The IVIS® Spectrum in vivo imaging system combines 2D optical and 3D optical tomography in one platform. The system uses leading optical technology for preclinical imaging research and development ideal for non-invasive longitudinal monitoring of disease progression, cell trafficking and gene expression patterns in living animals.
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An optimized set of high efficiency filters and spectral un-mixing algorithms lets you take full advantage of bioluminescent and fluorescent reporters across the blue to near infrared wavelength region. It also offers single-view 3D tomography for both fluorescent and bioluminescent reporters that can be analyzed in an anatomical context using our Digital Mouse Atlas or registered with our multimodality module to other tomographic technologies such as MR, CT or PET.
For advanced fluorescence pre-clinical imaging, the IVIS Spectrum has the capability to use either trans-illumination (from the bottom) or epi-illumination (from the top) to illuminate in vivo fluorescent sources. 3D diffuse fluorescence tomography can be performed to determine source localization and concentration using the combination of structured light and trans illumination fluorescent images. The instrument is equipped with 10 narrow band excitation filters (30nm bandwidth) and 18 narrow band emission filters (20nm bandwidth) that assist in significantly reducing autofluorescence by the spectral scanning of filters and the use of spectral unmixing algorithms. In addition, the spectral unmixing tools allow the researcher to separate signals from multiple fluorescent reporters within the same animal.
|Imaging Modality||Optical Imaging|
|Optical Imaging Classification||Bioluminescence imaging, Fluorescence Imaging|
|Product Brand Name||IVIS|
Pre-clinical in vivo imaging,Advances in the detection and quantification for 3D optical tomography of bioluminescent and fluorescent reporters to quantify in terms of either cell number or absolute pmol concentration will be discussed. These methods include enhancing the detected signal levels using slight compression which reduces the amount of tissue light propagates through. Calibration techniques to improve signal location by reducing the excitation light artifacts, the amount of detected autofluorescence and techniques to quantify 3D reconstruction results in terms of biological activity will be demonstrated.,Introduction,Optical tomography of bioluminescent and fluorescent reporters in pre-clinical animal models is an important technology for the translational study of disease and drug development. However, the quality of 3D reconstructions can be limited by the sensitivity of detection. 3D results often lack quantification with biologically relevant units, therefore, new methods to improve detection and to quantify the results in terms of absolute cell number or dye molecules have been developed.
Cerenkov Emission from radioisotopes in tissue,Optical imaging detects photons in the visible range of the electromagnetic,spectrum. PET and SPECT imaging instruments are sensitive to photons in the much,higher energy range of x-rays and gamma rays. While the PET and SPECT probes,which can generate Cerenkov light in tissue will continue to produce the relevant,gamma- and x-rays, visible photons will be produced from the Cerenkov emission,which the IVIS® will detect.,In beta decay emitters such as PET probes and isotopes such as 90Y, 177Lu, 131I and 32P,the beta particle will travel in the tissue until it either annihilates with an electron or,loses momentum due to viscous electromagnetic forces.,It is possible that the beta (electron or positron),is relativistic, traveling faster than the speed,of light in the tissue. While it is impossible,to travel than the speed of light in a vacuum,(c), the speed of light in tissue is v = c / n,where n is the tissue index of refraction and,n = 1. Cerenkov photons will be generated,by a relativistic charged particle in a dielectric medium such as tissue.
With the potential to treat a wide range of disease, from organ damage to congenital defects, stem cell research and tissue engineering form the underlying basis of regenerative medicine. Significant advances in the science of skin regeneration, for example, have now made it possible to develop and grow artificial skin grafts in a lab for treatment of burn victims. Other therapeutic applications include the use of stem cells to treat and repair central nervous system diseases such as ischemia and cerebral palsy, cardiovascular diseases, as well as autoimmune diseases including type I diabetes.
Optical-based in vivo imaging of vascular changes and vascular leak is an emerging modality for studying altered physiology in a variety of different cancers and inflammatory states. A number of fluorescent imaging probes that circulate with the blood, but have no target selectivity, have been used to detect tumor leakiness as an indication of abnormal tumor vasculature. Inflammation is also characterized by distinct vascular changes, including vasodilation and increased vascular permeability, which are induced by the actions of various inflammatory mediators. This process is essential for facilitating access for appropriate cells, cytokines, and other factors to tissue sites in need of healing or protection from infection. This application note investigates the use of three fluorescent imaging probes, to detect and monitor vascular leak and inflammation in preclinical mouse breast cancer models.
