Two-photon microscopy plays a significant role in biological applications, as it enables non-invasive, high-resolution imaging of thick tissue samples. This paper begins by elucidating fundamental concepts of two-photon microscopy to facilitate a deeper understanding of its luminescent mechanism. Subsequently, we provide a comprehensive overview of the imaging system, encompassing the light source, scanning unit, fluorescence collection, and detection components. As two-photon microscopy continues to evolve, We undertake assessments of cutting-edge optical instrumentation, emphasizing novel approaches for imaging across wider fields of view, at higher speeds, and with increased volumetric imaging capabilities. Furthermore, we summarize and compare the technical specifications of advanced two-photon imaging. Following this, the manuscript showcases the transformative applications of these innovative technologies in biological imaging, providing further evidence of the indispensable role of two-photon microscopy in biomedical imaging research.
SignificanceTwo-photon fluorescence microscopy (TPFM) excited by Gaussian beams requires axial tomographic scanning for three-dimensional (3D) volumetric imaging, which is a time-consuming process, and the slow imaging speed hinders its application for in vivo brain imaging. The Bessel focus, characterized by an extended depth of focus and constant resolution, facilitates the projection of a 3D volume onto a two-dimensional image, which significantly enhances the speed of volumetric imaging.AimWe aimed to demonstrate the ability of a TPFM with a sidelobe-free Bessel beam to provide a promising tool for research in live biological specimens.ApproachComparative in vivo imaging was conducted in live mouse brains and transgenic zebrafish to evaluate the performance of TPFM and Bessel-beam-based TPFM. Additionally, an image-difference method utilizing zeroth-order and third-order Bessel beams was introduced to effectively suppress background interference introduced by sidelobes.ResultsIn comparison with traditional TPFM, the Bessel-beams-based TPFM demonstrated a 30-fold increase in imaging throughput and speed. Furthermore, the effectiveness of the image-difference method was validated in live biological specimens, resulting in a substantial enhancement of image contrast. Importantly, our TPFM with a sidelobe-free Bessel beam exhibited robustness against axial displacements, a feature of considerable value for in vivo experiments.ConclusionsWe achieved rapid, high-contrast, and robust volumetric imaging of the vasculature in live mouse brains and transgenic zebrafish using our TPFM with a sidelobe-free Bessel beam.
Plasmonic nanoparticles have been widely used as substrates in surface-enhanced Raman scattering (SERS) due to their unique optical properties known as localized surface plasmon resonance (LSPR). In recent years, these nanomaterials have attracted much attention for their applications in chemical sensing, biosensing and imaging. In this study, we have synthesized Au@Ag core-shell nanoparticles with 1,4-benzenedithiol (1,4-BDT) sandwiched in between the Au-Ag gap of the core-shell structure, where the Raman signal from the 1,4-BDT molecules were greatly enhanced. This gap- enhanced Raman scattering signal was then used as an internal reference Raman signal. For this structure, detailed parameters, such as the core size and shape, shell thickness, were studied and optimized. Our results showed that the Raman intensity of 1,4-BDT would first increase to a maximum with the increasement in the thickness of the silver shell, while further increasement in the shell thickness would cause the SERS intensity to drop. We have then optimized the Raman tagged Au@Ag core-shell structure to obtain a probe with a stable reference Raman signal. These probes were then used for the trace detection of the pesticide of thiram through SERS. Using the reference peaks from 1,4-BDT, quantitative measurements of thiram were obtained with good linearity and reproducibility. Furthermore, due to the enhancement of the SERS effect, our probes were able to detect the pesticide at a low level of ~10-7 M at 559 cm-1 . In addition, combining the optimized core-shell Au@Ag nanoparticles and other metallic nanostructures would further enhance the detection sensitivity. These results show that SERS based on Au@1,4-BDT@Ag nanostructures could be a promising tool for chemical trace analysis.
