br bandpass emission filter Whole tissue images were acquire
605 ± 35 bandpass emission filter. Whole tissue images were acquired using Zeiss Axioscan Z.1 slide scanner (Carl Zeiss) at 20× magnifica-tion. The fluorescence signal was optimized at the start of the study so as to not oversaturate the signal. For a standardized comparison, the power of Colibri 7 light source and P22077 times of the Hamamatsu ORCA Flash4 camera (Hamamatsu Photonics) were kept constant for all samples. The imaging was performed on slides overnight after staining with identical image acquisition settings.
2.3. Statistical analysis
Statistical analysis was performed using MatLab. To quantify tumor-to-stroma pancreas tissue diagnostic detection, ROC curves were gen-erated for ductal adenocarcinoma versus stroma tissue. As a control, ROC curves were generated for healthy acinar tissue versus healthy acinar tissue. ROC curves and corresponding AUC measurements were calculated on a pixel-by-pixel basis by running a threshold value across individual pixel values for each tissue type and measuring the true-positive and false-negative fractions of adenocarcinoma ductal tissue over surrounding stromal tissue at each threshold increment. Histogram plots of the fluorescence image pixel values for ductal and stromal pixels were generated and plotted. Statistical significance between staining conditions was determined using the method described by Hanley and McNeil  with standard error measurements and cor-relation coeﬃcients for pixel intensity values for each condition used for z-score value calculations. Z-score values > 2 were considered statistically significant.
2.4. Original ex vivo direct fluorophore staining protocol
The initial wash to remove excess OCT was done by covering each tissue sample with PBS. After 2 min, it was removed with vacuum. The tissue samples were fixed with 2% PFA at rt. for 15 min and washed 3× using PBS with 0.1% Tween-20 for 5 min. Slides were incubated with fluorophore formulation, (10% dimethyl sulfoxide DMSO (control) or 10% DMSO fluorophore solution (dependent on specific concentra-tion of fluorophore), 5% Kolliphor EL, 85% of 75/25 serum/PBS) for 40 min at rt . Slides were covered to prevent photobleaching. Ex-cess fluorophore was removed through two 5 min rinses with PBS. Fluorophore post-fixing was done using 2% PFA for 15 min and washed 1× with PBS for 5 min. After removing excess PBS samples were mounted with fluoromount. The coverslip was placed onto tissue, making sure no air bubbles were present. The coverslips were sealed with colorless nail polish. The slides were stored at 20 °C. The total time for the staining protocol was 1 h 42 min.
2.5. Modified ex vivo direct fluorophore staining protocol
The initial rinse was done by flash washing each tissue sample with PBS with rapid removal with vacuum. The tissue samples were fixed with 2% PFA at rt for 5 min, rinsed rapidly 3× using PBS with 0.1% Tween-20. Slides were incubated with fluorophore formulation (5 min, rt). Slides were covered to prevent photobleaching. The excess fluor-ophore was removed by flash rinsing 2× with PBS. Fluorophore post-fixing was done using 2% PFA for 5 min and with a 1× wash with PBS. Excess PBS was removed and samples were mounted mounting with fluoromount. The coverslip was placed onto tissue, ensuring no air bubbles covered the samples. The coverslips were sealed with colorless nail polish. The slides were stored at 20 °C. The total time for the op-timized protocol was 15 min.
3. Results and discussion
3.1. Staining of human PDAC tissue
Our lab previously showed that 1 enabled the imaging of PDAC in a genetically engineered mouse model with high specificity both in vivo and ex vivo. Fig. 2 shows the ex vivo eﬃcacy of 1 in visualizing human PDAC, showing increased accumulation in the ductal adenocarcinoma epithelial tissue compared to the surrounding stroma tissue. In normal tissue, 1 shows a homogenous accumulation as also previously reported in our mouse model work .
To obtain a more complete picture of the inherent accumulation of 1 in ductal adenocarcinoma versus the stroma tissue, whole tissue images were obtained using Zeiss Axioscan Z.1 slide scanner (Fig. 3). The
I.R. Munhenzva, et al. Methods xxx (xxxx) xxx–xxx
Fig. 2. Ex vivo images from frozen sections of resected human tissue stained with com-pound 1. Normal tissue displayed homo-geneous staining in acinar cells. PDAC tissue displayed high contrast between the ductal and stroma tissue. White arrows are pointing to the selectively stained ducts with adenocarcinoma surrounded by blue
starred unstained stroma tissue regions of PDAC. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)