Ruben Zadoyan

Two-photon polymerization with variable repetition rate bursts of femtosecond laser pulses

We describe fabrication of microstructures by two-photon polymerization using bursts of femtosecond laser pulses. With the aid of an acousto-optic modulator driven by a function generator, two-photon polymerization is performed at variable burst repetition rates. We investigate how the time between the bursts of laser pulses influences the ultimate dimensions of lines written in a photosensitive resin. We observe that when using the same laser fluence, polymer lines fabricated at different burst repetition rates have different dimensions. In particular, the widths of two-photon polymerized lines become smaller with decreasing burst repetition rates. Based on the thermal properties of the resin and experimental writing conditions, we attribute this effect to localized heat accumulation.

Chemical mapping of three-dimensional microstructures fabricated by two-photon polymerization using CARS microscopy

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.

Microfabrication of three-dimensional filters for liposome extrusion

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.

Ophthalmic applications of ultrashort pulsed lasers

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.

Compact fixed wavelength femtosecond oscillators for multi-photon imaging

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.

High-resolution 3D printing for drug delivery

Liposomes are spherical particles comprising single or multiple concentric lipid bilayers commonly referred to as lamellae.1 Liposomes are used for drug delivery to specific sites in the body, and are usually fabricated by hydrating phospholipid films. Following hydration, liposomes can be loaded with reagents of interest, surface functionalized for targeted drug delivery, and tailored in size for more efficient delivery. The effectiveness of nanotherapeutic delivery strongly depends on particle size.2 The immune system can rapidly remove colloidal drug delivery particles by activating the complement system and by cells of the mononuclear phagocyte system. To evade such immune responses, nanoscale particles are required. Furthermore, the leading strategy for delivery to tumor sites is through the enhanced permeability and retention effect. That is, the vascular network surrounding a tumor site is known to be highly leaky because of enhanced requirements for oxygen and nutrients by the tumor. As a result, gaps between the thin layer of cells lining the interior surface of blood vessels can reach a couple of micrometers in size. Thus, nanoparticles are able to preferentially accumulate at tumor sites. An automated nanoparticle preparation platform— encompassing synthesis, functionalization, loading, and characterization in a total analytical system—would benefit drug delivery by liposomal nanocarriers.3 Miniaturizing such a device would indeed also improve performance in several ways from faster response times to reduced reagent consumption. Furthermore, such a device would expedite and stimulate the study of several aspects of liposome preparation and activity in both academic and industrial settings where fast prototyping is required. We have investigated the use of femtosecond laser direct writing for just such a device. In particular, we considered the advantages and disadvantages of two-photon polymerization (TPP) and femtosecond laser irradiation followed by chemical etching (FLICE).4, 5 Figure 1. Reproduction of Michelangelo’s David fabricated by two-photon polymerization (TPP).

Multimodal microscopy with high resolution spectral focusing CARS

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.

CARS module for multimodal 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.