Fig. 4.

In vivo NIRF imaging and PA imaging using DARPin-800CW conjugates. A NIRF-color merge and NIRF images of orthotopic HT-29_luc2 tumor-bearing mice at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. Images were captured using the clinical Artemis NIRF imager at an exposure time of 150 ms. “T” indicates the tumor localization, while “Cae’” indicates the corresponding background tissue (caecum). Mouse-specific TBRs are indicated in white in the right-upper quadrant of the NIRF images. B TBRs of orthotopic HT-29_luc2 tumors 24 h after intravenous administration of 6 nmol Ac2-800CW or Ec4.1-800CW as measured using the clinical Artemis NIRF imager. Mean TBRs are represented by the horizontal line together with their error bars representing the standard deviation. C Representative US and PA images of orthotopic HT-29_luc2 tumor-bearing mice at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. Images were captured using a penetration depth of approximately 1.5 cm. Tumors are delineated with a green line. D Biodistribution in orthotopic HT-29_luc2 tumors and healthy organs of mice at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. 1: lungs, 2: heart, 3: pancreas, 4: spleen, 5: stomach, 6: small intestine, 7: caecum, 8: rectum, 9: muscle, 10: brain, 11: skin, 12: liver, 13: kidneys, and 14: tumor. E Macroscopic fluorescence biodistribution of orthotopic HT-29_luc2 tumors and healthy organs at 24 h post-injection of Ac2-800CW or Ec4.1-800CW (Pearl imager). F HE staining, 800 nm heatmap and merge, as well as cytokeratin and EpCAM stainings of sequential tissue sections derived from orthotopic HT-29_luc2 tumors at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. Tumors are delineated by dashed white lines. HE-NIRF and cytokeratin-EpCAM images are taken at × 2 and × 15 magnification, respectively. Scale bars represent 100 µm. a.u, arbitrary units; HE, hematoxylin-eosin; MFI, mean fluorescence intensity; NIRF, near-infrared fluorescence; PA, photoacoustic; TBR, tumor-to-background ratio; US, ultrasound