Fig 1.
(A) The REMI system and (B) the channel-compression strategy.
(A) Schematic of the spectral-imaging system. A 785-nm laser is used to illuminate the NP-stained tissue, creating a 1 mm-diameter laser spot. Raman-scattered photons from illuminated NPs are collected by 27 multimode fibers and transmitted to a customized spectrometer, where they are dispersed onto a cooled deep-depletion spectroscopic CCD. (B) The camera is used in full-vertical binning (FVB) mode, in which the signal from all 27 collection fibers are binned together by the camera, with 1024 pixels (spectral channels) used to resolve the grating-dispersed wavelength axis. In this study, the 1024-channel data set is further binned along the wavelength axis (horizontal binning) to examine the effects of spectral compression.
Fig 2.
(A-D) Linearity test for 5-flavor NP mixtures and (E,F) the limit of detection (LOD) as the number of bins (spectral channels) is reduced.
(A-D) Five NP flavors were mixed in an equimolar concentration ratio (1:1:1:1:1) or a concentration ratio of 3:2:1:1:1 (S420:S421:S440:S481:S493). Various dilutions of these NP mixtures were prepared in the range of 0.5-100 pM. The NP concentrations were measured with the REMI system to calculate their concentration ratios. (a,b) Linearity plots for the 1:1:1:1:1 NP mixture. (C,D) Linearity plots for the 3:2:1:1:1 NP mixture. Error bars represent the standard deviation across 5 repeated experiments. Note that the “sample concentration” refers to the concentration of the S440 NP (which serves as the negative-control NP). (E,F) The limit of detection (LOD) is defined as the concentration at which the error in the measured concentration ratio exceeds 10%. The shaded curves indicate the standard deviation in the LOD measurements from 5 repeated experiments.
Fig 3.
REMI approach performed on an A431 tumor xenograft (EGFR++, HER2+) stained with a 5-flavor NP mixture (EGFR-, HER2-, Control-S481-, Control-S493-, and isotype-NPs, 150 pM/flavor).
(A) Ratiometric images of the tumor xenograft. From top to bottom, each row shows the ratio of EGFR/isotype-NP, HER2/isotype-NP, unconjugated-S481/isotype-NP, and unconjugated-S493/isotype-NP, respectively. From left to right, each column shows the ratiometric image obtained with a decreasing number of spectral channels. (B) A photograph of the A431 tumor xenograft. (C) H&E-stained pathology section of the tumor xenograft. (D) Average error (%) in the measured NP ratios when using spectral compression in comparison with the gold-standard images (full 1024 channels). Error bars represent the standard deviation amongst all pixels in the image.
Fig 4.
REMI approach performed on a human breast tissue specimen stained with a 5-flavor NP mixture (EGFR-, HER2-, CD24-, CD44-, and isotype-NPs, 150 pM/flavor).
(A) Ratiometric images of a human breast tissue specimen. From top to bottom, the rows display ratiometric images of EGFR/isotype-NP, HER2/isotype-NP, CD24/isotype-NP and CD44/isotype-NP. From left to right, the columns display ratiometric images obtained with a decreasing number of spectral channels. (B) A photograph of the tissue specimen. (C) H&E histology of the specimen, with higher magnification views of fat (left), normal breast tissue (middle), and tumor (right). Unlabeled scale bars represent 200 μm. (D) Average error (%) in the measured NP ratios when using spectral compression in comparison to the gold-standard images (full 1024 spectral channels). The error bars represent the standard deviation amongst all pixels in the image.