The advantage of this approach is the complete absence of any micromanipulation of optical elements by the end-user with the significant possibility of automating the measurements. The rationale of the chip for optical uncaging is illustrated in Figure 1(a). It is based on a 500-��m-thick fused silica substrate with a 2 mm-square well in the center, representing the region for the cell culture.Figure 1.(a) Rationale of the chip for optical uncaging. Several optical waveguides drive excitation light in to the cell culture region (central well). The culture will take place on a thin glass, allowing high quality optical imaging from the bottom. (b) Top …This well is closed by a 180-��m-thick borosilicate coverslip, previously glued on the bottom surface of the chip.
In this way, the culture is grown on a thin glass allowing to achieve high quality optical imaging with oil-immersion objectives. The square well is then optically addressed from the sides by several optical waveguides, each bonded to an optical fiber which is in turn connected to the excitation laser source for optical uncaging in different positions of the well. In the following sections we will describe how such device has been fabricated by using only femtosecond lasers as microfabrication tools. In addition, preliminary characterization of its operation will be given for monolithic optical uncaging.2.?Chip fabricationThe experimental setup used for the fabrication of the device is shown schematically in Figure 2.
It is based on femtosecond laser radiation focused by a microscope objective inside the fused silica substrate (Lithosil, Schott AG, Germany), which is suitably moved by 3D computer-controlled translation stages (Physik Instrumente, Germany). The femtosecond laser micromachining Carfilzomib capabilities used in the present work are the fabrication of microcuts through the glass slab and the direct writing of optical waveguides (see inset of Figure 2). Two different lasers were used, one for fabricating the microcuts and a second Cilengitide one for writing the optical waveguides. For the microcutting we used a diodepumped cavity-dumped Yb:KYW laser, generating 350-fs pulses at 1,030-nm wavelength with pulse energies up to 1 ��J at a repetition rate of 600 kHz. The waveguide fabrication was performed by means of a regeneratively amplified Ti:sapphire laser generating 150-fs, 500-��J pulses at 1 kHz and 800 nm.