ABSTRACT
Appendix A: Parts and Vendors for the TPLSM .................................................... 109 Mechanical .................................................................................................... 109 Optical .......................................................................................................... 109 Scanners ........................................................................................................ 109 Photomultiplier Tubes ................................................................................... 110 Electronics .................................................................................................... 110 Laser and Ancillary Equipment .................................................................... 110
Appendix B: Basics of Interferometric Autocorrelation ........................................ 111 Acknowledgments .................................................................................................. 112 References .............................................................................................................. 112
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 amplied ultrashort laser pulses.
