Quantum dot thermometry.

Qdot 655 ITK amino polyethylene glycol-coated quantum dots (Invitrogen) were nanoinjected (40 nl, 1 μM) into anterior visual cortex. A cranial window was implanted 1 wk after injection, and spectrometry was performed 1 wk after window implant. The microscope (920-nm excitation) was focused 250 μm below the dura, and detected light was passed through an emission filter (HQ675-70m-2P; Chroma) and coupled to the optical fiber input of a spectrometer (Acton SP2300i; Princeton Instruments). Laser scanning started at least 3 min before spectrum acquisition onset to ensure the temperature in the focus reached steady state. Control spectra to determine spectral shifts with temperature (shift: 0.119 nm/°C, r² = 0.998) and illumination power (shift: 1.41 nm/W, r² = 0.983) were obtained in a sealed glass well (∼50 μl) immersed in water. Qdot 655 solution (750 nM) was pumped through the well with a syringe (1 ml/30 s) while the emission spectrum was measured. Temperature within the well was monitored with an embedded thermocouple. Spectra were acquired with exposure times of 30 s (in vitro) and 120-1,000 s (in vivo). For temperature calibrations, the dye temperature ranged between 28.5°C and 44.8°C. Illumination power calibrations were obtained at 28.3°C.