We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ∼81 and ∼44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.
Increasing interest in the role of lipids in cancer cell proliferation or resistance to drug therapies has motivated the need
to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive
chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell
studies. Methods that allow the analysis of lipid content in live cells is therefore of great importance for research. Using
Raman micro-spectroscopy we investigated whether the female hormone medroxyprogesterone acetate (MPA) and the
synthetic androgen R1881 affect the lipid expression in breast (T47D) and prostate (LNCaP) cancer cells. Differences
were noted in the spectral regions at 830-1800 cm-1 and 2800-3000 cm-1 when comparing different drug treatments.
Significant changes were noticed for saturated (1063 - 1125 cm-1, 1295 cm-1 and 1439 cm-1), unsaturated (1262 cm-1 and
1656 cm-1, and 1720 - 1748 cm-1) chemical bonds, suggesting that the composition of the lipid droplets was changed by
the hormone treatments. Also, significant differences were observed in the high frequency regions of lipids and proteins
at 2851 cm-1 and around 2890 cm-1. Principal component analysis with Linear Discriminant Analysis (PCA-LDA) of the
Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881 or vehicle (P < 0.05).
Future work includes analysis to determine exact lipid composition and concentrations as well as development of clinical
techniques to characterize differences in patient tumor lipid profiles to determine response to drug treatment and
prognosis.
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