Deep-Tissue Imaging of a Zebrafish Brain Using a Three-Photon Fluorescence Microscope

Abstract

Source: Hontani, Y., et al. Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain. J. Vis. Exp. (2022)

This video demonstrates the setup and execution of three-photon imaging in an anesthetized zebrafish brain. The protocol enables deep-tissue visualization of fluorescently labeled neurons, revealing real-time neuronal activity and structures for neuroscience research applications.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intravital imaging in the zebrafish brain
   NOTE: To properly locate the fish brain under the objective lens, a secondary charge-coupled device (CCD) camera is used on the same path as the excitation light for widefield imaging.
   
   1. Set up the microscope and calibrate the power.
   2. Place a low magnification (typically 4x) objective on the microscope.
   3. Place the Petri dish containing the fish and the tubes under the microscope.
   4. Use a light-emitting diode (LED) light source to illuminate the Petri dish.
   5. Open the Camera mode of the image acquisition software (Figure 1).
   6. Click Live.
   7. Choose channel A on the right side of the screen.
   8. Adjust the histogram settings to see the image clearly.
      NOTE: These need to be updated as needed.
   9. Set the motor setting on the motor controller to Base.
   10. Lower the objective until the fish is visible.
       NOTE: Ensure that the objective lens does not make any physical contact with the head.
   11. Place the center of the fish head at the center of the field of view.
   12. Move the objective up and away from the fish head.
   13. Replace the low magnification objective lens with the high numerical aperture (NA) objective lens for 3PM (three-photon microscopy).
       NOTE: The low magnification lens and the high NA objective lens do not need to be parfocal but must be close enough to ensure the fish is within the field of view of the high NA objective lens.
   14. Slowly lower the objective lens, ensuring that the objective does not make any physical contact with the head. In the CCD camera software, stop moving the objective when the top of the head is visible. Set the z location to 0 μm.
       NOTE: Ensure that there are no air bubbles under the objective lens when using a water-immersion objective.
   15. Turn off the LED light source and close the dark curtain around the system.
   16. Set the imaging acquisition software to Multiphoton GG mode for 3P imaging and set the power under the objective lens to less than 1 mW (with ~1 MHz pulse repetition rate).
   17. Change the motor setting button from Base to Objective on the motor controller.
   18. Turn down the lights in the room.
   19. Turn on the photomultiplier tubes (PMTs) and open the 3PM excitation source shutter. Ensure that an outline of the bone appears in the fluorescence signal channel due to autofluorescence and in the third harmonic generation (THG) signal channel due to THG of the bone (Figure 1C).
   20. Perform imaging at different depths by increasing the power levels when imaging deeper.

Results

Figure 1: Representative screenshots for intravital Imaging in fish (protocol section 1). (A

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5% Povidone-iodine	Amazon	NDC 67818-155-32	Aceptical cleaning of surgical areas
70% Ethanol	Thermo Fisher Scientific	CAS 64-17-5	Aceptical cleaning of surgical areas
Agarose	Sigma	A4718-256	Preparing zebrafish chamber
Bergamo II	Thorlabs		Multiphoton Imaging Microscope
Bupivacaine	Cornell Veterinary Care
GaAsP Amplified PMT	Thorlabs	PMT2100	PMT detector
Heater (800 W)	Finnex		Aquarium heater for zebrafish water)
Kimwipes	Kimtech		Laboratory tissue for preparing zebrafish
Nanofil syringe (10 micrometer) with 36 G needle	WPI	NANOFIL + NF36BV	Syringe and needle for injection of pancuronium bromide
Peristaltic Pump	Elemental Science	ESI MP2	Water pump for zebrafish setup
Polyethylene tubing (I.D. 0.58 mm., O.D. 0.965 mm.)	Elemental Science	MP2 pump tubing	Tubing that goes in the mouth of the zebrafish
SR400	Stanford Research Systems	SR400	Photon counter
Standard Photodiode Power Sensor	Thorlabs	S122C	Power detector
Thin strip boxing wax	Corning Rubber Co., Inc.		Holding tubing in place in zebrafish chamber
ThorImage	Thorlabs		Image acquisition software
Tricaine (Ethyl-m-aminobenzoate methanesulfonate salt)	MP	103106	Zebrafish anesthesia and euthanasia
Tygon tubing (I.D. 1/16 in., O.D. 1/8 in.)	Tygon		Tubing for water flow for zebrafish preparation
VaporGuard	VetEquip	931401	For recycling isoflurane
Vetbond tissue adhesive	3M	1469SB	To glue the glass window on the mouse skull, and to glue the laboratory tissue when preparing the fish.
XLPLN25XWMP2	Olympus		Multiphoton Excitation Dedicated Objective