We developed a module for dual-output, dual-wavelength lasers that facilitates multiphoton imaging and spectroscopy experiments and enables hyperspectral imaging with spectral resolution up to 5 cm−1. High spectral resolution is achieved by employing spectral focusing. Specifically, two sets of grating pairs are used to control the chirps in each laser beam. In contrast with the approach that uses fixed-length glass rods, grating pairs allow matching the spectral resolution and the linewidths of the Raman lines of interest. To demonstrate the performance of the module, we report the results of spectral focusing CARS and SRS microscopy experiments for various test samples and Raman shifts. The developed module can be used for a variety of multimodal imaging and spectroscopy applications, such as single- and multi-color two-photon fluorescence, second harmonic generation, third harmonic generation, pump-probe, transient absorption, and others.
Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.
Liposomes play a relevant role in the biomedical field of drug delivery. The ability of these lipid vesicles to encapsulate and transport a variety of bioactive molecules has fostered their use in several therapeutic applications, from cancer treatments to the administration of drugs with antiviral activities. Size and uniformity are key parameters to take into consideration when preparing liposomes; these factors greatly influence their effectiveness in both in vitro and in vivo experiments. A popular technique employed to achieve the optimal liposome dimension (around 100 nm in diameter) and uniform size distribution is repetitive extrusion through a polycarbonate filter. We investigated two femtosecond laser direct writing techniques for the fabrication of three-dimensional filters within a microfluidics chip for liposomes extrusion. The miniaturization of the extrusion process in a microfluidic system is the first step toward a complete solution for lab-on-a-chip preparation of liposomes from vesicles self-assembly to optical characterization.
In recent years two-photon microscopy with fixed-wavelength has raised increasing interest in life-sciences: Two-photon (2P) absorption spectra of common dyes are broader than single-photon ones. Therefore, excitation of several dyes simultaneously with a single IR laser wavelength is feasible and could be seen as an advantage in 2P microscopy. We used pulsed fixed-wavelength infrared lasers with center wavelength at 1040 nm, for two-photon microscopy in a variety of biologically relevant samples, among these a mouse brain sample, a mouse artery (within the animal, acute preparation), and a preparation of mouse bladder. The 1040 nm laser proved to be efficient not only in exciting fluorescence from yellow fluorescent protein (YFP) and red fluorescent dyes, but also for second harmonic generation (SHG) signals from muscle tissue and collagen. With this work we demonstrate that economical, small-footprint fixedwavelength lasers can present an interesting alternative to tunable lasers that are commonly used in multiphoton microscopy.
In this work we describe a device that extends capabilities of multiphoton microscopes based on dual wavelength output femtosecond laser sources. CARS with 17cm-1 spectral resolution is experimentally demonstrated. Our approach is based on spectral focusing CARS. For pulse shaping of the pump and Stokes beams we utilize transmission gratings based stretcher. It allows the dispersion of the stretcher to be continuously adjusted in wide range. The best spectral resolution is achieved when the chirp rates in both pump and Stokes beam are matched. The device is automated. Any change in the beam path lengths due to the stretcher adjustment or wavelength tuning is compensated by the delay line. We incorporated into the device a computer controlled beam pointing stabilization system that compensates the beam pointing deviation due to dispersion in the system. High level of automation and computer control makes the operation of the device easy. We present CARS images of several samples that demonstrate high spectral resolution, high contrast and chemical selectivity.
Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.
We describe a stand alone CARS module allowing upgrade of a two-photon microscope with CARS modality. The
Stokes beam is generated in a commercially available photonic crystal fiber (PCF) using fraction of the power of
femtosecond excitation laser. The output of the fiber is optimized for broadband CARS at Stokes shifts in 2900cm-1
region. The spectral resolution in CARS signal is 50 cm-1. It is achieved by introducing a bandpass filter in the pump
beam. The timing between the pump and Stokes pulses is preset inside the module and can be varied. We demonstrate
utility of the device on examples of second harmonic, two-photon fluorescence and CARS images of several biological
and non-biological samples. We also present results of studies where we used CARS modality to monitor in real time the
process of fabrication of microstructures by two-photon polymerization.
We describe a modular ultrafast spectroscopy setup based on standard commercially available components that can
easily be configured to conduct multi-beam and multi-color multimodal pump-probe ultrafast experiments.
Multimodality of the setup is demonstrated on examples of transient absorption and different types of four-wave mixing
time resolved experiments in colloidal nanoparticles, neat liquids and solutions.
Two-photon polymerization (TPP) is an enabling technology that allows fast prototyping of parts with sub-100 nm
resolution. Due to its ability to fabricate microstructures with arbitrary three-dimensional geometries, TPP has been
employed in diverse fields such as photonics, microelectronics, microelectromechanical systems, and microfluidics.
However, no information is available to date that microscopically correlates the experimental conditions used in TPP
with the properties of the ultimate microstructure. We present a study where the distribution of polymer cross-linking in
three-dimensional microstructures fabricated by TPP is visualized by means of nonlinear microscopy. In particular,
coherent anti-Stokes Raman scattering (CARS) microscopy is employed to image polymer microstructures with
chemical specificity. The characterization of the microstructures based on the acquired images permits rational
optimization of the TPP process.
We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10 to 20 µm beneath the surface for both sources. The increased penetration depth at 1300 nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-µm excitation in nonlinear optical microscopy.
Ultrashort laser pulses can be used to create high precision incision in transparent and translucent tissue with minimal damage to adjacent tissue. These performance characteristics meet important surgical requirements in ophthalmology, where femtosecond laser flap creation is becoming a widely used refractive surgery procedure. We summarize clinical findings with femtosecond laser flaps as well as early experiments with other corneal surgical procedures such as corneal transplants. We also review laser-tissue interaction studies in the human sclera and their consequences for the treatment of glaucoma.
Femtosecond laser pulses focused inside liquid helium initiate an excitation sequence that leads to formation of molecular Rydberg states He2 detected by fluorescence spectroscopy. Unlike in the case of longer laser pulses, the excitations may be created in a controllable way, at light intensities below dielectric breakdown. The initial step is ionization of He atoms, as demonstrate by charge separation in external electric field. A sequence of the subsequent processes is proposed, which accounts for rapid production of He2, in less than 10 ns, observed by nanosecond time- resolved laser induced fluorescence following the excitation pulse. the lowest triplet state excimers He2(3a), probed in the latter experiment, are long-lived and survive in concentrations of the order of 1011-1012 cm-3 on a millisecond time scale. Femtosecond time- resolved spectroscopy was performed on He2* molecules in liquid He, using the pump-probe sequence He2*(3a) + 790 nm yields He2*(3c), He2*(3c) + 790 nm yields He2*(3f). The observed decay of the transient signals with characteristic time 3.5 ps is thought to be due to solvent motion corresponding to the relaxation of the liquid helium 'bubble' around the intermediate He2*(3c) state.
Understanding many-body dynamics on a molecular level is a major aim in condensed phase photodynamical research. Much can be learned about this general field through studies of molecular photodissociation in model systems, namely crystalline rare gas solids. The aim of this presentation is to illustrate this proposition by highlights drawn from a variety of related investigations. Under the title of photodissociation in solids, several related processes can be categorized: charge transfer induced 1 radiative dis sociation ,2 atomic photomobility,3 are examples.
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