Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

The overall goal of the following experiment is to produce fluorescence images of epidermal growth factor receptor expression in an orthotopic glioma mouse model. This is achieved by inoculating the left cerebral hemisphere of an A IIC mouse with green fluorescent protein expressing human U2 51 glioma cells. After the tumor has grown for two weeks, the mouse is injected with a cocktail of two fluorescent tracers.

The mouse is then imaged on a small animal x-ray system to collect anatomical data necessary for accurate image reconstruction. The data is used to anatomically position the source and detect locations of the optical tomography system, and customized software is used to collect the fluorescence data and to construct the final image. Ultimately, this method can construct an image of a tumor based on its overexpression of epidermal growth factor using both fluorescence and x-ray imaging, the accuracy of which can be compared with contrast enhanced magnetic resonance imaging.

The main advantage of this technique over existing methods like charge couple, device based small animal fluorescence tomography is that single photon counting detection using photo multiplier tubes offers a much higher sensitivity and dynamic range for light detection. This method can ultimately be used to monitor, gauge the success of molecular therapies by measuring the expression of their targets While subcutaneous tumors are readily imaged with non tomographic methods using surface imaging. This system is designed to image through tissue up to several centimeters thick, making it especially well suited to image, contrast, agent delivery, and leakage into brain tumors.

Though this method can provide insight into cancer biomarker expression, it can also be applied to other studies of disease such as infection, obesity, or aging elements such as Alzheimer's. Generally, individuals new to this method will struggle because of the diffuse nature of light propagation in biological tissue and the difficulty in using x-ray tomography for the diffuse light reconstruction. Visual demonstration of this method is critical.

As the data collection and image reconstruction steps can be difficult to learn, ensuring that the data collected from the fluorescent system is well calibrated is important. The near fast software package had been designed to read in optical data and use the x-ray tomography images to create a finite element mesh, which is used as a spatial template for fluorescence reconstruction. To begin place an anesthetized athymic nude mouse onto a sterile surgical drape and confirm the depth of anesthesia before proceeding clean and disinfect the skin over the incision area.

Using Betadine place a sterile surgical drape over the incision site using a scalpel, make a small incision slightly left of the midline and 0.5 centimeters posterior to the intraocular line. Clean the surface of the skull by clearing away any connective tissue so that landmarks can be identified. Once the skull is exposed, use a high speed drill with a sterile one millimeter bit to make a hole that is two millimeters left of the central line and two millimeters behind the bgma.

Load a Hamilton Micros syringe with a blunt ended 27 gauge needle with the cells to be implanted. Next, place the mouse onto a stereotaxic frame. Then confirm a deep surgical and aesthetic plane with a toe pinch and replace the surgical drape on the animal.

Next, secure the loaded syringe onto the stereotaxic frame. Position the syringe over the hole in the skull and insert the tip of the needle three millimeters below the skull surface. Next, withdraw the needle one millimeter to create a pocket.

The next step is to slowly inject the cells into the left cerebral hemisphere over a five minute period. Once the injection is complete, withdraw the needle and swab the hole with Betadine. To prevent cells from growing outside of the injection site, remove the mouse from the stereotaxic frame and fill the exposed hole with prewarm bone wax.

Lastly, close the incision site with sterile five oh nylon suture. Return the mouse to a heated cage and monitor until recovered after recovery, inject the mouse with 130 microliters of 0.1 milligram per kilogram buprenorphine IP and allow the tumor to grow for 14 days prior to imaging. On the day of mouse imaging, initiate the system to allow the lasers and light detectors to warm up for approximately 20 minutes.

To avoid drifts and system sensitivity, place a 100 degree by four degree engineered line diffuser at the direct center of the imaging gantry to disperse the excitation lasers amongst the system's detection channels in equal intensity. Adjust the angle of the diffuser by hand to maximize the amount of signal detected by all five light collection channels. Next place optical density.

Two neutral density filters in front of all fluorescence detection photomultiplier tubes called PMTs and optical density. One neutral density filters in front of all transmittance detection. PMTs collect 100 temporal pulse spread functions or T psfs of the laser each with a one second integration time for each laser sequentially, normalize each TPSF by the laser reference correct for temporal drift in the laser reference and average over all iterations for each detector and each laser separately.

