ReZolve-L1^™ responds to altered autophagy.

<p>Confocal micrographs of <i>Drosophila</i> fat body tissue stained with the ReZolve-L1^™ (red) or LysoTracker^® Green to detect autophagic activity. (<b>A</b>) Representative images from control or <i>Tor</i>^<i>TED</i> larvae at -8 h PF, fed on standard feeding media (-8 h PF Fed). (<b>B</b>) Representative images from control or Atg9^RNAi larvae at -4 h PF, fed on standard feeding media (-4 h PF Fed) (<b>C</b>) or exposed to 4 hours of amino acid and sugar starvation (-4 h PF Starved). LD indicates lipid droplets; arrows indicate co-location between ReZolve-L1^™ and LysoTracker^® Green; arrow heads indicate ReZolve-L1^™ only compartments. Scale = 10 μm. (<b>D</b>) Histogram showing the number of ReZolve-L1^™ autophagic compartments per 100 μm² of cell area. Mean ± s.e.m. presented for control or <i>Tor</i>^<i>TED</i> larvae at -8 h PF, fed on standard feeding medium (-8 h PF, Fed) and control or Atg9^RNAi larvae at -4 h PF, fed on standard media (-4 h PF, Fed) or exposed to 4 hours of amino acid and sugar starvation (-4 h PF, Starved). Groups are compared by ANOVA with Tukey post-hoc test, different letters indicates significance difference between means, P < 0.05.</p

ReZolve-L1^™ detects autophagic compartments in <i>Drosophila</i> fat body tissue during metamorphosis.

<p>(<b>A</b>) Confocal micrographs of fat body cells explanted from wild type <i>Drosophila</i> larvae and pupae, at either -4 h PF, 0 h PF, or +2 h PF and stained with ReZolve-L1^™ (red) and LysoTracker^® Green. Scale bars = 10 μm. (<b>B</b>) Schematic of the relationship between <i>Drosophila</i> developmental progression, autophagic activity and polar lipid content in the lipid droplets of fat body tissue.</p

ReZolve-L1^™ interacts with polar lipids.

<p>A ReZolve-L1^™ lipid overlay demonstrates an interaction between the molecular probe and six polar lipid species (left hand side of the Fig) and no detectable interaction with either four neutral lipid species, fatty acid species or methanol controls (right hand side of the Fig). Blanks show the detection of the lipid background, without incubation with ReZolve-L1^™.</p

Raman map of ReZolve-L1^™ in 3T3 adipocytes relative to lipid species.

<p>(<b>A</b>) Transmitted light image of a 3T3 cell on which Raman mapping was performed. Black box indicates area scanned. (<b>B</b>) Raman maps showing the distribution of <b>ν</b>(C = O) lipid esters (blue), lipid backbone <b>ν</b>(C = C) (green) and ReZolve-L1^™ (red). Raman maps were generated by calculating the area under the following spectral regions; 1720–1765 cm^-1, 1625–1675 cm^-1 and 1610–1620 cm^-1, respectively. (<b>C</b>) Spectra extracted from locations <b>a, b</b> and <b>c</b> shown in panel (<b>B</b>).</p

ReZolve-L1^™ is detected in lipid droplets in fed larvae, but co-locates with Atg8aGFP autophagic compartments upon combined amino acid and sugar starvation.

<p>(<b>A</b>) Confocal micrographs of <i>Drosophila</i> fat body tissue from larvae at -4 h PF, stained with ReZolve-L1 (red) and expressing the autophagic marker <i>Atg8a-GFP</i> (green). Tissue was explanted from fed larvae or larvae exposed to 4 hours of amino acid, sugar or amino acid and sugar starvation. (<b>B</b>) Confocal micrographs of <i>Drosophila</i> fat body tissue from larvae at -4 h PF, stained with Oil Red O and expressing the autophagic marker <i>Atg8a-GFP</i>. Tissue was explanted from fed larvae or larvae exposed to 4 hours of amino acid, sugar or amino acid and sugar starvation. LD indicates lipid droplets. Scale bars = 10 μm.</p