It’s simple: More information means more understanding,For today’s researchers in oncology, infectious diseases, inflammation, neuroscience, stem cells,and other disciplines, there’s an increasing need for in vivo imaging that enables you to visualize,multiple events simultaneously and to extract the maximum amount of information from each,subject – leading to greater biological understanding.,Multimodal imaging enables a better understanding of disease biology. By utilizing in vivo,optimized bioluminescent and fluorescent agents and radioactive probes, researchers can,measure depth, volume, concentration, and metabolic activity, providing a wealth of information,for untangling the mysteries of disease.,Coregistration allows researchers to overlay images from multiple imaging modalities, providing,more comprehensive insight into the molecular and anatomical features of a model subject.,For example, optical imaging data can be used to identify and quantify tumor burden at,the molecular level and, when integrated with microCT, provides a quantitative 3D view of,anatomical and functional readouts.,At PerkinElmer, we’ve developed industry leading imaging technology for preclinical research.,Our technology integrates 3D optical and PET modalities with microCT to provide a better,understanding of disease. And that means better monitoring of disease progression, earlier,detection of treatment efficacy, and deeper understanding of metabolic changes that take place,throughout disease development.
Researchers trust our in vivo imaging solutions to give them reliable, calibrated data that reveals pathway characterization and therapeutic efficacies for a broad range of indications. Our reagents, instruments, and applications support have helped hundreds of research projects over the years. And our hard-earned expertise makes us a trusted provider of pre-clinical imaging solutions— with more than 9,000 peer reviewed articles as proof.
Colorectal cancer patients often develop liver metastases and thus, frequently have poor prognosis. There additionally appears to be a vast heterogeneity in their liver metastatic disease, a characteristic that hasn’t been adequately explored in animal models of colorectal cancer. While bioluminescence imaging (BLI) has been widely used to non-invasively monitor colorectal cancer and liver metastastic development in vivo, a study specifically emphasizing their growth rates and colonization efficiencies within the liver microenvironment hasn’t been attempted until now.
The IVIS Spectrum advanced pre-clinical optical imaging system combines high throughput and full tomographic optical imaging in one platform. The system uses leading optical imaging technology to facilitate non-invasive longitudinal monitoring of disease progression, cell trafficking and gene expression patterns in living animals. Take full advantage of bioluminescent and fluorescent reporters across the blue to near infrared wavelength region using optimized set of high efficiency filters and spectral unmixing. It also offers true 3D tomography for both fluorescent and bioluminescent reporters that can be analyzed in anatomical context against a Digital Mouse Atlas or registered with other tomographic technologies such as MR, CT or PET through the multimodality module.
Adaptive Fluorescence Background Subtraction Pre-clinical in vivo imaging technical note for IVIS Imaging Systems. Instrument background occurs when excitation light leaks through the emission filter. This occurs more frequently when the excitation and emission filters are narrowly separated. The ring you see is a result of non specific light reflecting off of the stage at an incident angle and passing through the filter causing what appears as leakage around the edges.
Auto-exposure technical note for IVIS pre-clinical imaging systems
DLIT setup and acquisition IVIS pre-clinical imaging systems. Bioluminescence Tomography or Diffuse Light Imaging Tomography (DLIT) utilizes the data obtained from a filtered 2D bioluminescent sequence in combination with a surface topography to represent the bioluminescent source in a 3D space. Utilizing DLIT, you can determine the depth of sources in your animal and calculate the absolute intensity of that source.
DLIT 2 Topography technical note for IVIS Spectrum imaging system. The IVIS Spectrum has a laser galvanometer that we routinely use to project the FOV onto the surface of the instrument. It produces the green outline you see on the stage when the door is opened. We utilize this laser to project a series of parallel lines across your subject. We acquire a photographic image (the Structured Light Image) when the lines are projected across the animal and from that image we can calculate the height at points on the back of your subject based on the curvature of these laser lines as they cross over the subject. This height map allows us to reconstruct a shell or isosurface of your animal. This shell is referred to as the Surface Topography and is used in calculating bioluminescent signal depth and intensity during the DLIT 3D source reconstruction.
DLIT 3 Reconstruction technical note for IVIS Spectrum imaging systems
Determine Saturation for IVIS imaging systems - technical note
Technical notes for Drawing ROIs for IVIS in vivo imaging systems. The circle, square, free draw, or grid (for well plates) can be used to draw your ROIs. ROI selections,are user-specific and are dependent on the model being analyzed. It is irrelevant which shape that is used for a particular ROI.
