Single-cell Photoconversion in Living Intact Zebrafish

Summary

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Abstract

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos⁺ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Introduction

Multiple distinct cells interact to build and maintain complex animal and plant tissues. These cells are often intercalated and difficult to distinguish from neighbors at a single cell level without high resolution microscopy that require fixation of tissue. However, to understand how these tissues form, are maintained, and become diseased, it has been essential to investigate how single cells within the tissue are interacting over time. Ideally, these experiments require the labeling of single cells within a tissue in a non-invasive manner without the requirement of fixation. Scientists have now developed numerous techniques to accomplish this task^1,2,3,4.

The discovery and implementation of the jellyfish green fluorescent protein (GFP) was one exciting approach that allowed for labeling of distinct cells in a tissue environment¹. Using cell-specific promoters, it is possible to genetically select a subset of cells that are labeled¹. Alternatively, viral induced expression of GFP can be utilized for user-selected expression of GFP^3,4. Although quite useful, genetic mediated expression of GFP does not allow user-selected expression within a subset of cells in the tissue; and viral expression of GFP, although advantageous, can be invasive. With the advent of GFP derivatives and clever techniques like Brainbow to express distinct fluorescent proteins more sparsely within tissues, it has become possible to visualize single cells and the interactions among them in complex tissue^2,5. However, these approaches label cells in a random fashion. If the desired experiment requires visualization of a single cell or population of cells that is defined by the experimenter, they are therefore limited. With such experiments, it would be advantageous to have a genetically expressed fluorescent protein that can be manipulated to distinguish, in a single cell fashion, it from other fluorescent and non-fluorescent cells.

To achieve this goal and visualize the cell biology of single cells within a complex living tissue, the scientific community uses single cell photoconversion of distinct fluorescent proteins^6,7,8. Using genetically controlled expression of a photoconvertible protein (i.e., eos, kaede, etc.) that transitions from a green to red fluorescent state when exposed to UV (488 nm) light, we can distinguish a single cell from its fluorescently labeled neighbors^6,7,8. This approach utilizes an apparatus attached to our confocal microscope which can direct light from a laser stack to a diffraction-limited region of interest. With this technique, we can either label single cells or larger populations in a user-defined manner^9,10,11. The technique is minimally invasive compared to single cell injections of viral GFP. As a proof of concept, we show that we can photoconvert single cells within a ganglion in the peripheral nervous system and photoconvert larger populations like cells located on the ventral side of the spinal cord^9,10,11,12. We then can visualize these photoconverted cell populations 24 h later to gain insight into their movement and differentiation during development.

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Protocol

All animal studies were approved by the University of Notre Dame Institutional Animal Care and Use Committee.

1. Preparation of Zebrafish Specimen

1. Place one adult male and one adult female Tg(convertible protein) into a mating chamber per standard procedures¹³. In this manuscript, use Tg(sox10:eos) fish⁹ because of access but other transgenic lines with photoconvertible protein can be equally used. Set up more than one chamber in case fish do not lay. Allow the fish to remain in the chamber overnight.
2. The next morning, collect eggs in 100 mm Petri dishes. Allow eggs to mature to 24 h post-fertilization (hpf) before screening.
3. If animals above were heterozygotes for the transgene, screen 24 hpf dishes for Tg(sox10:eos)+ embryos with a 488 nm light source and GFP filter sets on a dissecting microscope. Isolate Tg(sox10:eos)+ embryos and allow to mature to 48 hpf.
4. Dechorionate embryos manually with a needle or tweezers.
5. Prepare and microwave 5 mL of 0.8% low-melting point agarose solution.
   1. Once agarose is cool to the touch, place 3-4 anesthetized Tg(sox10:eos)+48 hpf fish in the center of a 10 mm glass-coverslip bottom Petri dish.
   2. Add enough agarose to cover the surface of the coverslip, approximately 1 mL. Use a probe needle to arrange fish on their sides. Allow agarose to solidify to ensure mounting of animals. It may be necessary to continually re-arrange the zebrafish until the agar solidifies¹⁴. Solidification takes approximately 2 min.
6. After the agarose has solidified for 2 min, slowly add embryo medium containing 0.02% aminobenzoic acid ester (Tricaine) to dish until the bottom surface of the agar and dish is submerged. This should be approximately 3mL.

2. Microscope Mounting and Pre-conversion Imaging

1. Open confocal software and select the capture and focus windows [Figure 1]. Under the capture window, select the lab-specific conversion imaging setting under the capture setting drop down tab [Figure 1]. Here, the lab-specific setting is called "Fish Imaging."
2. Place specimen on confocal scope and bring into focus using course and fine adjustment knobs.
3. Open the focus window and locate the desired region of interest (i.e. dorsal root ganglia).
4. Select the c488  laser under the Filter Set menu. Set the exposure to 300 ms, laser power to 5, and intensify to 75 [Figure 2].
5. Check the 3D box under the capture type section. In the 3D Capture section, select use current position and check range around current. In the same section, set the range to 35, the number of planes to 36, and the step size to 1. The range can be increased or decreased to accommodate for the depth of the imaging area. For the spinal cord a range value between 35-40 stacks typically is sufficient [Figure 5].
6. Select current location [Figure 5].
7. Click start at the bottom of the capture window to acquire image.

