A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons.
A laser-induced native fluorescence detection system optimized for analysis of indolamines and catecholamines by capillary electrophoresis is described. A hollow-cathode metal vapor laser emitting at 224 nm is used for fluorescence excitation, and the emitted fluorescence is spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: 250-310 nm, 310-400 nm, and >400 nm. A separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. The instrument provides more information than a single-channel system, without the complexity associated with a spectrograph/charge-coupled device-based detector. With this instrument, analytes can be separated and identified not only on the basis of their electrophoretic migration time but also on the basis of their multichannel signature, which consists of the ratios of relative fluorescence intensities detected in each wavelength channel. The 224-nm excitation channel resulted in a detection limit of 40 nmol L-1 for dopamine. The utility of this instrument for single-cell analysis was demonstrated by the detection and identification of the neurotransmitters in serotonergic LPeD1 and dopaminergic RPeD1 neurons, isolated from the central nervous system of the well-established neurobiological model Lymnaea stagnalis. Not only can this system detect neurotransmitters in these individual neurons with S/N>50, but analyte identity is confirmed on the basis of spectral characteristics.
Lapainis
T
T
Department of Chemistry and the Beckman Institute, University of Illinois, 600 S. Matthews Ave., Box 63-5, Urbana, IL 61801, USA.
Scanlan
C
C
Rubakhin
S S
SS
Sweedler
J V
JV
D000818:Animals; D001048:Aplysia; D002395:Catecholamines / Q000032:analysis; D002490:Central Nervous System / Q000737:chemistry / Q000166:cytology; D004298:Dopamine / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D008195:Lymnaea; D009474:Neurons / Q000737:chemistry* / Q000166:cytology; D018377:Neurotransmitter Agents / Q000032:analysis*; D009655:Octopamine / Q000032:analysis; D012701:Serotonin / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D002395:Catecholamines; D018377:Neurotransmitter Agents; D009655:Octopamine; D012701:Serotonin; D014443:Tyrosine; D014364:Tryptophan; D004298:Dopamine