Search Results (16)

A two-photon fluorescence microscope employing line-shaped illumination is presented. This type of excitation is commonly expected to bring about the degradation of axial resolution because of the weaker focusing of the illuminating beam in just one direction. On the basis of a detailed theoretical investigation of the beam shaping performed by cylindrical lenses when inserted in conventional point-scanning systems, we design and implement a microscope set-up making use of readily available optical components. The experimental results show that the proper choice and arrangement of the cylindrical lenses that we have devised is able to preserve the optical-sectioning capability at the video-rate acquisition speed. Full article

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti–sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5–1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging. Full article

The coupling of the spin-orbit angular momentum of photons in a focused spatial region can enhance the localized optical field’s chirality. In this paper, a scheme for producing a superchiral optical field in a 4$\pi$ microscopic system is presented by tightly focusing two counter-propagating spiral wavefronts. We calculate the optical forces and torques exerted on a chiral dipole by the chiral light field and reveal the chiral forces by combining the light field and dipoles. Results indicate that, in addition to the general optical force, particles’ motion would be affected by a chiral force that is directly related to the particle chirality. This chiral mechanical effect experienced by the electromagnetic dipoles excited on a chiral particle could be characterized by the behaviors of chirality density and flux, which are, respectively, associated with the reactive and dissipative components of the chiral forces. This work facilitates the advancement of optical separation and manipulation techniques for chiral particles. Full article

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution. Full article

The evolution of laser technologies and the invention of ultrashort laser pulses have resulted in a sharp jump in laser applications in life sciences. Developmental biology is no exception. The unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of transparent material without disrupting the surrounding tissues makes ultrashort lasers a versatile tool for precise microsurgery of cells and subcellular components within structurally complex and fragile specimens like embryos as well as for high-resolution imaging of embryonic processes and developmental mechanisms. Here, we present an overview of recent applications of ultrashort lasers in developmental biology, including techniques of noncontact laser-assisted microsurgery of preimplantation mammalian embryos for oocyte/blastomere enucleation and embryonic cell fusion, as well as techniques of optical transfection and injection for targeted delivery of biomolecules into living embryos and laser-mediated microsurgery of externally developing embryos. Possible applications of ultrashort laser pulses for use in Assisted Reproductive Technologies are also highlighted. Moreover, we discuss various nonlinear optical microscopy techniques (two-photon excited fluorescence, second and third harmonic generation, and coherent Raman scattering) and their application for label-free non-invasive imaging of embryos in their unperturbed state or post-laser-induced modifications. Full article

To improve the detection sensitivity of a porous silicon optical biosensor in the real-time detection of biomolecules, a non-spectral porous silicon optical biosensor technology, based on dual-signal light detection, is proposed. Double-light detection is a combination of refractive index change detection and fluorescence change detection. It uses quantum dots to label probe molecules to detect target molecules. In the double-signal-light detection method, the first detection-signal light is the detection light that is reflected from the surface of the porous silicon Bragg mirror. The wavelength of the detection light is the same as the wavelength of the photonic band gap edge of the porous silicon Bragg mirror. CdSe/ZnS quantum dots are used to label the probe DNA and hybridize it with the target DNA molecules in the pores of porous silicon to improve its effective refractive index and enhance the detection-reflection light. The second detection-signal light is fluorescence, which is generated by the quantum dots in the reactant that are excited by light of a certain wavelength. The Bragg mirror structure further enhances the fluorescence signal. A digital microscope is used to simultaneously receive the digital image of two kinds of signal light superimposed on the surface of porous silicon, and the corresponding algorithm is used to calculate the change in the average grey value before and after the hybridization reaction to calculate the concentration of the DNA molecules. The detection limit of the DNA molecules was 0.42 pM. This method can not only detect target DNA by hybridization, but also detect antigen by immune reaction or parallel biochip detection for a porous silicon biosensor. Full article

Dark field scattering microscopy can create large hyperspectral data sets that contain a wealth of information on the properties and the molecular environment of noble metal nanoparticles. For a quick screening of samples of microscopic dimensions that contain many different types of plasmonic nanostructures, we propose a multivariate analysis of data sets of thousands to several hundreds of thousands of scattering spectra. By using non-negative matrix factorization for decomposing the spectra, components are identified that represent individual plasmon resonances and relative contributions of these resonances to particular microscopic focal volumes in the mapping data sets. Using data from silver and gold nanoparticles in the presence of different molecules, including gold nanoparticle-protein agglomerates or silver nanoparticles forming aggregates in the presence of acrylamide, plasmonic properties are observed that differ from those of the original nanoparticles. For the case of acrylamide, we show that the plasmon resonances of the silver nanoparticles are ideally suited to support surface enhanced Raman scattering (SERS) and the two-photon excited process of surface enhanced hyper Raman scattering (SEHRS). Both vibrational tools give complementary information on the in situ formed polyacrylamide and the molecular composition at the nanoparticle surface. Full article

