Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Summary

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Abstract

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Protocol

We describe a protocol of cell labeling using caged fluorescein, however, other photo-activatable dyes and proteins can be similarly used.

1. Injection of Caged Fluorescein

1. Prepare 5% stock solution (5mg caged-fluorescein / 100 μL 0.2M KCl) of Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic, Invitrogen, molecular probes, Carlsbad, CA, cat. no. D-3310). Aliquot and store in -20°C. Note that DMNB-caged fluorescein is sensitive to light and should be kept in the dark.
2. Prepare an injection trough made of 1% agarose (Sigma, St. Louis, MO, cat. no. A9539) as described in....

Discussion

Photo-activatable compounds are molecules whose function is masked until they are illuminated with a specific wavelength (usually UV), inducing a photochemical reaction that converts the molecules into a biologically or chemically active state. These probes provide very powerful tools in cell biology research, since the activation can be precisely controlled temporally and spatially by limiting their exposure to light.

The significant advantage of multi-photon microscopy is its relatively d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic)	Invitrogen	D-3310	molecular probes
Agarose for injection trough and coated plates	Sigma-Aldrich	A9539
Thin Wall Glass Capillaries with filament	World Precision Instruments, Inc.	TW100F-6
Micropipette puller	Sutter Instrument Co.	P-97
Microloader tip	Eppendorf	5242 956.003
Pneumatic picopump	World Precision Instruments, Inc.	PV820
Phenylthiourea (PTU)	Sigma-Aldrich	22290-9
Low melting point agarose for embryo mounting	Ultra Pure LMP agarose	16520100
Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments	Roche Group	11426338910
Fast Red	Roche Group	11496549001