Chapter 14 - Optimizing Fluorescence Excitation and Detection for Intravital Two-Photon Microscopy
Section snippets
Purpose
Two-photon excitation cross-sections are often measured by physics laboratories and can vary considerably depending on the method and laboratory. Therefore, it is imperative that biologists optimize the fluorescence excitation for the conditions under which they typically perform their experiments. The purpose of this chapter is to provide practical tips and guidelines for biologists to acquire excitation spectral fingerprints for intravital two-photon microscopy in deep tissue. The protocol
Theory
Two-photon microscopy provides an ideal platform to image dynamic biological processes in deep tissue of live animals with minimal perturbation to the underlying physiology (Helmchen & Denk, 2005; Zipfel, Williams, & Webb, 2003). Recent improvements in ultrafast laser technology have made it possible for biologists to operate two-photon microscopes without specialized training in laser physics. Practical guidelines are therefore needed to facilitate the uptake and application of two-photon
Equipments
Below is a list of the equipments needed to perform excitation lambda scans and the key points to look for.
Materials
Fluorescent reporter mice: There are a number of genetically encoded fluorescent reporter mice that can be used for two-photon microscopy. In this protocol, we will describe the two-photon excitation properties of green and red species of the optical highlighter KikGR in a novel genetically modified mouse line established by Kanagawa and Tomura (Tomura et al., manuscript in preparation).
RPMI
VetBond tissue glue
Silicone grease
Protocol
Duration Time Preparation 1 h Protocol 25–50 min Preparation Clean and sterilize surgical instruments. Caution Class 4 laser safety. Tip Laser power calibration curves can be performed in advance and stored until required for the excitation lambda scan.
See the flowchart in Fig. 1.
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