Figure 1. Imaging depth in intravital microscopy. (A) Jablonski diagram of fluorophore excitation by single and multiphoton microscopy. The energy gap between two energy levels (ground state, E₀ and excited state, E_*) can be filled by one, two or three photons. After thermal decay, the system relaxes to the ground state by emitting a photon. (B) All the photons generated by the imaging area are emitted, and collected by the detection system. (C) When the imaging area is located in solid tissue both the excitation beam (blue arrows) and the emitted photons (green arrows) are scattered. The deeper the targeted area the lower is the probability to excite a transition or to detect the emitted photons. (D) Diagram illustrating imaging modality, resolution and optics as a function of imaging depth. (E) Diagram illustrating alternative approaches to extend the imaging depth by MPM. (F) Diagram illustrating GRIN lenses and micro prisms inserted in the tissue to image up to 1–2 mm in depth. (G) Effect of the clearing agent scale/e. A portion of the quadriceps was excised from a mouse expressing soluble GFP, fixed in 2% formaldehyde and incubated for 3 d in the clearing agent scale/e.⁴⁰ A z-scan was acquired by two-photon microscopy (excitation wavelength 930 nm) using water immersion 25x lens (XLPL25XWMP, from Olympus). Notably, cleared tissue was imaged up to 1.2 mm and the muscle fibers were nicely resolved, whereas usually non-cleared tissue cannot be imaged beyond 500 µm.