When performing in vivo imaging of live samples, it is a big challenge to penetrate thick tissues while still maintaining high resolution and a large field of view because of the sample-induced aberrations. These requirements can be met by combining the benefits of two-photon excitation, beam modulation and adaptive optics in an illumination path. However, the relationship between aberrations and the performance of such a microscopy system has never been systematically and comprehensively assessed. Here, two-photon Gaussian and Bessel beams are modulated as illumination beams, and how aberrations affect the thickness of the illumination beams is evaluated. It is found that the thickness variation is highly related to the azimuthal order of Zernike modes. The thickness of the two-photon Gaussian beam is more sensitive to Zernike modes with lower azimuthal order, while the thickness of the two-photon Bessel beam is more sensitive to the higher-azimuthal-order Zernike modes. So, it is necessary to design a new strategy to correct aberrations according to the effects of different Zernike modes in order to maximize the correction capability of correctors and reduce the correction errors for those insensitive Zernike modes. These results may provide important guidance for the design and evaluation of adaptive optical systems in a two-photon excitation microscope. Full article

Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues. Full article

For the first time, the time-resolved two-photon excited autophosphorescence of non-labeled biological specimens was investigated by phosphoresce lifetime imaging with microscopic spatial resolution. A modified multiphoton tomograph was employed to record both photoluminescence contributions, autofluorescence and autophosphorescence, simultaneously, induced by two-photon excitation using an 80 MHz near infrared femtosecond-pulse-laser scanning beam, an acousto-optic modulator, and a time-correlated single-photon counting module for lifetime measurements from the picosecond to the microsecond range. In particular, the two-photon-excited luminescence of thermally altered bones was imaged. A strong dependence of the phosphorescence intensity on exposure temperature, with a maximum emission for an exposure temperature of approximately 600 °C was observed. Furthermore, the phosphorescence lifetime data indicated a bi-exponential signal decay with both a faster few µs decay time in the range of 3–10 µs and a slower one in the range of 30–60 µs. The recording of fluorescence and phosphorescence allowed deriving the relative signal proportion as an unbiased measure of the temperature dependence. The measurements on thermally altered bones are of particular interest for application to forensic and archeological investigations. Full article

The development of lanthanide-based luminescent probes with a long emission lifetime has the potential to revolutionize imaging-based diagnostic techniques. By a rational design strategy taking advantage of computational predictions, a novel, water-soluble Eu³⁺ complex from a cyclen-based ligand bearing 1,3-disubstituted benzo[h]isoquinoline arms was realized. The ligand has been obtained overcoming the lack of reactivity of position 3 of the isoquinoline moiety. Notably, steric hindrance of the heteroaromatic chromophore allowed selective and stoichiometry-controlled insertion of two or three antennas on the cyclen platform without any protection strategy. The complex bears a fourth heptanoic arm for easy conjugation to biomolecules. This new chromophore allowed the sensitization of the metal center either with one or two photons excitation. The suitability as a luminescent bioprobe was validated by imaging BMI1 oncomarker in lung carcinoma cells following an established immunofluorescence approach. The use of a conventional epifluorescence microscope equipped with a linear structured illumination module disclosed a simple and inexpensive way to image confocally Ln-bioprobes by single photon excitation in the 350–400 nm window, where ordinary confocal systems have no excitation sources. Full article

Special integrated photonic surface structures composed of a dielectric semicircle ridge and a dielectric block placed on a metal substrate are proposed for the investigation of surface plasmon polariton (SPP) reflection and transmission effects. A fabrication method called microscope projection photolithography was employed for the preparation of the structures. Leakage radiation microscopy was applied for the excitation and observation of surface plasmon polaritons (SPPs). It was observed that SPPs exhibit a remarkable decrease in intensity when impinging onto the rectangular dielectric block. Nevertheless, the transmitted wave out of the dielectric block was always observable. The propagation behavior of both the reflected waves at two boundaries (air/dielectric and dielectric/air) and the transmitted wave inside the dielectric block were demonstrated for different SPP incident conditions. The variation of the angles of reflection and transmission with respect to the incident angle was analytically and experimentally investigated. An agreement between the calculated results and the experimental results was obtained. Our findings might allow for novel applications in sensing and analytics once the structures will be functionalized. Full article

Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community. Full article

Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications. Full article

An overview is given of the molecular quantum electrodynamical (QED) theory of resonance energy transfer (RET). In this quantized radiation field description, RET arises from the exchange of a single virtual photon between excited donor and unexcited acceptor species. Diagrammatic time-dependent perturbation theory is employed to calculate the transfer matrix element, from which the migration rate is obtained via the Fermi golden rule. Rate formulae for oriented and isotropic systems hold for all pair separation distances, R, beyond wave function overlap. The two well-known mechanisms associated with migration of energy, namely the R⁻⁶ radiationless transfer rate due to Förster and the R⁻² radiative exchange, correspond to near- and far-zone asymptotes of the general result. Discriminatory pair transfer rates are also presented. The influence of an environment is accounted for by invoking the polariton, which mediates exchange and by introducing a complex refractive index to describe local field and screening effects. This macroscopic treatment is compared and contrasted with a microscopic analysis in which the role of a neutral, polarizable and passive third-particle in mediating transfer of energy is considered. Three possible coupling mechanisms arise, each requiring summation over 24 time-ordered diagrams at fourth-order of perturbation theory with the total rate being a sum of two- and various three-body terms. Full article