How does two photon microscopy work and what advantages does it have ?
Two-photon microscopy is a fluorescence imaging technique that uses near-infrared (NIR) pulsed lasers to excite fluorescent molecules only when two photons arrive at the molecule simultaneously.
Instead of one high-energy photon exciting a fluorophore, two lower-energy photons (usually NIR) combine their energies.
•This “two-photon absorption” is extremely unlikely unless the light intensity is very high — which only happens at the focal point of the laser.
As a result, fluorescence occurs only in a tiny focal volume, giving:
—> Intrinsic 3D sectioning (no need for a pinhole like in confocal microscopy).
—> Much deeper tissue penetration (NIR scatters less).
—> Less photodamage and photobleaching outside the focus
Important: always turn the photomultipliers off when opening the curtain of the microscope so you dont break them
What role does the camera play in the setup of the microscope?
Camera: placed to visualize the stage and moniture the mice´s breathing and the anesthesia
How was the mouse modell prepared for the experiment?
the mice undergo a surgery where a 4 mm window is placed in their head by removing a part of the skull and pulling the muscle aside to insert the window right on the brain. The cranial window is secured by a titanium ring.
after two weeks the wound is fully healed and the mouse can be used for imaging
the mice used in the practical coures were GMOs with tagged Monocytes (red) and Microglia (green)
What happens to the mice during the imaging process?
mouse is placed in a specialized induction chamber connected to a calibrated vaporizer that mixes 5% isoflurane with oxygen
after that mouse is placed on a heating pad to keep the body temperature at a physiological level and put in Front of a mask. The amount of isoflurane supplied through the mask is reduced to 2-3% to not suffocate the mouse. oxygen is also supplied through the mask. It is also importat to cover the mice´s eyes with eyecream since they can not blink anymore
the mouse is ingected with a lectin dye into one of the tail veins that taggs the walls of aterioles to make them visual in the brain of the mouse
--> an alternitive to the injection in the tail vein is a retro-orbital injection
the mouse is then placed under the microscope where the anesthesia is sustained by another mask
What was the point of the performed experiment?
the main point of the experiment is to monitor the migration of microglia in the brain after an induced lesion with a laser in ateriols in the brain
to that end the mice have tagged microglia and monocytes to track the movements of microglia in the brain and the migration of monocytes from the lesion into the brain. Additionally the aterioles are stained with lectin to visualize the structures the lesion can be induced in
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