Gabriele Marchi

Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses

Laser-induced cell transfer has been developed in recent years for the flexible and gentle printing of cells. Because of the high transfer rates and the superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of laser based cell printing. Therefore alternative approaches using deep UV laser sources and protein based acceptor films for energy absorption, have been introduced. Nevertheless, deep UV radiation can introduce DNA double strand breaks, thereby imposing the risk of carcinogenesis. Here we present a method for the laser-induced transfer of hydrogels and mammalian cells, which neither requires any sacrificial material for energy absorption, nor the use of UV lasers. Instead, we focus a near infrared femtosecond (fs) laser pulse (λ = 1030 nm, 450 fs) directly underneath a thin cell layer, suspended on top of a hydrogel reservoir, to induce a rapidly expanding cavitation bubble in the gel, which generates a jet of material, transferring cells and hydrogel from the gel/cell reservoir to an acceptor stage. By controlling laser pulse energy, well-defined cell-laden droplets can be transferred with high spatial resolution. The transferred human (SCP1) and murine (B16F1) cells show high survival rates, and good cell viability. Time laps microscopy reveals unaffected cell behavior including normal cell proliferation.

Force and temperature characteristics of a fs-laser machined locally micro-structured FBG

A locally micro-structured fiber Bragg grating (LMFBG) was manufactured by forming a circumferential groove in the middle of a type I fiber Bragg grating (FBG). The groove was directly ablated using a fs-laser and had a length of 86μm, a depth of 27μm and steep side walls. Due to the precisely machined geometry of the structure the reflection spectra can be accurately described with a fairly simple theoretical model. At several constant temperatures in the range from 5°C to 45°C this structure was exposed to various compressive loads in the range from 0N to -1.42N. Here the force and temperature sensitivity of the LMFBG are presented. This structure can be used for miniaturized compressive force sensing at variable temperatures, which is of particular interest for many bio-medical applications.

Femtosecond Laser Machined Micro-Structured Fiber Bragg Grating for Simultaneous Temperature and Force Measurements

A new fabrication method for a locally micro-structured fiber Bragg grating (LMFBG) is proposed and demonstrated. With this new type of LMFBG, simultaneous sensing of compressive force and temperature is possible. The LMFBG consists of a circumferential groove of ~86 μm length and ~27 μm depth in the middle of a type I FBG. Direct femtosecond (fs) laser ablation was applied to manufacture the groove with constant depth and steep side walls. Axial compressive force acting on the LMFBG results in the occurrence of a pass band in the reflection spectrum due to a stress-induced phase shift in the structured part of the LMFBG. Temperature changes instead induce shifts of the whole spectrum without influencing its shape. A theoretical model that can describe the LMFBG reflection spectra is presented. It consists of a product of three transfer matrices for uniform FBGs with external force, temperature, and the optical power used to interrogate the LMFBG as free parameters. The model-based theoretical line shapes showed very good agreements with measured LMFBG spectra at various force and temperature values in the ranges of 5-45 °C and 0 to -1.44 N. The ability to describe the LMFGB as a combination of three uniform FBGs is due to the precise shape of the engraved structure which is a unique feature of fs laser machined LMFBGs. The difference between the LMFBG-based force and temperature values and the reference values are <;0.03 N for force and <;2.0 K for temperature.

Laser structured fibre Bragg gratings as enhanced force sensors

The production and characterisation of a micro-structured FBG force sensor is described. Employing femtosecond laser micro machinery a circumferential ditch of about 30 μm depth and 40 μm width is engraved in the clad of an optical fibre at the centre of a 3 mm long type I fibre Bragg grating (FBG). The purpose of the structure is the enhancement of the force sensitivity characteristics for the measurement of sub-mN forces. Phase-shift spectra occur when axial stress is applied to the fibre. Exploiting this phenomenon experimental tests show a 10% improvement in the sensitivity performance when compared to an unstructured FBG.

Cartilage microindentation using cylindrical and spherical optical fiber indenters with integrated Bragg gratings as force sensors

Fiber optic microindentation sensors that have the potential to be integrated into arthroscopic instruments and to allow localizing degraded articular cartilage are presented in this paper. The indenters consist of optical fibers with integrated Bragg gratings as force sensors. In a basic configuration, the tip of the fiber optic indenter consists of a cleaved fiber end, forming a cylindrical flat punch indenter geometry. When using this indenter geometry, high stresses at the edges of the cylinder are present, which can disrupt the tissue structure. This is avoided with an improved version of the indenter. A spherical indenter tip that is formed by melting the end of the glass fiber. The spherical fiber tip shows the additional advantage of strongly reducing reflections from the fiber end. This allows a reduction of the length of the fiber optic sensor element from 65 mm of the flat punch type to 27 mm of the spherical punch. In order to compare the performance of both indenter types, in vitro stress-relaxation indentation experiments were performed on bovine articular cartilage with both indenter types, to assess biomechanical properties of bovine articular cartilage. For indentation depths between 60 μm and 300 μm, the measurements with both indenter types agreed very well with each other. This shows that both indenter geometries are suitable for microindentation measuremnts . The spherical indenter however has the additional advantage that it minimizes the risk to damage the surface of the tissue and has less than half dimensions than the flat indenter.

Characterization of bovine cartilage by fiber Bragg grating-based stress relaxation measurements

A fiber-based device for testing mechanical properties of cartilage is presented within this study. The measurement principle is based on stepwise indentation into the tissue and observing of corresponding relaxation of the stress. The indenter tip is constituted of a cleaved optical fiber that includes a fiber Bragg grating which is used as the force sensor. Stress relaxation measurements at 25 different positions on a healthy bovine cartilage sample were performed to assess the behavior of healthy cartilage. For each indentation step a good agreement was found with a viscoelastic model that included two time constants. The model parameters showed low variability and a clear dependence with indentation depth. The parameters can be used as reference values for discriminating healthy and degenerated cartilage.

Laser induced forward transfer of living ceils using femtosecond laser puises

Laser induced forward transfer (LIFT) has been used in recent years for the rapid 3D-bioprinting of cells. Because of the high transfer rates and superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of LIFT based cell printing [1].

Stress tuneable phase shifts of femtosecond-laser microstructured FBG for indentation measurements of biological tissue: experimental and theoretical investigation

An automated fs-laser machining procedure was developed to engrave circumferential grooves into the cladding of optical fibres. The grooves are positioned centrally to type I fibre Bragg gratings (FBG) and form locally micro structured FBGs. The grooves realized so far were ~30μm deep and were 48 μm to 200 μm long. These devices show the occurrence of a phase shifted spectrum when axial stress is applied. It is shown in this paper that this property can be used to achieve higher force sensitivities when compared to conventional FBGs. These devices are advantageous for the investigation of tissue by indentation-type elasticity measurements. An experimental and theoretical investigation of the dependence of the force sensitivity on the length of the structure is reported.