar and normal skin and changes before and after treatment.Results:Pathological scars and normal skin differed markedly regarding the epidermal morphological structure and the spectral characteristics of collagen fibers.Five indexes were employed to distinguish between normal skin and scar tissue.Statistically significant differences were found in average depth(t=9.917,P<0.001),thickness(t=4.037,P<0.001),occupation(t=2.169,P<0.050),orientation of collagen(t=3.669,P<0.001),and the DEJ contour ratio(t=5.105,P<0.001).Conclusions:Use of portable handheld TPM can distinguish collagen from skin tissues;thus,it is more suitable for scar imaging than reflectance confocal microscopy.Thus,a TPM may be an auxiliary tool for scar treatment selection and assessing treatment efficacy.
基金Singapore National Research Foundation Competitive Research Program(No.R279-000-483-281)National Research Foundation Investigatorship(No.R279-000-444-281)the National University of Singapore(No.R279-000-482-133)资助
基金supported by the Science and Technology Project of Jiangsu Province(BZ2022056)the Ministry of Education,Singapore(Tier 1 A-8000013-00-00)。
文摘The low signal-to-noise ratio(S/N)and single functionality of fluorescence imaging agents have limited their practical applications.Bright two-photon excitation(2PE)imaging probes are highly desirable in vivo with larger imaging depth,minor autofluorescence background,and less photodamage.Herein,we developed responsive aggregated gold nanoparticles(Au NPs)as high contrast 2PE imaging agents,capable of emitting red fluorescence upon excitation of near-infrared(NIR)laser and possessing a high signal-to-noise ratio(S/N of 2,475).By forming aggregates in situ inside tumor tissue,doxorubicin hydrochloride(DOX)-loaded mix-charged gold nanoparticles(DOX-MC-Au NPs)were utilized to act as selective fluorescence imaging probes and precise therapy agents.These high-contrast theranostic agents offer a promising potential for precise cancer imaging and therapy,which might open a new venue to multifunctional and noninvasive theranostics.
基金supported by National Key Research and Development Program of China(No.2022YFA1207600)National Natural Science Foundation of China(Nos.22178395,62175262 and 62005294)。
文摘To overcome the conflict between the long-wavelength excitation and high singlet oxygen quantum yield of photosensitizers,we conjugated a two-photon fluorophore,tetrahydroquinoxaline coumarin(TQ),and an efficient photodynamic therapeutic agent,benzo[a]phenothiazinium(NBS-NH_(2)),through a hexamethylene linker to build a two-photon photosensitizer,TQ-NBS.In TQ-NBS,TQ served as an energy donor and NBS-NH_(2) acted as an energy acceptor;and TQ-NBS was a F?rster resonance energy transfer(FRET)cassette with a 92.8%efficiency.The large two-photon absorption cross-section of TQ allowed photosensitizer TQ-NBS to work in a 900 nm two-photon excitation(TPE)mode,which greatly benefited the deep tissue penetration in PDT treatment.Meanwhile,the excellent phototoxicity and near-infrared fluorescence of NBS-NH2was kept in TQ-NBS under a TPE mode via a FRET process.Photosensitizer TQ-NBS exhibited a high phototoxic efficacy in living cells and tumor-bearing mice.
文摘With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in traditional fluorescence microscopes have limited their imaging applications mostly to superficial tissues. To overcome these disadvantages, researchers have developed the graded-index （GRIN） probes with small diameters for imaging internal organs of small animals in a minimally invasive fashion. However, dynamic imaging based on GRIN lens has not been studied extensively. Here, this paper presented a fluorescence endoscopic imaging system based on GRiN lenses using one-photon and two-photon excitation. GRIN lenses with 1.15 mm diameter and 7.65 mm length were used in the system. The images were acquired by a compact laser scanning imaging system with a resonant galvo-mirror system to scan the laser beam and a photomultiplier tube （PMT） to detect fluorescence signals. Experimental results showed that this system using two-photon excitation could implement dynamic fluorescence microendoscopic imaging and monitor the movement of blood flow beneath the skin in anesthetized mice while producing images with higher contrast and signal to noise ratio （SNR） than those using one photon excitation. It would be a useful tool for studying biological processes of small animals or plants in vivo.
