Longitudinal Two-Photon Imaging of Fluorescently Labeled Neurons in a Live Mouse Hippocampus

Abstract

Source: Ulivi, A. F., et. al. Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice. J. Vis. Exp. (2019)

This video demonstrates longitudinal two-photon imaging of fluorescently-labeled pyramidal neurons in a live mouse, showcasing the preparation, cannula alignment, and imaging of dendrites and dendritic spines to study neuronal structure in the hippocampal CA1 region.

Protocol

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

1. Preparation of the imaging session
2. Turn on the imaging setup in advance and let the laser to warm up and stabilize, if necessary.
3. Anesthetize the mouse. Place the mouse in the anesthesia induction chamber at 3% isoflurane in 1 L/min O₂ and wait until it loses consciousness. Assess anesthesia using the toe pinch reflex test.
4. Use forceps to carefully remove the adhesive film from the head holder plate. Hold the mouse in a hand and the head holder plate with the fingers. Remove the film gently, to avoid damaging the preparation.
5. Position the mouse under the microscope, over the heating carpet, and secure the head plate to the holder (Figure 1D).
6. Position the nose cone to cover the snout and decrease isoflurane to 1.5-2%.
7. Apply ophthalmic ointment to the mouse's eyes.
8. Clean the imaging cannula by rinsing it with deionized water. Use a syringe and a thin needle to drop water into the cannula and a vacuum pump to remove it.

2. Imaging session

1. Use low magnification and long working distance objectives to visually check the cannula for residual water, dirt, integrity, and the presence of fluorescence.
2. Align the cannula to the optic axis by adjusting the angles of the head holder arms.
3. Switch to the 25X 1.0 NA (numerical aperture), 4 mm WD (working distance), or the 40X 0.8 NA, 3 mm WD, water immersion objectives. Add enough deionized water to fill the cannula and maintain excess water on top of the cannula. Avoid the formation of air bubbles.
4. Use two-photon (2P) excitation and image fluorescent signals.
   NOTE: The imaging protocol is highly dependent on the time and spatial scales to be imaged. Please refer to the section's representative results and discussion for details about the imaging settings we have used to produce the images shown in this article.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stainless steel tube Ø 3,0 x 0,25 mm (Inner Ø 2,5 mm ) L = 500 mm	Sawade	R00303	Stainless steel tube for the cannula metal ring
Microscope Cover glass (4 mm round)	Engelbrecht Medizin and Labortechnik		Glass coverslips for the cannula glass
Isofluran CP, Flasche 250 ml	Henry Schein VET GmbH	798932	Liquid isoflurane for anesthesia
Harvard Apparatus Isoflurane Funnel-Fill Vaporizer	Harvard Apparatus GmbH	34-1040	Isoflurane vaporizer
Lab Active Scavenger	Gropper Medizintechnik	UV17014	Isoflurane scavenger system
CMA 450 Temperature Controller	Hugo Sachs Elektronik - Harvard Apparatus GmbH	8003770	Heating blanket
Bepanthen Augen- und Nasensalbe	Bayer AG		Ophtalmic ointment
Head plates	Custom made		30 mm x 10 mm size; 8 mm diameter hole, titanium
Head plate clamp	Custom made		Head plate holder
Pedestal post holders	Thorlabs	PH20E/M	Head plate holder
Stainless steel post	Thorlabs	TR30/M	Head plate holder
Stainless steel post	Thorlabs	TR75/M	Head plate holder
Stainless steel post	Thorlabs	TR150/M	Head plate holder
Post connector clamps	Custom made		Head plate holder
Aluminum Breadboard, 300 mm x 450 mm x 12.7 mm, M6 Taps	Thorlabs	MB3045/M	Microscope stage
7" x 4" Lab Jack	Thorlabs	L490/M	Microscope stage
Low profile face mask small mice	Emka Technologies	VetFlo-0801	Anesthesia facemask holder
Power Meter Model 1918-R	Newport		Power meter
X-Cite 120Q	Excelitas Technologies		Fluorescence lamp
Two-photon microscope	Bruker	Ultima IV	Two-photon microscopes
Two-photon microscope	Thorlabs	Bergamo	Two-photon microscopes
Plan N 4x/0.10 ∞/-/FN22	Olympus		Objectives
Plan N 10x/0.25 ∞/-/FN22	Olympus		Objectives
LMPlan FLN 20x/0.40 ∞/-/FN26.5	Olympus		Objectives
XLPlan N 25x/1.00 SVMP ∞/0-0.23/FN18	Olympus		Objectives
Ultafast tunable laser for 2P excitation	Spectraphysics	Mai Tai Deep See	Excitaiton lasers
Ultafast tunable laser for 2P excitation	Spectraphysics	InSight DS+ Dual beam	Excitaiton lasers
Ultafast tunable laser for 3P excitation	Coherent	Monaco	Excitaiton lasers