PMC

Comparison of filter cube images of anti-PSA–COIN and anti-PSA–Alexa on adjacent sections of formalin-fixed paraffin-embedded (FFPE) prostate tissue. Images are acquired using conventional filter set imaging (×5 objective, Calcium Crimson filter set, exposure 5 sec). (A) Brightfield image of a prostate tissue section. (B) Fluorescence emission from anti-PSA–Alexa 568 on FFPE prostate tissue. (C) Raman emission from anti-PSA–COIN for the same region as B in the adjacent slide. Both stains report PSA expression in the epithelium of prostate glands but not in the stroma, and the COIN and Alexa show comparable brightness (true intensities shown). Bar = 500 μm.

Direct quantitative comparison between anti-PSA–Alexa and anti-PSA–COIN in parallel plate assays and tissue assays. (A) Direct comparison of intensities reported by anti-PSA–Alexa and anti-PSA–COIN for parallel wells in a plate-binding assay. Each data point is the average of three experiments conducted using the same COIN and Alexa reagents on different days, with corresponding error bars. Pearson correlation coefficient for the entire set is 0.996. (B) Direct comparison of intensities reported by anti-PSA–Alexa or anti-PSA–COIN from the same glands in adjacent tissue sections. COIN intensities are normalized by the intensity of the corresponding direct binding assay for each COIN preparation. COIN and Alexa intensities are reported as the signal-to-background ratio for each gland. Data include experiments performed the same day on adjacent slides (black circles), experiments using the same COIN on different days (black circles and cross-hatched circles), and experiments using completely different COIN preparations (black circles and white circles). Black symbols represent measurements using three different COIN preparations applied to adjacent slides and measured on the same day, and error bars represent the precision of replicate measurements. Pearson correlation coefficient for the entire set is 0.85.

Illustration of spectral analysis for COIN and Alexa stains on adjacent tissue sections. (A) Adjacent sections of FFPE prostate tissue were stained either with anti-PSA–COIN or with anti-PSA–Alexa. Spectra were acquired at each point (boxes) in a raster pattern spanning the stroma, epithelium, and lumen of a single gland. (B) Representative spectra from anti-PSA–Alexa Fluor 568 measured in the epithelium (red) and stroma (gray). The upper spectrum (red) was also used as the reference spectrum for subsequent Alexa spectral fitting. Nonspecific Alexa staining (gray spectrum) occurred uniformly across the stroma at ∼20% of positive signal. (C) Representative spectra from anti-PSA–COIN measured in the epithelium (red) and stroma (gray). The upper spectrum (red) was also used as the reference spectrum for subsequent COIN spectral fitting. (D,E) Intensities use a linear scale from black (zero) to red, as shown by the intensity bar; some high-intensity values are truncated. (F,G) Binary results for Alexa and COIN where each pixel is classified as positive (red) or negative (gray). The lumen region is rejected from the binary analysis due to the difficulty of accurately identifying this boundary in brightfield images as well as the occasional presence of stained debris in the lumen. Note the presence of stained tissue debris in D. Bar = 50 μm.