These average TPSs are the detector specific instrument response functions used in the optical image reconstruction 12 hours prior to the imaging procedure. Inject the test mouse with a cocktail of fluorescent tracers. Accurate calibration of the fluorescent tomography system and restricting animal motion are the most difficult aspects of this procedure.

The imagery reconstruction is so ill posd that even slight errors in data collection can lead to significant errors in the reconstructed image. To ensure accurate data is collected, we immobilize the mouse as best as possible and repeat the calibration before and after scanning to improve the chance of getting an accurate calibration When ready. Place the anesthetized animal onto the fiberglass supports of the imaging bed.

After positioning the head into the nose cone for continuous gas anesthesia, secure the teeth on the bite bar and tape down the animal. The mouse should be placed at the approximate center of the imaging gantry. This positioning can be guided by rotating the excitation laser 180 degrees about the mouse, ensuring that the focal point of the laser illuminates a point roughly on the center of the mouse from the perspective of the laser at all angles.

Once positioned properly, carefully transferred the imaging bed and the mouse to the micro CT scanner and collect anatomical information at a resolution of 93 micrometers isotropic for the whole head of the mouse. Visualize the CT image stack and choose the slices to be imaged with the fluorescence tomography system. Carefully transfer the imaging bed and mouse back to the fluorescence tomography system.

Choose the number of source positions for each imaging slice the integration time for each TPSF measurement, the number of iterations for each source position, and the position and number of desired imaging slices from the CT image stack created earlier. Next place filters in front of the fluorescence detection PMTs to block out all excitation light and optical density to neutral density filters in front of the transmittance detection PMTs. To avoid saturation of those detectors, run the data acquisition software collecting fluorescence and transmittance t psfs at each defined source detector position at both excitation wavelengths.

For every set of T psfs collected, monitor and record the laser intensity with a reference PMT channel. Determine the outer surface of the mouse and the location of the imaging bed. Support rods from the CT images and create masks that cover the confines of the mouse and the imaging rod.

Separately, use the mouse mask to produce a finite element mesh of the animal using the near fast software. Localize the source and detector positions from the fluorescence tomography system on the surface of the mesh based on micro CT and fluorescent spatial registration coordinates. Remove optical data points associated with source or detector positions that interact with the location of the imaging bed.

Support rods normalize data collected at each source detector position by the laser reference correct for temporal drift in the laser reference and correct for filter sensitivities, which were determined by experimental testing at time of purchase. Take the born ratio of the data for each source detector position and multiply with a forward model simulation of transmittance based on the finite element animal mesh for uniform optical properties. This is done to mitigate errors associated with source or detector tissue coupling to calibrate the data to the model and to adjust data for other aspects of model data mismatch construct a data vector composed of the scaled difference of the born ratio data collected at both wavelengths.

The scaling factor is chosen to maximize EGFR binding contrast, perform time domain image reconstruction with the calibrated difference data using the TPSF for each detection channel as an input and create fluorescence maps of the contrast enhanced targeted tracer seen. Here is an example of a fluorescence reconstruction overlaid with a coregistered CT anatomical image from a mouse with a U2 51 orthotopic glioma tumor. The center of mass of the glioma determined by the fluorescence reconstruction was within one millimeter of the tumor center of mass, determined by contrast enhanced magnetic resonance imaging.

The software for data acquisition is custom built for this device, but most of the image processing techniques can be done using the near fasts software available@nearfasts.org. So while attempting this procedure, it's important to ensure that both the system and the data are calibrated accurately before the image reconstruction. This calibration requires that the sensitivity and temporal differences between the detectors are accounted for Following image reconstruction of a biomarker targeted tracer.

Similar imaging of an untargeted tracer provides a means of correcting for non receptor mediated uptake. This allows the amount of tracer binding as well as the in vivo receptor density to be quantified After its development. This technique inspired other researchers to design x-ray imaging guided fluorescence tomography systems, and paved the way for whole body imaging of larger animals.

After watching this video, you should have a good understanding of how to carry out a diffuse fluorescence tomography experiment, which combines x-ray anatomical imaging and optical imaging systems for cancer biomarker imaging.