基金Supported by the National Natural Science Foundation of China (Grant Nos. 90508003 and 30700215)
文摘Combined with the fluorescence labeling technique, two-photon microscopy excited with femtosecond pulse laser has become an important tool for neuroscience research. In this research, the calcium signals from neurons in rat cortex slice were monitored by a custom-built two-photon microscopy, and the spontaneous calcium signals and the pharmacological responses as well as the responses to femtosecond pulse laser stimulation were recorded. The results showed that the amplitude of the cal-cium signals increased in direct proportion to the corresponding electrical activities. Glutamate induced a calcium transient, but continuous application resulted in smaller response. Simultaneous monitoring of neuronal populations distinguished the neurons of different microcircuits. The femtosecond pulse laser induced local or global calcium signals in the pyramidal neurons. The approach of interrogation and control of neural activities using femtosecond pulse laser is non-contact, nondestructive, repeatable, and without any additional substrates, which will contribute to the development of neuroscience.
基金Supported by the National Natural Science Foundation of China (Grant Nos. 20773139, 20825314, and 20833008)State Key Project for Fundamental Research (Grant Nos. 2006CB806000 and 2007CB815200)the Chinese Academy of Sciences (Grant No. KJCX2.Y.W.H06)
文摘We report a newly synthesized polymer of a star-shaped porphyrin compound(TPA-FxP) with four oligofluorene arms at its meso positions with the pronounced enhancement of the two-photon properties and the generation of singlet oxygen by utilizing the two-photon excited fluorescence resonance energy transfer.The steady-state spectra and transient triplet-triplet absorption spectra give evidence that the enhanced two-photon absorption cross section results from not only the through-space energy transfer(Frster) but also the through-bond energy transfer between conjugated peripheral oligofluorene arms and the porphyrin core.The two-photon absorption cross section at 780 nm up to 3360 GM(1 GM = 10-50 cm4·s/photon) of TPA-FxP was obtained,which is comparable to the highest values reported from other similar chemically modified porphyrin core compounds.Furthermore,the enhanced production of singlet oxygen under two-photon absorption conditions is also reported.
基金supports from the National Key Research and Development Program of China(2017YFC0110200)Program 973(2015CB755502)+4 种基金the National Natural Science Foundation of China(NSFC)(81571724,81701744,81822023)the Natural Science Foundation of Guangdong Province(2014A030312006,2017A 030310308)the Scientific Instrument Innovation Team of Chinese Academy of Sciences(GJJSTD 20180002)the Shenzhen Science and Technology Program(JCYJ20170818164343304,JCYJ20170818155006471,JCYJ20160608214524052,JCYJ20180507182432303)the SIAT Innovation Program for Excellent Young Researchers(201821).
文摘Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuorescence lifetime imaging microscopy(TP-FLIM)allows non-invasive,label free,three-dimensional,high-resolution imaging of living tisues with not only histological but also biochemical characterization ability in both qualitative and quantitative way.Benefiting from these advantages,this technology is protmising for clinical diagnosis of digestive tract tumors.In recent years,many efforts have'been made in this field and some remarkable progress has been achieved.In this paper,we overview the recent progress of TP-FLIM-based researches on digestive tract tumor detection.Among them,our latest results on the gastric cancer and esophageal cancer are elaborately depicted.Finally,we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.
基金financial support of the present work by the National Natural Science Foundation of China(Nos.51773056,51603067,21705040)Natural Science Foundationof Hunan Province,China(No.2018JJ3143)+3 种基金China Postdoctoral Science Foundation(Nos.2017M622571,2017M622568 and 2018T110824)Open Project Program of State Key Laboratory of Chemo/Biosensing and Chemometrics(No.2016019)Open Fund of the State Key Laboratory of Luminescent Materials and Devices(South China University of Technology)(No.2019-skllmd-09)Natural Science Foundation of Guangdong Province(No.2017A030313299)。
文摘Direct,in situ selective detection of intracellular formaldehyde(FA)is of great significance for understanding its function in FA-related diseases.Herein,red carbon dots(RCD)are reported as label-free two-photon fluorescent nanoprobes for detecting and imaging of FA.Upon addition of FA,the-NH2 groups of RCD could quickly and specially react with aldehydes to form Schiff base and then the strong fluorescence of RCD with blue-shift emission is recovery due to the destruction of the hydrogen bond interaction between RCD and water.In addition,the nanoprobes exhibit outsta nding photo stability,rapid response(<1 min),high sensitivity(~9.9μmol/L)and excellent selectivity toward FA over other aldehyde group compounds.Notably,owing to the good cell-membrane permeability and biocompatibility,as well as the large two-photon absorption cross-section,the as-prepared RCD can be used as label-free nanoprobes for selectively detecting and imaging FA in living cells and zebrafishes through one-photon and two-photon excitation.Moreover,RCD could stain the tissue of zebrafishes at depths interval of up to 240μm under two-photon excitation.This research implied that RCD are promising tools for directly and in situ imaging FA in vivo,thus providing critical insights into FA-related pathophysiological processes.