Fluorescence Tomography – Setup and Sequence Acquisition
Fluorescence Tomography – Source Reconstruction and Analysis - FLIT Reconstruction
Acquisition of High Resolution Images. This quick reference guide is for those researchers who wish to perform analysis that requires high resolution including in vitro studies when one may want to discern aspects about cell layers, ex vivo tissue imaging, or imaging of tissue slices. You will not need this resolution in most in vivo studies.
Not only is it possible to load multiple images as a group, for example multiple days of a longitudinal study, but it is also possible to load multiple images and Overlay them i.e. bioluminescent tumor with fluorescent targeted drug acquired in two separate images.
It is possible to copy 3D sources (voxels) from one 3D reconstruction into another. For example, superimposing DLIT or FLIT signals is easy. However, the two combined sources must be based upon the same surface topography to produce meaningful information. Therefore it is imperative that the mouse remain completely still between acquisition of the DLIT and FLIT images.
Acquiring the most accurate quantitation of your bioluminescent sources requires a close understanding of the underlying kinetics involved in producing and capturing the detected light. After injection, the substrate for your bioluminescent probe will diffuse through the body of your subject eventually coming in contact with the luciferase enzyme in your target cells. In the case of Firefly luciferase, D-luciferin is catalyzed to oxyluciferin in the presence of ATP, Mg2+, and oxygen producing light as a consequence. It is this light that is measured and used to accurately quantify your samples in vivo. The diffusion of D-luciferin is dependent on several factors including but not limited to method of injection, metabolism, and tissue localization of your source.
For many studies, it may be desirable to open a group of images together, for example, analyzing multiple days of longitudinal study side by side using the same scale.
This guide will walk you through the steps of manually entering your sequences for the spectral unmixing procedure. The Living Image 4.3.1 software version includes an Autoexposure setting and an Imaging Wizard. For questions on how to use these two features please see the respective quick reference guide associated with these workflows. You can also find information pertaining to the use of these features in the Spectral Unmixing Wizard Setup reference guide. These features are designed to make setting up your sequences as easy as possible and we highly recommend that you take advantage of them when performing these steps.
Subject ROI using IVIS imaging systems
Transillumination is a 2D fluorescence imaging technique that utilizes an excitation light source located below the stage. Transillumination is superior to epi-illumination at detection of red-shifted, deep tissue fluorescent sources due to the transilluminator’s concentrated delivery of excitation light into the subject via a 2 mm beam and lower autofluorescence levels attained due to the position of the animal in relation to the excitation light.
In order to facilitate faster transillumination imaging, with Living Image 4, we have incorporated raster scanning capabilities. With raster scanning, the shutter remains open as the transillumination excitation source moves underneath the animal. This results in a single image and faster imaging times.,Note: This Transillumination Fluorescence – Raster Scan Tech Note was designed as a supplement to Transillumination Fluorescence Tech Note 14a. For information about setup of your 2D transillumination fluorescence sequences, please first consult that tech note.
Normalized transmission fluorescence is a technique that allows us to subtract background light leakage through thin tissue from transillumination images utilizing an extra image captured with a neutral density (ND) filter. The ND filter dampens the intensity of the halogen lamp to 1/100th of the source intensity but does not filter out specific wavelengths. Light of all wavelengths is allowed to pass through the animal and the image is collected with the emission filter of your choice.
Bioluminescence and Fluorescence tomography allows a user to determine not only the depth and anatomical localization of their source(s) but also the intensity of those sources expressed as either photons/second (DLIT) or total fluorescent yield (pmol/M cm) (FLIT). It is possible to extrapolate the number of cells in a DLIT source or the number of dye molecules or cells in a FLIT source if a quantification database is available. The database is derived from an analysis of images of known serial dilutions of luminescent or fluorescent cells, dye molecules, or labeled compounds.
Working with Image Math. Image Math is a rudimentary but effective method for Spectrum and Lumina users to subtract background from images without performing Spectral Unmixing.
The primary goal of preclinical imaging is to improve the odds of clinical success and reduce drug discovery and development time and costs. Advances in non-invasive in vivo imaging techniques have raised the use of animal models in drug discovery and development to a new level by enabling quick and efficient drug screening and evaluation. Read this White Paper to learn how preclinical in vivo imaging helps to ensure that smart choices are made by providing Go/No-Go decisions and de-risking drug candidates early on, significantly reducing time to the clinic and lowering costs all while maximizing biological understanding.