Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes.

Mice Lrat −/− and Rpe65 −/− mice with a C57BL/6J background were crossed with C57 albino wild–type (WT) C57BL/6J– Tyr C–2J mice to produce albino Lrat −/− and Rpe65 −/− animals. Albino Abca4 −/− Rdh8 −/− double knockout mice were generated by crossing Abca4 −/− Rdh8 −/− mice with a mixed 129Sv and C57BL/6J background with 129/SvJ albino WT mice 17 , 35 . PCR genotyping of these mice was performed as previously described 17 , 35 – 37 . Animals were housed and crossbred in the Case Western Reserve University (CWRU) Animal Resource Center Facility where they were maintained under either a 12 hr light (10 lux)/dark illumination cycle or in complete darkness. Manipulations in the dark were performed under dim red light transmitted through a filter (transmittance 560 nm; No. 1 Safelight; Eastman Kodak, Rochester, NY). Mice were provided free access to a standard chow diet and water. Prior to experimental analyses they were anesthetized by intraperitoneal injection with 10 μl/g body weight of 6 mg/ml ketamine and 0.44 mg/ml xylazine, and then euthanized by cervical dislocation. All animal procedures were approved by the CWRU Animal Care and Use Committee and complied with the American Veterinary Medical Association Guidelines on Euthanasia. Mice were gavaged with 9– cis –retinyl acetate according to a previously published protocol 38 .

Retinoid and A2E Analyses

All experimental procedures related to extraction, derivatization, and separation of retinoids from dissected mouse eyes were carried out as previously described 17 , 35 . A2E was synthesized from all– trans –retinal and ethanolamine, and purified by HPLC 35 . Quantification of A2E after HPLC was accomplished by comparison with known concentrations of pure synthetic A2E standards 35 .

Multi–photon excitation microscopy

TPM imaging was done with a Leica TCS SP2. A Ti:Sapphire laser (Coherent Chameleon XR) delivered