Imaging biophotonic activities (emissions) after the application of glutamate in mouse coronal brain slices.

<p>(<b>A–E</b>) A representative regular image of a coronal brain slice (<b>A</b>). The dynamic change of biophotonic activities in this slice was demonstrated by relative gray values (RGVs, <b>B</b>) and biophoton numbers (BPNs, <b>C</b>). Representative biophoton gray images (<b>D</b>) and corresponding biophoton number images (<b>E</b>) at the selected time periods indicated in <b>B</b> (digit:1–7) after the application of 50 mM glutamate. Each image in <b>D</b> or <b>E</b> was obtained from the merger of 25 continuously processed original gray images or biophoton number images (1 min imaging time for each original image, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085643#pone.0085643.s001" target="_blank">Figure S1</a>). The time points are indicated in <b>B</b> for the first and second application of 50 mM glutamate (arrow) and slice washing (arrowhead). (<b>F, G</b>) The sum of the time course of the average change of RGVs (<b>F</b>) and BPNs (<b>G</b>) (blue line, n = 6), and the effects of early slice washing (pink arrowhead) and the second application of 50 mM glutamate (pink arrow) (pink line, n = 5) are much less relative to the late treatments (blue arrowhead and arrow, see also in <b>B</b>). (<b>H</b>) The sum of the time course of the average change of RGVs in <b>F</b> from 25 continuously processed original gray images. (<b>I–K</b>) Dose-dependent changes of biophotonic activities (<b>I</b> and <b>J</b>) and the sum of the time course of the average change of RGVs from the 25 continuously processed original gray images (<b>K</b>); no obvious effect was found at the concentrations of 12.5 mM (n = 4), the time to reach the maximal effect was longer, and the amplitude of the maximal effect was significantly less at 25 mM than that at a concentration of 50 mM (<b>K</b>, 232.3±7.4 versus 91.5±7.9 min; 211.4±22.4 versus 410.2±30.9 RGVs, p<0.001, n = 6 for 25 or 50 mM). 1 min imaging time for each time point in <b>B</b>, <b>C</b>, <b>F</b>, <b>G</b>, <b>I</b> and <b>J</b>. Data show mean±s.e.m. n = the number of slices from the same number of mice.</p

Schematic drawing of the biophoton imaging system.

<p>EM-CCD camera (1); EM-CCD camera supporter and up-down adjuster (2); Lens (3); Conical light isolation cover (black cotton cloth) for lens and sample (4); Sample stage (5); Perfusion chamber and sample (6); Sample stage supporter and adjuster (7); Micromanipulator (8); Pressure control perfusion system: solution delivery device (9); Cooling water circulation pump (10); Bent stainless steel tube (11); EM-CCD controller (12); Computer (13); Pressure control perfusion system (14); Electrical heating controller (15); Electrical heater (16); Input micropump for chamber perfusion (17); Output micropump for chamber perfusion (18); Output micropump for local perfusion (19); Collective beaker for local perfusion (20); Glass bottle for chamber perfusion medium (21); Membrane oxygenator (22); Gas cylinder (23); Ion layer of dark box (24); Lead plate layer of dark box (25); Black cotton cloth layer of dark box (26); Dark box (90 cm×70 cm×110 cm) (27).</p

Imaging biophotonic activities and transmission in mouse sagittal brain slices.

<p>(<b>A</b>) A representative regular image of sagittal brain slices and the regions of interest (ROIs), including the cerebral cortex, corpus callosum and thalamus, are marked by red lines, which were selected for the analysis of biophotonic activities. (<b>B–I</b>) Representative biophoton gray images at the selected time periods before (<b>B</b>–<b>E</b>) and after (<b>F</b>, <b>G</b>) the application of Protein Phosphatase 2 inhibitor (OA). Each image was obtained from the merger of 100 continuously processed original grey images. The sum of the time course of the average change of RGVs (<b>H</b>, 1 min imaging time for each time point) and the sum of time course of average change of RGVs from the 100 continuously processed original gray images (<b>I</b>) in the cerebral cortex, corpus callosum and thalamus before and after the application of OA (n = 7 slices from the same number of mice). The significant increase of biophotonic activities in the corpus callosum and the thalamus (<b>C–E, H, I</b>) tended to decay after the application of OA (<b>F–I</b>). Arrow and arrowheads in <b>H</b> and <b>I</b> indicate the time points for the long-lasting application of 50 mM glutamate and 200 nM OA, respectively. Data show mean±s.e.m. <b>CX</b>: cerebral cortex; <b>CC</b>: corpus callosum; <b>Tha</b>: thalamus. * <b>CC</b> or <b>Tha</b> group (corresponding color) versus <b>CX</b> group at the same time periods; + the effects after application of OA versus the maximum effect (arrowhead) before in each group. * or + P<0.05, ** or ++ P<0.01. (<b>J</b>) A schematic explanation for the origin of biophotonic activities in the corpus callosum and thalamus in a sagittal brain slice. The cut end of axons in the corpus callosum (red spots) and the cut end of axonal terminals in the thalamus (blue spots) originate from the cortical projection neurons (red and green, respectively). Such anatomic projection patterns of cortical projection neurons indicate that the detected biophotonic activities in the corpus callosum and thalamus originate from the cortical projection neurons via the biophotonic transmission along their axons or axonal terminals.</p