In vivo imaging of small animals for pre-clinical research using fluorescent markers is a novel approach to visualize cellular processes occurring at the micron level. There is growing interest in understanding what occurs at the level of tumor metastasis through direct visualization. For example, studies show that it is possible to use green fluorescent protein (GFP) and red fluorescent protein (RFP) labeled tumor cells to observe their behavior in the vasculature in vivo, such as tumor shedding and extravagation[i].  The iBox Explorer Imaging Microscope, for visualization of the tumor microenvironment both accurately and reliably, will expand the understanding of the mechanism of metastasis. The ability to view fluorescence ranging from the organ level down to the cellular level can add valuable information of in vivo processes. 

In a study, a pancreatic tumor tissue sample was imaged at two magnifications: 2.5x and 8.8x. A red fluorescent protein (RFP) filter highlights the RFP-tagged cytoplasm and observes the distinct morphologic characteristics of individual cells as well as tissue microenvironment ex vivo. Use of the iBox Explorer can aid in the visualization of many cellular processes through fluorescence technology. Visualization can occur across a wide range of magnifications and spanning the visible to near infrared spectrum.   

XPA1 dual-color mouse pancreatic cancer cell line (AntiCancer, Inc.) was grown orthotopically, surgically resected, sectioned with a microtome at a 20-micron thickness, and transferred to a slide for imaging. Two tissue slices were selected from the center of the bulk tumor and placed side by side.  The sample was then stored in a -20°C freezer until it was ready for imaging.

BioLite^TM Xe MultiSpectral Light Source (UVP, LLC), part of the iBox Explorer, provides luminous excitation with a high level of intensity. For excitation of RFP flurophore, a filter (peak wavelength 525nm and bandpass 502-547nm) was used. A darkroom emission filter (peak wavelength 605nm and bandpass of 580-630nm) highlights the RFP-labeled cytoplasm. Images were captured using the iBox Explorer. Illumination was directed coaxially and via side-lighting through the iBox Explorer's optics. Captured images were edited with the background fluorescence removed using histogram adjustment. Images were pseudo-colored according to filtration specifications (RFP/GFP).  

Figure 1 shows 2.5x magnification of an illuminated specimen. Cellular orientation, tissue architecture and cytoplasmic morphology can be clearly delineated using an RFP emission filter.  Further magnification (8.8x) of the same sample (Figure 2) reveals more details of cytoplasmic structure, specifically areas of high concentration of RFP in addition to cytoplasmic extensions and cellular orientation.   

This application illustrates the iBox Explorer for imaging fluorescently-labeled histology samples of tumor tissue. Using the iBox Explorer's high-specification, cooled camera and RFP filters, very crisp images of tumor cells at two different magnifications were obtained. These images clearly delineate cell and tissue borders and cellular morphology. The applications of the iBox Explorer Imaging Microscope can expand to include other aspects of cancer study, such as elucidating the nature of the tumor microenvironment, hematogenous trafficking of metastases, tumor angiogenesis and tumor shedding. With a resolution of 2.28 pixels per micron, the use of the iBox Explorer has the capability to image in vivo applications on the microscopic level.

iBox and VisionWorks are registered trademarks of UVP, LLC. Explorer and BioLite are trademarks of UVP, LLC.

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