In Vivo Two-Photon Laser Scanning Microscopy with Concurrent Plasma-Mediated Ablation Principles and Hardware Realization

Abstract

Many biological processes of current interest occur below the surface layers of accessible tissue. It is often the case that the surface layers cannot easily be removed without adversely affecting the physiology and function of the deeper layers. A variety of imaging techniques have been developed to perform sectioning deep to the surface using optical, electrical, and magnetic contrast agents and recording methods. Two-photon laser scanning microscopy (TPLSM) with ultrashort (i.e., order 100-fs) pulsed laser light provides optically sectioned images from depths of 500 microns or more below the surface in highly scattering brain tissue [1–5]. This method is unique in that it can provide images with submicrometer lateral resolution and micrometer axial resolution on the millisecond time-scale [6], as is required for the study of many dynamic biological processes [1,7–11].A strength of TPLSM is the ease with which this technique may be combined with electrical measurements of physiological parameters and with other optical techniques. These include one-photon uncaging with gated continuous laser light, two-photon uncaging with ultrashort pulsed laser light and, of particular interest in this work, plasma-mediated ablation through the nonlinear absorption of amplified ultrashort laser pulses.A nonlinear interaction that is of particular relevance for the ablation of submicrometer volumes of neuronal [12] and vascular [13] tissue deep in the brain is plasma-mediated ablation [14–16]. Here, the optical field of the ultrashort pulse leads to a high rate of tunneling by electrons to form an electron plasma [17]. The density of this plasma rapidly builds up by virtue of field-driven collisions between free electrons and molecules, while the thickness of the plasma approaches a value that is much less than the depth of the focal volume. The absorption of laser light by this thin layer leads to highly localized ablation and the explosive removal of material. One critical feature of plasma-mediated ablation is that light outside of the focal volume has little detrimental effect on the specimens; this limits the thermal build-up and ensuing collateral damage in the targeted tissue.This chapter provides an overview of the technical considerations relevant to the design and assembly of a system for TPLSM that is particularly suitable for in vivo imaging and histology, either alone or in combination with plasma-mediated ablation. It extends an earlier technical presentation [18] and complements other designs of upright systems [19–21]. The conversion of commercially available confocal microscopes for use in TPLSM, rather than the de novo construction of a system, have been described [22–26]. Commercial systems for TPLSM are available from Prairie (Ultima; www.prairie-technologies.com ) and Leica (TPS MP; www.leica.com ), and it is likely that the commercial market will continue to mature. Commercial systems that incorporate amplified ultrashort laser pulses do not exist at this time, although it should be simple to adapt current systems to support two beams.