In vitroimaging using laser photostimulation with flavoprotein autofluorescence

Brian B. Theyel, Daniel A. Llano, Naoum P. Issa, Atul K. Mallik, S. Murray Sherman

Research output: Contribution to journalArticlepeer-review


Imaging of 300-500 mm mouse brain slices by laser photostimulation with flavoprotein autofluorescence (lFpa) allows the rapid and sensitive mapping of neuronal connectivity. It is accomplished using uV laser-based photo-uncaging of glutamate and imaging neuronal activation by capturing changes in green light (˜520 nm) emitted under blue light (˜460 nm) excitation. this fluorescence is generated by the oxidized form of flavoprotein and is a measure of metabolic activity. lpFaoffers several advantages over imaging techniques that rely on dye loading. First, as flavoprotein imaging measures endogenous signals, it avoids the use of heterogeneously loaded and potentially cytotoxic dyes. second, flavoprotein signals are large (1-20% above baseline), obviating the need for averaging. third, the use of photostimulation ensures orthodromic neuronal activation and permits the rapid interrogation of multiple stimulation sites of the slice with a high degree of precision (˜50 mm). Here we describe a step-by-step protocol for the incorporation of lpFainto virtually any slice rig, as well as how to do the experiment.

Original languageEnglish (US)
Pages (from-to)502-508
Number of pages7
JournalNature Protocols
Issue number4
StatePublished - Apr 2011

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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