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In vitro imaging using laser photostimulation with flavoprotein autofluorescence

Nature Protocols volume 6, pages 502–508 (2011)Cite this article

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Abstract

Imaging of 300–500 μm 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. LPFA offers 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 μm). Here we describe a step-by-step protocol for the incorporation of LPFA into virtually any slice rig, as well as how to do the experiment.

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Figure 1: LPFA imaging setup.
Figure 2: Brightness calibration example.
Figure 3: 'Double-bubble' pattern.
Figure 4: Examples of synaptic activation following photostimulation.

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Author notes

  1. Daniel A Llano

    Present address: Present address: Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (D.A.L.).,

Authors and Affiliations

  1. Department of Neurobiology, University of Chicago, Chicago, Illinois, USA

    Brian B Theyel, Naoum P Issa, Atul K Mallik & S Murray Sherman

  2. Department of Neurology, University of Chicago Medical Center, Chicago, Illinois, USA

    Daniel A Llano

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  1. Brian B Theyel
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Contributions

B.B.T. and D.A.L. generated data shown in the figures. B.B.T. generated a draft of the manuscript. All authors contributed to editing the manuscript and development of laser-scanning photostimulation.

Corresponding author

Correspondence to Brian B Theyel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Method 1

Text file for "imageproc_streampix" (TXT 1 kb)

Supplementary Method 2

Text file for "imageproc_laser" (TXT 2 kb)

Supplementary Method 3

Text file for "march" (TXT 1 kb)

Supplementary Method 4

Text file for "deltamarch" (TXT 1 kb)

Supplementary Method 5

Zip file containing the programs as Matlab files (ZIP 3 kb)

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Theyel, B., Llano, D., Issa, N. et al. In vitro imaging using laser photostimulation with flavoprotein autofluorescence. Nat Protoc 6, 502–508 (2011). https://doi.org/10.1038/nprot.2011.315

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