Fig 1.
(a) Schematic of the QDAC-DDSI tissue staining protocol. (b) Photograph of the hyperspectral specimen imaging system with major components labeled. (c) Schematic of the image processing steps. Pixel-wise spectral fitting of the hyperspectral image stack extracts the two QDAC channels, which are then normalized to a calibration volume and used to compute the DDSI image.
Fig 2.
Linearity of response of the hyperspectral imaging system to co-localized quantum dot concentration in tissue-simulating phantoms: (a) Mean values of fluorescence signal plotted as a function of QD-605 concentration (0 to 40 nM) while QD-655 was maintained at 20 nM. In (b), similar data for the reverse case (QD-605 constant with changing QD-655) are shown.
Fig 3.
Confirmation of receptor-specific binding in vitro: (a) HER2 expression of two breast cancer cell lines (MCF7-parent line, termed HER2(-) and a transfected MCF7 line expressing HER2, termed HER2(+)), determined by flow cytometry. (b) Representative fluorescence micrographs of HER2(+) and HER2(-) cells stained in vitro with QDAC-605-targeted (QD-605 conjugated to trastuzumab) and QDAC-655-targeted (QD-655 conjugated to trastuzumab). The red channel represents quantum dot fluorescence and blue DAPI staining. (c) Fluorescence micrographs of both cell lines stained with the untargeted QDAC counterparts (QD-605 or QD-655 conjugated to mouse-IgG). Scale bars on all images are 10 μm. Quantitative analysis of images in (b) and (c) are provided in (d) and (e), respectively.
Fig 4.
Representative specimen images after topical staining/washing for all four staining conditions investigated.
Columns labeled “Untargeted” and ‘Targeted” represent images of the tissue after pixel-by-pixel spectral fitting of the hyperspectral data and normalization to the calibration volume, and thus show the fluorescence of the targeted and untargeted QDAC channels. The DDSI column shows the processed DDSI images. “N” and “T” refer to normal and tumor tissue, respectively. Each panel presents four specimen samples from each staining condition: (a) Incubation in a 10 nM stain solution at 37 °C, (b) Incubation in a 10 nM stain solution at 22 °C, (c) Incubation in a 100 nM stain solution at 37 °C, (d) Incubation in a 10 nM stain solution at 37 °C with the quantum dot labels reversed.
Fig 5.
Receiver operating characteristic curves and AUC values plotted for each condition corresponding to the panel arrangement in Fig 4: (a) Incubation in a 10 nM stain solution at 37 °C (N = 4 tumor specimens), (b) Incubation in a 10 nM stain solution at 22 °C (N = 4 tumor specimens), (c) Incubation in a 100 nM stain solution at 37 °C (N = 6 tumor specimens), (d) Incubation in a 10 nM stain solution at 37 °C with the quantum dot labels reversed (N = 5 tumor specimens).
Fig 6.
Images of DDSI, H&E and HER2-IHC for each tissue specimen for all staining conditions investigated: (a) Incubation in a 10 nM stain solution at 37 °C, (b) Incubation in a 10 nM stain solution at 22 °C, (c) Incubation in a 100 nM stain solution at 37 °C, (d) Incubation in a 10 nM stain solution at 37 °C with the quantum dot labels reversed. DDSI images represent the surface of fresh specimens while H&E and HER2-IHC are from tissue sections as close to the surface as possible.