Computational protocol: Near Infrared Fluorescent Nanoparticles Derived from Hyaluronic Acid Improve Tumor Contrast for Image-Guided Surgery

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Protocol publication

[…] Breast tissue mimicking phantoms were constructed as described elsewhere ,. Briefly, 10% gelatin powder (Spectrum; New Brunswick, NJ), was dissolved in 50 mM Tris-HCl with 15 mM NaN3. This was dissolved by stirring at 50 °C, followed by cooling to 33-37 °C. Dried bovine erythrocytes (Sigma; St. Louis, MO) were then added to achieve 17 μM hemoglobin, followed by 1 % (v/v) Intralipid 20% (Sigma; St. Louis, MO). This mixture was then aliquoted into tissue culture dishes to achieve thicknesses of 2, 3, 4, 5, or 6 mm, and placed at 4 °C until ready for use. Tumor fluorescent signal penetration through tissue phantoms was analyzed using FIGSS. Initial readings with no phantoms were taken. Phantoms which measured between 2-6 mm thick in 1 mm increments were then placed sequentially over the tumors and imaged using 30 mW laser excitation. Images taken using the IGS system were rated with a qualitative “detection factor” (DF) using a strong (+++), moderate (++), weak (+), and absent (-) scale. Color and NIR channel images were saved separately followed by processing with ImageJ (NIH; Bethesda, MD). All images were processed identically. [...] Tumor tissue from image-guided surgery studies was immediately placed in OCT mounting media gel and rapidly frozen in liquid nitrogen. These samples were then cut and processed by the UNMC Tissue Sciences Facility. Sections from each sample were stained with hematoxylin and eosin (H&E) or were left unstained for NIR fluorescence microscpy. Samples were analyzed with an Olympus IX73 Inverted Microscope with a xenon excitation source and imaged with an Olympus DP80 Digital Camera and cellSens Dimension software. H&E-stained tissue samples were visualized and imaged with brightfield microscopy. Unstained tissue samples were visualized and imaged for autofluorescence (FITC filter cube) and NIR fluorescence (ICG filter cube). Exposure times of each channel were manually adjusted to maintain clear image resolution. Background signal from images in the autofluorescence and NIR channels were black-balanced on an area that contain tissue using an automated algorithm provided by cellSens Dimension software. NIR signal was pseudo-colored yellow, but with no additional signal processing, using ImageJ software (https://imagej.nih.gov/ij/). […]

Pipeline specifications

Software tools ImageJ, cellSens
Applications Bright-field microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Homo sapiens/Mus musculus xenograft, Mus musculus
Diseases Breast Neoplasms, Neoplasms
Chemicals Hyaluronic Acid, Indocyanine Green