Cryo-Fluorescence Tomography (CFT) is a method to interrogate whole organs and animals for molecular probes, chiefly fluorescent markers, in 3D, at very high resolution and very high dynamic range. Xerra is a device from Emit Imaging which uses CFT to do tracking of gene expression, immunologics and early stage PK/PD and biodistribution studies.
CFT is an ex vivo Molecular Tissue Imaging (MTI) technique where we generate 3D fluorescence and 3D anatomical images from individual 2D sections. CFT is a meso-scopic technique that can visualize cell clusters up to large organs within samples from isolated organs to whole intact animals and it is not exclusive of other techniques so fits into any research workflow. CFT is a powerful validation tool for invivo imaging, and can also facilitate more accurate histology and pathology studies like auto-rad, IHC, H&E etc.
CFT uses LED laser excitation light with narrow band emission filters to produce a stack of 2D block face images to reconstruct into a 3D volume.
Adeno-associated virus vectors (AAV) are emerging tools in gene therapies, however, in their development, it can be very difficult to map the full extent of the gene delivery.
CFT has been used to map Her2 expressing tumor cells which co-express Tdtomato, novel probes with a Her-2 protein targeting antibody labeled with Alexa Fluor
IVIS data does not show non specific binding or renal clearance and is not good for longitudinal studies. Histology is also difficult and very labor intensive. Xerra gives much better data on probes kinetics, targeting and non-specific binding.
Researchers can label macrophages with a near infrared probe like Texas Red, Cy7 IRdye 800, IRdye 700, Cyanine based dyes, Rhodamine dyes, BODIPY dyes, Quantum Dots etc. to try to visualize inflammatory response. Marcrophages and tumor associated macrophages in response to tumor growth are a great use of CFT. Xerra produces 16 bit images for large dynamic range and high resolution data.
Macrophages can be seen in long bone and in the bone marrow along with circulation and heterogeneous uptake in the tumor and lymph nodes and liver and spleen.
Xerra multi channel data gives us an excellent view of the tumor micro environment and can visualize Green Fluorescent Protein (GFP), Cerulean (CFP), Anthozoan fluorescent proteins, Yellow Fluorescent protein (YFP), Red fluorescent protein RFP, mCherry, TagRFP, etc.
Fluorescent protein reporter genes in the Adeno associated virus vectors, AAVs, are a great application for CFT which can visualize AAV propagation through out the whole animal. Since we are imaging the top surface we are not hindered by short wavelengths.
3D maximum intensity projections (MIPs) of Patient Derived Xenograft, PDx, tumors with a novel molecular probe injected IV prior to surgical resection can be imaged.
Vascular images can be made with Indocyanine green (ICG), Evans Blue, Fluorescein and CFT can image Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence
CFT is invaluable in immune oncology targets like PD1, PDL1, CD4, CD8 probes etc and can also image labeled T cells and B cells that have been injected after flow cytometry. CFT is a great precursor to Radio labeling
CFT has been used to track Exosomes, antisense oligonucleotide (ASOs), mRNA and other gene therapies.
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