Chapter 14 - Optimizing Fluorescence Excitation and Detection for Intravital Two-Photon Microscopy

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Abstract

Commercial two-photon microscope systems incorporating turnkey ultrafast lasers have made the technology more user-friendly and accessible to nonspecialized biology laboratories. This has been accompanied by the development of an exciting range of new fluorescent proteins and dyes such as near-infra-red fluorescent proteins and optical highlighters. However, the two-photon absorption properties of these fluorescent molecules are not widely available and cannot be reliably predicted from their single photon absorption spectra. Furthermore, the spectral characteristics of fluorescent proteins in vivo can be affected by the local environment and light scattering by deep tissue and can vary greatly from one laboratory to the next. Here, we describe a simple protocol for determining the two-photon excitation peaks of fluorescent reporters that can be tailored to the relevant tissue samples to suit the imaging goals of individual biological laboratories.

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

DurationTime
Preparation1 h
Protocol25–50 min
PreparationClean and sterilize surgical instruments.
CautionClass 4 laser safety.
TipLaser 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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