Tunable Excitation Wavelength Research Articles

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

Temporal‐Focusing Multiphoton Excitation Single‐Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores

AbstractSingle‐molecule localization microscopy (SMLM) based on temporal‐focusing multiphoton excitation (TFMPE) and single‐wavelength excitation is used to visualize the three‐dimensional (3D) distribution of spontaneously blinking fluorophore‐labeled subcellular structures in a thick specimen with a nanoscale‐level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out‐of‐focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single‐wavelength TFMPE achieves wide‐field and axially confined two‐photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two‐dimensional TFMPE‐SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE‐SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid‐beta peptide deposits.

Towards applicable photoacoustic micro-fluidic pumps: Tunable excitation wavelength and improved stability by fabrication of Ag-Au alloying nanoparticles

Photoacoustic micro-fluidic pumps (PMPs) based on localized surface plasmon resonance (LSPR) of nanoparticles (NPs) can convert photo energy into kinetic energy of fluidic jetting therefore attracted considerable attentions in microfluidics and optofluidics. In this work, PMPs are fabricated using Ag-Au alloy NPs. Through solid-state-dewetting, alloy NPs are prepared of variable component ratios for tunable LSPR absorption between 440 and 530 nm. The relationship between NP morphology and optical properties are studied with the assistant of finite-difference time-domain (FDTD) simulation. Photoacoustic jets are observed through the excitation of both 450 and 532 nm lasers. Noticeably, with 450 nm laser, long lasting jets over 1 h are excited on Ag0.9Au0.1, which sharply contrast to the duration within 1 min on Ag0.2Au0.8 NPs with 532 nm laser, indicating the importance of material selection in performance improvement. Further investigation on jetting stability is performed according to the morphological evolution by laser striking, suggesting the enhanced stability can be related to the increased melting-point and thermal-stability of alloys.

Simulation design of wide-field temporal-focusing multiphoton excitation with a tunable excitation wavelength

The optical parameters of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable of achieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to have good excitation performance. For this purpose, the approaches were adopted to quantitatively evaluate the grating groove density, focal length of the collimating lens and objective, and different excitation wavelengths. A grating with a groove density of 830 lines/mm enables the TFMPEM system to achieve a wavelength range of 700–1000 nm by adjusting the incident angle of the ultrafast laser on the grating; a diffraction efficiency of 81 ± 3% was obtained at this wavelength range. By using the 830 lines/mm grating, a collimating lens with a 500 mm focal length and a 60× water immersion objective, we achieved a large excitation area and a better filling effect of the spectrum band of the pulse on the back focal plane of the objective; these parameters ensure high optical sectioning and small variation in the illumination power density within this wavelength range. The variation ranges of the excitation area, optical sectioning, and illumination power density of 4272 ± 798 μm2, 2.6 ± 0.3 μm, and 71 ± 29%, respectively, were obtained in two-photon excitation fluorescence imaging at 700–1000 nm excitation wavelengths.

A CuO-functionalized NMOF probe with a tunable excitation wavelength for selective detection and imaging of H2S in living cells.

Recently, fluorescent nanoscale metal-organic frameworks (NMOFs) have been proven to be useful probes for the detection and imaging of active biomolecules in living cells. However, the excitation wavelengths of these NMOF fluorescence probes are mostly in the ultraviolet region, which unavoidably results in reduced cell activity, limited tissue penetration depth and inevitable biological background interference. Herein, to solve this problem, a CuO functionalized NMOF probe with a tunable excitation wavelength based on Förster resonance energy transfer (FRET) for selective detection and imaging of the third important gaseous signaling molecule hydrogen sulfide (H2S) in living cells as an example is presented. In the energy transfer system, NMOF confines the luminophore organic dye thiazole orange within its intrinsic porous matrix as the energy donor, in which the excitation wavelength of the NMOF can be tuned simply from UV to Vis through the choice of dye molecules, and the H2S-responding site copper oxide nanoparticle (CuO NP) is the acceptor. After the surface functionalization of CuO NPs onto the NMOF, the fluorescence of the NMOF can be efficiently quenched based on the FRET. When H2S appeared, the fluorescence of the nanoprobe is recovered due to the interruption of FRET. This facile yet powerful strategy not only provides an instantaneous fluorescence probe for selective H2S detection in living cells but also offers a valuable approach for using porous NMOFs to sense other biological species.

Lanthanide-based near-infrared emitting metal-organic frameworks with tunable excitation wavelengths and high quantum yields.

We report here two isoreticular ytterbium (Yb) 2D metal-organic frameworks (MOFs) emitting near-infrared (NIR) luminescence with 1.26(2)% quantum yield values, which are among the highest values reported to date for MOFs incorporating NIR-emitting lanthanide cations. The excitation wavelength of the YbIII MOFs can be red-shifted to the visible range by introducing an amino group to the organic chromophore. Density functional theory (DFT) calculations were performed to gain insights into the photophysical properties of this new family of MOFs.

White-Emitting Protein Nanoparticles for Cell-Entry and pH Sensing

Facile synthesis of white-emitting, protein-based, metal-free, stable, nontoxic, and pH sensitive, advanced functional nanoparticles (GlowDots), as alternatives to quantum dots, is reported here. Controlled cross-linking of bovine serum albumin resulted in facile formation of spherical nanoparticles of 35 nm in diameter with a sharp size distribution (±10 nm), which were then conjugated with specific dyes to produce white-emitting particles with tunable excitation wavelengths. Chemical novelty is that the particle size, size distribution, stability, surface chemistry, and emission properties are under full chemical control where the size and absorption/emission properties are independently tuned. Up to 100 dye molecules were attached to each particle, on an average, and hence, particles acquired strong absorption cross-sections as well as high brightness. White fluorescence of GlowDots is strongly sensitive to pH over a range of pH 2–11, and pH-induced emission changes are fully reversible. The particles readily entered HeLa cells and emission color depended on particle location in the live cells, which is most likely due to the local environment surrounding the particles. These are the very first reports of white-emitting advanced functional nanoparticles that are biodegradable, sensitive to pH, and amenable for live cell imaging to probe the subcellular compartments.

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.

In situ measurement of AlAs and GaAs refractive index dispersion at epitaxial growth temperature

In situ molecular beam epitaxy control of III–V optoelectronic device growth has been achieved by dynamic optical reflectometry with tunable excitation wavelength, through the use of a titanium:sapphire laser light source. This new multiwavelength reflectometry method was used to determine the values of the AlAs and GaAs refractive indices at growth temperature (600 °C). Index dispersion between 760 and 960 nm is presented and found to be in good agreement with the existing models.

Picosecond kinetics and transient spectra of pseudoisocyanine monomers and J-aggregates in aqueous solution

Bleaching of monomer and J-aggregate bands of pseudoisocyanine was observed using tunable excitation wavelengths. Both bleached bands decrease with (15 ± 3) ps. A residual J-band bleaching exists up to 1 ns. A new transient absorption in superposition with the J-band was round to consist of two bands with different time behaviour.