All-optical, in-situ histology of neuronal tissue with femtosecond laser pulses

Goal We describe the application of femtosecond laser pulses to image and ablate neuronal tissue for the purpose of automated histology. The histology is accomplished in-situ by serial two-photon imaging of labeled tissue and removal of the imaged tissue with amplified, approximately 100 fs duration pulses, as illustrated schematically in figure 1A. The ablation of tissue with femtosecond, infrared laser pulses requires large optical fluences, in excess of 1 J/cm 2. In the past, such high fluences have been achieved with commercially available ~ 100 MHz laser oscillators with ~ 100 fs pulse duration, whose 1 to 10 nJ pulse energies can be focused to a less than 1 mm 2 spot size with high numerical aperture (i.e., tight focusing) objectives. Such beams have been used for fine-scale ablation of subcellular structures (Tirlapur and Konig, 2002). More typically, femtosecond laser pulses have been amplified with an optical amplifier to obtain pulses with comparable duration and microjoule energies, albeit at repetition rates of only 1 to 10 kHz. These amplified pulses have been used to ablate a wide variety of tissues, In this chapter, we describe the coupling of femtosecond ablation with two photon laser scanning microscopy (TPLSM)