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.
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.
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
2. Embryo Mounting
3. Photoactivation
4. Detection of the Uncaged Fluorescein
5. Fluorescent Visualization of the Uncaged Area
6. Solutions
100 x E3 | 174mM NaCl, 21mM KCl, 12mM MgSO4, 18mM Ca(NO3), 15mM HEPES, pH=7.4. Sterilize by filtration and store at 4°C |
20 x E2 | 0.3M NaCl, 10mM KCl, 20mM CaCl2*2HOH, 20mM MgSO4*7HOH, 3mM KH2PO4, 0.8mM Na2HPO4*2HOH, filtrate and store in room temperature |
1 x E2 | Dilute 20 x E2 in double distilled water and add freshly made NaHCO3 (to final concentration of 0.7mM), Penicillin and Streptomycin (dilute pen strep solution to a final concentration of 1 x which contains 100U of penicillin and 100mg streptomycin per mL (Invitrogen, Carlsbad, CA, cat. no. 15140-122)) |
PBST | 0.1% TWEEN-20 in PBS |
PBSTr | 0.3% Triton in PBS |
Blocking solution | 10% BSA, 0.3% Triton, 1% DMSO in PBS |
TNT | 100mM Tris-HCl pH-7.5, 150mM NaCl, 0.5% TWEEN-20 in double distilled water |
Prestain solution | 100mM Tris-HCl pH-8.2, 0.4M NaCl, 0.1% TWEEN-20 in double distilled water |
7. Representative Results
An example of the use of two-photon microscopy to photoactivate agents in a living zebrafish embryo is presented. Using a similar approach we previously traced the lineage of the photoactivated labeled neural progenitors in the zebrafish brain2,3. Figure 1 shows photoactivation of a caged fluorescein conjugated tracer dye in anterior neural plate of zebrafish embryos carrying a neurog1::gfp reporter transgene, which served as a live intrinsic landmark.
Embryos were injected with caged fluorescein at the one cell stage and embedded in agarose at bud stage. At 3- 5-somite stage the spatial coordinates were measured in the neurog1::gfp transgenic embryo to determine the region of interest (ROI) for uncaging (Figure 1A, A’). We uncaged the fluorescein lineage tracer in a specific domain of the neural plate (Figure 1B, B’). Subsequently, the fate of this given labeled neural progenitor subdomain was determined at prim5 (24 hours post fertilization) stage by immunostaining of the uncaged fluorescein (Figure 1C, C’).
The extent of two-photon laser uncaging, i.e. the intensity of the uncaged fluorescence and the thickness of the photoactivated domain (z-span), can be controlled by adjusting two parameters: laser intensity and duration. The latter can be controlled by adjustment of the iteration number and scan speed (see part 3, step 9; Russek-Blum, 2009). In the example shown in Figure 1, we used relative laser power of 12% AOM transmission, 20 iterations and scan speed of 25.6 μsec/ pixel. Using these settings we labeled a ROI containing 9-10 cells and a photoactivated z- span of 30μm (~1-2 cell rows).
Figure 1. A Representative result of photoactivation of caged fluorescein in live zebrafish embryo using two-photon microscopy.
Schematic illustration (A-C) and representative images (A’-C’) of the photoactivation process. Live zebrafish embryo (A, A’) expressing GFP under the control of neurogenin-1 promoter (neurog1::gfp) that was injected with caged fluorescein tracer dye at the 1 cell stage. At the 3-5- somite stage, a discrete area of the forebrain primordium (diencephalon) was photoactivated and the uncaged fluorescein tracer dye could be detected (B, B’). Subsequent to the photoactivation procedure, the embryo was incubated at 28.5°C and brain cells containing the uncaged fluorescein were traced by anti-Fluorescein immunostaining at 24 hours post fertilization (hpf; C, C’). Dien., Diencephalon; Tel., Telencephalon.
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 deep optical penetration and reduced nonspecific phototoxicity. For the activation process, two photons of low energy must be absorbed by the photo-activatable compound at the same time. As the probability of such an event is dependent on the photon density, the activation remains restricted to the focal plane. The activation region can therefore be selectively manipulated in a defined volume within the tissue.
We present a protocol for photoactivation of caged fluorescein using two-photon microscopy in live zebrafish embryos. In the specific example shown herein we use two-photon microscopy to induce caged-fluorescein photolysis in order to mark cells at early embryonic stages and thereafter trace the lineage of the labeled cells in the developing zebrafish brain. In comparison to photoactivation by laser and flash-lamp, which results in the activation of the tracer dye in a few tens of cells and lack of resolution in the z axis4,5, two-photon-based photoactivation can reach a spatial resolution of a few micrometers obtaining axial resolution of a proximally one to two cell rows 2,3.
The procedure reported herein can be utilized for the activation of a variety of compounds at a single cell resolution in live specimens. For example, the Kaede protein can be similarly used to label cells following its photoconversion from a green to red fluorescenct protein6,7. Other light-activated proteins that can be applied include the light-gated ion channel, Channelrhodopsin, which can modulate neuronal activity8 and photosensitizers such as KillerRed, which induces cell death upon light irradiation9.
A variety of caged molecules have been reported, allowing modulation of physiological processes by manipulation of extracellular and intracellular compounds10. These include, active neurotransmitters (e.g. glutamate11,12) and second messengers (e.g. calcium13) and steroid hormones (e.g. retinoic acid14). Lastly, photo-mediated control of gene activation and silencing in zebrafish has been introduced. Two-photon- based photoactivation of caged antisense oligonucleotides (morpholinos) and RNAs can be used for gene knockdown and gain-of-function in restricted cell population of a live zebrafish embryo15,16.
In sum, two- photon- based photo-activation in live zebrafish embryos is: 1) Precise- one to two cell rows along x,y,z axes. 2) Quantitative- the extent of photoactivation can be controlled. 3) Versatile- may be applied for a variety of photo-convertible proteins.
Thanks are due to Genia Brodsky for figure graphics; Vyacheslav Kalchenko, Douglas Lutz, and Leonid Roitman for technical advice and assistance with the two-photon uncaging; Maayan Tahor and Suliman Elsadin for technical assistance; Uwe Strahle for kindly providing the neurogenin1 reporter line and Amos Gutnick for comments on this manuscript. The research in the Levkowitz lab is supported by the German-Israeli Foundation (grant number 183/2007); Israel Science Foundation (grant number 928/08) and the Harriet&Marcel Dekker Foundation. G.L. is an incumbent of the Tauro Career Development Chair in Biomedical Research.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 | A9539 | ||
Thin Wall Glass Capillaries with filament | World Precision Instruments | TW100F-6 | ||
Micropipette puller | Sutter Instrument | P-97 | ||
Microloader tip | Eppendorf | 5242 956.003 | ||
Pneumatic picopump | World Precision Instruments | PV820 | ||
Phenylthiourea (PTU) | Sigma | 22290-9 | ||
Low melting point agarose for embryo mounting | Ultra Pure LMP agarose | 16520100 | ||
Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments | Roche | 11426338910 | ||
Fast Red | Roche | 11496549001 |