3. Single-cell Photoconversion

1. Open confocal software and select the capture and focus windows [Figure 1]. Under the capture window, select the lab-specific conversion imaging setting under the capture setting drop down tab [Figure 1]. Here, the lab-specific setting is called "Fish ablate full chip."
2. Select the c488 and c541 laser under the Filter Set menu. If not using the same microscope software, find the menu to select different lasers and select the 488 nm and 541 nm lasers. Set the exposures to 300 ms, laser power to 5, and intensify to 75 [Figure 2]. These laser settings are selected based on producing enough fluorescent signal without causing photobleaching or toxicity. If toxicity or photobleaching is visualized, reduce laser power or exposure.
3. Open the focus window and click on the photomanipulation tab in the focus window. Adjust laser parameters accordingly. Change the laser stack power to 2, and then click Go. Change the Raster block size to 1 and click Set. Change the Double-click size to 4. Change the laser line to v405 [Figure 3].
4. Open the advanced capture settings in the capture window. Select the photomanipulation tab and change the Double-click repetitions to 2. Click OK [Figure 4].
5. Select the XY tab in the focus window. Double check the laser parameters from step 2 in the photomanipulation tab [Figure 3, Figure 4]. Set laser settings to photoconvert the cell of interest without photoconversion of surrounding cells.
   1. If photoconversion of adjacent cells is present, reduce laser power. If photoconversion of cells does not occur, laser powers can be increased. Optimally, set laser power to photoconvert only the region of interest and not surrounding areas.
6. Check the timelapse box under capture type. Then, click Start [Figure 6A].
7. Once the live timelapse window opens, select the circle tool on the top toolbar [Figure 7]
8. Draw a circle in the centermost region of the cell. Right click the drawn circle, select FRAP region, and wait 3 seconds. The selected area should become dimmer [Figure 8]. Click stop capture.
9. If imaging more than one animal, go back to the XY tab in the focus menu and select position 2. Then, repeat steps 4.1-4.6.
10. To convert a population of cells, follow the protocol and laser parameters for steps 4.1-4.4 except instead of drawing a circle to FRAP the region of interest, use the line tool. Draw a line on the region of interest and FRAP the region using the same parameters listed above.

4. Post-photoconversion Imaging

1. Once all points are photoconverted. Select the lab-specific standard image stack setting described in the precoversion imaging step 3. This is found under the capture setting drop down tab in the capture window [Figure 5].
2. Select the c488 laser and set the exposure to 300ms, laser power to 5, and intensify to 75 [Figure 2].
3. Select the c541 laser found under the same menu as the c488 laser. Set the exposure to 500 ms, laser power to 10, and intensify to 75 [Figure 9].
4. Check the 3D box under the capture type section. In the 3D Capture section, select use current position and check range around current. In the same section, set the range to 35, the number of planes to 36, and the step size to 1. The range number may increase or decrease to accommodate the desired imaging depth [Figure 5].
5. If there are multiple points in the XY focus tab, select the multipoint list option in the capture window. If not, select current location.
6. Click start at the bottom of the capture window to acquire image.

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Results

Photoconversion of fluorescent proteins can be used to label distinct cells within a tissue⁶. To demonstrate this, Tg(sox10:eos) fish⁹ were used to express the photoconvertible protein Eos under the regulatory sequences of sox10. The Tg(sox10:eos) animals at 48 hpf were first mounted, and then imaged to detect any non-specific photoconversion that may have occurred. The Eos unconverted fluorescent signal with littl...

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Discussion

In complex tissues, distinct cell-types organize into specific domains. Recently techniques have been utilized to label individual cells within these large tissue structures^1,2,3. Here we demonstrate two techniques that can similarly be utilized to visualize both single cell interactions and cell population interactions within complex tissues. The advantage of the photoconversion technique is the spatial control the scientist ha...

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Materials

Name	Company	Catalog Number	Comments
Tg(sox10:eos) zebrafish animals			Fish were obtained and crossed from the fish facility at the University of Notre Dame
100 X 15 mm petri dish	VWR	25384-302
Embryo medium			Embryo medium is made weekly and provided by the fish facility at the University of Notre Dame containing 5L RO water, 30uL methylene blue, and 200mL salt stock.
0.8% Low Melting Point Agarose	dot scientific inc	9012-36-6
35 X 10 mm glass-coverslip bottom petri dish	Ted Pella Inc.	14021-20
Needle dissecting probe
0.002% 3-aminobenzoic acid ester (Tricaine)	Fluka analytical	A5040-250G
Fluorescent Dissecting Microscope with GFP filters	Zeiss Axiozoom
Confocal microscope with lasers to excite GFP and RFP filter sets	3i spinning disk confocal
UV light source (laser) for photoconversion	405 nm laser
Slidebook software	3i
Methylene blue	Kordon