Da-Ting Lin, Ph.D.

Department of Neuroscience / HHMI

Johns Hopkins University School of Medicine

Email: dlin13@jhu.edu

Dendritic spines are the site of glutamergic synapses. Two-photon uncaging of glutamate could evoke EPSC that closely mimics mEPSC. By combining two-photon imaging and two-photon glutamate uncaging, one could directly examine the functional state of individual spines. To achieve this goal, I have set up two-photon imaging and two-photon uncaging of glutamate on a Zeiss LSM 510 upright system. The two-photon imaging laser is a Coherent Chameleon HP (690 nm -1040nm) and the two-photon uncaging laser is a Coherent Chameleon set at 720 nm. An APE FentoControl was used to pre-chirp the uncaging laser pulse so the laser pulse was restored to 130 fs at the image plane as measured using CARPE from APE. An AOM was used to control the imaging laser power, and a pockel cell from Conoptics was used to control the uncaging laser. The pockel cell was directly control by the analogue output of electrophysiology amplifier. Evoked response from glutamate uncaging was similar to that of endogenous mEPSC. Earlier work was done by collaboration with Dr. Jean-Claude Beique, an former postdoc in Dr. Huganir's lab. Click here for reference.

The setup: (front view)

The setup: (side view with laser cover off)

(Green, dendritic morphology; Red: uncaging site)

(Black trace: mEPSC; Red trace: EPSC evoked by two-photon uncaging)