High-contrast optical imagi ng is achievable using phosphoresce nt labels to suppress the short-lived background due to the optical backscatterand autofluoresce nee.However,the long-lived phosphorescence is generally ...High-contrast optical imagi ng is achievable using phosphoresce nt labels to suppress the short-lived background due to the optical backscatterand autofluoresce nee.However,the long-lived phosphorescence is generally incompatible with high-speed laser-scan ning imaging modalities.Here,we show that upc on versi on nan oparticles of structure NaYF4:Yb co-doped with 8%Tm(8T-UCNP)in combi nation with a commerciallaser-scanning multiphoton microscopy are uniquely suited for labeling biological systems to acquire high-resolution images with the enhancedcon trast.In comparison with many phosphoresce nt labels,the 8T-UCNP emission lifetime of-15μs affords rapid image acquisition.Thehigh-order optical nonlinearity of the 8T-UCNP(n=4,as confirmed experimentally and theoretically)afforded pushing the resolution limitattain able with UCNPs to the diffraction-limit.The contrast enha nceme nt was achieved by suppressing the backgro und using(i)ban dpassspectral filtering of the narrow emission peak of 8T-UCNP at 455-nm,and(ii)time-gating implemented with a time-correlated single-photon counting system that demonstrated the contrast enhancement of>2.5-fold of polyethyle neimine-coated 8T-UCNPs take n up by huma nbreast adeno carcinoma cells SK-BR-3.As a result,discrete 8T-UCNP nan oparticles became clearly observable in the freshly excised splee ntissue of laboratory mice 15-min post in trave nous injectio n of an 8T-UCNP solution.The dem on strated approach paves the way forhigh-contrast,high-resoluti on,and high-speed multiphot on microscopy in challe nging envir onments of i ntense autofluorescence,exogenous staining,and turbidity,as typically occur in intravital imaging.展开更多
Fluorescence microscopy is a powerful tool for scientists to observe the microscopic world,and the fluorescence excitation light source is one of the most critical components.To compensate for the short operation life...Fluorescence microscopy is a powerful tool for scientists to observe the microscopic world,and the fluorescence excitation light source is one of the most critical components.To compensate for the short operation lifetime,integrated light sources,and low excitation efficiency of conventional light sources such as mercury,halogen,and xenon lamps,we designed an LED-integrated excitation cube(LEC)with a decentralized structure and high optical power density.Using a Fresnel lens,the light from the light-emitting diode(LED)was effectively focused within a 15 mm mounting distance to achieve high-efficiency illumination.LEC can be easily designed in the shape of fluorescence filter cubes for installation in commercial fluorescence microscopes.LECs’optical efficiency is 1–2 orders of magnitude higher than that of mercury lamps;therefore,high-quality fluorescence imaging with spectral coverage from UV to red can be achieved.By replacing conventional fluorescence filter cubes,LEC can be easily installed on any commercial fluorescence microscope.A built-in LEC driver can identify the types of LEDs in different spectral bands to adopt the optimal operating current and frequency of pulses.Moreover,high-contrast images can be achieved in pulse mode by time-gated imaging of long-lifetime luminescence.展开更多
文摘High-contrast optical imagi ng is achievable using phosphoresce nt labels to suppress the short-lived background due to the optical backscatterand autofluoresce nee.However,the long-lived phosphorescence is generally incompatible with high-speed laser-scan ning imaging modalities.Here,we show that upc on versi on nan oparticles of structure NaYF4:Yb co-doped with 8%Tm(8T-UCNP)in combi nation with a commerciallaser-scanning multiphoton microscopy are uniquely suited for labeling biological systems to acquire high-resolution images with the enhancedcon trast.In comparison with many phosphoresce nt labels,the 8T-UCNP emission lifetime of-15μs affords rapid image acquisition.Thehigh-order optical nonlinearity of the 8T-UCNP(n=4,as confirmed experimentally and theoretically)afforded pushing the resolution limitattain able with UCNPs to the diffraction-limit.The contrast enha nceme nt was achieved by suppressing the backgro und using(i)ban dpassspectral filtering of the narrow emission peak of 8T-UCNP at 455-nm,and(ii)time-gating implemented with a time-correlated single-photon counting system that demonstrated the contrast enhancement of>2.5-fold of polyethyle neimine-coated 8T-UCNPs take n up by huma nbreast adeno carcinoma cells SK-BR-3.As a result,discrete 8T-UCNP nan oparticles became clearly observable in the freshly excised splee ntissue of laboratory mice 15-min post in trave nous injectio n of an 8T-UCNP solution.The dem on strated approach paves the way forhigh-contrast,high-resoluti on,and high-speed multiphot on microscopy in challe nging envir onments of i ntense autofluorescence,exogenous staining,and turbidity,as typically occur in intravital imaging.
基金the Shenzhen Science and Technology Program(KQTD20170810110913065 and 20200925174735005).
文摘Fluorescence microscopy is a powerful tool for scientists to observe the microscopic world,and the fluorescence excitation light source is one of the most critical components.To compensate for the short operation lifetime,integrated light sources,and low excitation efficiency of conventional light sources such as mercury,halogen,and xenon lamps,we designed an LED-integrated excitation cube(LEC)with a decentralized structure and high optical power density.Using a Fresnel lens,the light from the light-emitting diode(LED)was effectively focused within a 15 mm mounting distance to achieve high-efficiency illumination.LEC can be easily designed in the shape of fluorescence filter cubes for installation in commercial fluorescence microscopes.LECs’optical efficiency is 1–2 orders of magnitude higher than that of mercury lamps;therefore,high-quality fluorescence imaging with spectral coverage from UV to red can be achieved.By replacing conventional fluorescence filter cubes,LEC can be easily installed on any commercial fluorescence microscope.A built-in LEC driver can identify the types of LEDs in different spectral bands to adopt the optimal operating current and frequency of pulses.Moreover,high-contrast images can be achieved in pulse mode by time-gated imaging of long-lifetime luminescence.
