Time-gated fluorescence imaging:Advances in technology and biological applications

Time-gated(TG)fluorescence imaging(TGFI)has attracted increasing attention within the biological imaging community,especially during the past decade.With rapid development of light sources,image devices,and a variety of approaches for TG implementation,TGFI has demonstrated numerous biological applications ranging from molecules to tissues.The paper presents inclusive TG implementation mainly based on optical choppers and electronic units for synchronization of fluorescence excitation and emission,which also serves as guidelines for researchers to build suited TGFI systems for selected applications.Note that a special focus will be put on TG implementation based on optical choppers for TGFI of long-lived probes(lifetime range from microseconds to milliseconds).Biological applications by TG imaging of recently developed luminescent probes are described.

Deep learning enhanced NIR-Ⅱ volumetric imaging of whole mice vasculature

Fluorescence imaging through the second near-infrared window(NIR-Ⅱ,1000–1700 nm) allows in-depth imaging.However, current imaging systems use wide-field illumination and can only provide low-contrast 2D information, without depth resolution. Here, we systematically apply a light-sheet illumination, a time-gated detection, and a deep-learning algorithm to yield high-contrast high-resolution volumetric images. To achieve a large Fo V(field of view) and minimize the scattering effect, we generate a light sheet as thin as 100.5 μm with a Rayleigh length of 8 mm to yield an axial resolution of 220 μm. To further suppress the background, we time-gate to only detect long lifetime luminescence achieving a high contrast of up to 0.45 Icontrast. To enhance the resolution, we develop an algorithm based on profile protrusions detection and a deep neural network and distinguish vasculature from a low-contrast area of 0.07 Icontrast to resolve the 100μm small vessels. The system can rapidly scan a volume of view of 75 × 55 × 20 mm3and collect 750 images within 6mins. By adding a scattering-based modality to acquire the 3D surface profile of the mice skin, we reveal the whole volumetric vasculature network with clear depth resolution within more than 1 mm from the skin. High-contrast large-scale 3D animal imaging helps us expand a new dimension in NIR-Ⅱ imaging.

Resolution and contrast enhancement of laser-scanning multiphoton microscopy using thulium-doped upconversion nanoparticles

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.

Contrast-enhanced fluorescence microscope by LED integrated excitation cubes

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.