Photothermal therapy is a tumor target therapy, which can induce temperature increase by laser in tumor tissue to eliminate cancer cells, without affecting surrounding healthy tissue. The temperature distribution in the tumor tissue directly determine the therapeutic efficacy. Therefore, real-time temperature monitoring is particularly important during photothermal therapy. However, accurate real-time temperature measurements in deep tissue still face many obstacles. This paper shows the feasibility of photoacoustic technology for real-time temperature monitoring with or without nanomaterials as photoacoustic contrast agents.
As a near-infrared photosensitizer, IR780 is a promising agent for photodynamic therapy. However, its clinical application was limited by its poor aqueous solubility and chemical stability. Therefore, the purpose of this study was to prepare IR780 encapsulated nanoemulsion (IR780-NE) to enhance the solubility and chemical stability of IR780, using a low-energy emulsion inversion point (EIP) method, which could avoid complex procedure and IR780 degradation during preparation process. The IR780-NE presented a homogeneous and clear appearance, as well as a mean droplet diameter of 38.17 ± 3.59 nm, as determined by dynamic light scattering. The IR780-NE greatly enhanced the chemical stability of IR780, with more than 80% IR780 retained after storage at 25 °C for 4 days. The IR780-NE also showed satisfactory physical stability, with no alteration of mean droplet diameter during storage at 25 °C for 4 days or 4 °C for 30 days. The zeta potential of the IR780-NE was nearly zero (-0.27 ± 2.06 mV), and the ultraviolet-visible light absorption spectrum showed that the near-infrared light absorbing ability of IR780 was not influenced. In conclusion, the IR780-NE is a potential delivery system for the application of IR780 in biological systems.
In this paper, we proposed a photoacoustic (PA) method for detecting liquid viscosity based on frequency-resolved measurement. A negative correlation was investigated theoretically between the liquid viscosity and the full width at half maximum (FWHM) of the PA frequency spectrum. To test the feasibility of this method, water mixed with different concentrations of ink and glycerol was measured. The results indicate that liquids with higher viscosity will lead to a higher FWHM reduction in the PA frequency spectrum and the frequency spectrum was independent from the absorption coefficient of the liquid, which demonstrate that this technique has future potential clinical applications for monitoring the viscosity changes in subcutaneous microvasculature.
In this work, gold nanoparticles coated with CTAB were used as probes for studying the interactions between nanoparticles and cells. The dark field scattering spectra from cells were obtained at different time points after they were treated with gold nanoparticles. By analyzing the results, we find that the dark field scattering spectrum changes at different time points, which is of certain significance in studying the interactions between cells and gold nanoparticles.
Two-photon excitation fluorescence imaging (TPEFI) is widely used in biomedicine because it has advantages of low cytotoxicity, high resolution and deep imaging depth. In this experiment, OVCAR-3 ovarian cancer cells were labelled with the squaraine dye and detected by confocal laser scanning microscope ( Leica TCS SP8 )for single-photon and two-photon imaging. The results showed that the squaraine dye can be used well to two-photon imaging.
We present our recent work on the applications of fluorescence lifetime imaging microscopy(FLIM), including the monitoring of macromolecule dynamic changes in the nucleolar compartments and the auxiliary diagnosis of H and E-stained sections. We demonstrated the capability of FLIM to measure protein concentration in the specific cellular compartments in live cells. We proposed to use FLIM to monitor changes in intracellular protein concentration caused by various factors e.g. cell cycle progression, drug treatment etc. In the future, FLIM technology is expected to be combined with super-resolution optical imaging. FLIM with molecular resolution will have the potential to serve as a powerful tool for discovering new phenomena and revealing new mechanisms in biomedical research, which will effectively promote the development of life science.
We report the observation of a supercontinuum (SC) in aqueous CdTe/ZnSe quantum dots (QDs) pumped by 800-nm femtosecond laser pulses based on simulations and experiments. For an average nanocrystal of 5.5 nm in size and a weight concentration of 5 mg/mL, the SC generated with a low-input energy range exhibits high optical to optical conversion efficiency. The measured SC spectrum exhibits spectral broadening that is significantly influenced by the nonlinear coefficient of the QDs. This nanoparticles-based SC effect may have special applications for optical coherence imaging and ultrashort pulse compression.
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