The overall goal of this procedure is to detect and analyze the in vivo systemic delivery of the tumor targeted nanoparticle HeroX using multimode optical imaging. First, the nanoparticle is injected into the tail vein of a tumor bearing mouse and fluorescence images are acquired using a multi-mode optical imaging system. Following euthanasia, the tumor and organs are harvested and imaged again to detect fluorescent particles within tumor cells.
In situ confocal imaging is then performed. Analysis of the resulting images demonstrates quantifiable targeting and penetration of her docs into tumors after systemic delivery. A domain of advantages of multimodality imaging techniques over the existing techniques such as single imaging mode is providing multiple and complementary information that can enhance analysis and quantification of tumor delivery.
This method can help answer key questions in the drug delivery field, such as whether a targeted drug is not only delivered to tumors but actually penetrates into tumor cells. Demonstrating the procedure will be Dr.J Yun Wang, a former postdoctoral fellow in my lab, and Janet Markman, a current graduate student In this protocol. Six to eight week old.
New, new mice bearing subcutaneous bilateral flank xenograft tumors are used. Prepare for systemic delivery by mixing enough HeroX with sterile saline to equate 0.2 milliliters of a 0.004 milligram per kilogram dose of HeroX per injection. Gently draw 0.2 milliliters of the HeroX mixture into a three 10th cc insulin syringe fitted with a 29 gauge needle.
Take care to ensure that there are no bubbles following anesthesia. Inject the entire mixture into the tail vein following the injection. Pinch the tail to prevent blood loss from the injection site.
After the bleeding stops, clean the injection site with a sterile swab and return it to the cage. Repeat injection on the same mouse for six more sequential days. Once per day, the accumulation of HeroX fluorescence in tumors can be detected by the last day of injection.
Day seven, using a multi-mode imager. The procedures here employ the use of an optical imaging system with fluorescence intensity, spectral fluorescence, lifetime, and intra vital confocal imaging capabilities. Shown here is the in vivo optical imager prototype.
The cooled high sensitivity camera and high power laser lines incorporated in this system yield higher contrast fluorescence images compared to commercial optical imaging systems, especially for the in vivo detection of doxorubicin fluorescence. Turn on the multimode in vivo optical imager in the software. Select an emission band pass filter suited for doxorubicin fluorescence detection.
Turn on the laser and place an excitation band pass filter at the laser optical path. After anesthetizing a mouse in the anesthetizing chamber with isoflurane, transfer it to the imaging chamber of the multimode in vivo optical imager. Place a nose cone over the nose of the mouse and open the flow to administer continuous anesthesia.
During image acquisition, acquire fluorescence images using an exposure time of five to 15 seconds. Her doc's fluorescence can be imaged in tumors and specific organs, including the liver, kidney, spleen, heart, and skeletal muscle harvested from euthanized mice 24 hours after the final day of injection. Turn on the multi-mode in vivo optical imager.
As before, then place the tumors and specific organs arranged on a Petri dish. Inside the imaging chamber of the multimode in vivo optical imager acquire fluorescence images of the tissues using an exposure time of five to 15 seconds. Then repeat the same acquisitions using an empty Petri dish, which will serve as the background following acquisition.
Perform image analysis and processing, including background correction or contrast adjustment. In situ confocal imaging allows detection and analysis of herd herd's tumor accumulation at the cellular level. Using a confocal microscope.
Select 488 nanometer laser light for excitation of doxorubicin and 560 nanometer to 620 nanometer emission wavelengths. For doxorubicin fluorescence detection, select a 40 x or 63 x objective and place a drop of immersion oil on it. Place the tumors from the euthanized mouse on a Petri dish to avoid tissue degradation.
Then transfer the tumors to a delta T chamber for confocal imaging. Acquire confocal images of the tumors at sequential focal depths with a step size of one micron and thickness of 20 microns. An example of sequentially acquired images along the Z axis is shown here.
Generate maximum intensity Z projections of the images. Then using image J or a similar program, calculate the mean fluorescence intensities of the maximum intensity Z projection images for the overall field of view ratio. Metric spectral imaging and analysis allows discrimination between doxorubicin, fluorescence and autofluorescence.
Using a laser scanning fluorescence confocal microscope acquire 15 images of the HeroX treated and untreated tumors at a specified depth within the spectral range of 510 to 650 nanometers with a step size of 10 nanometers and excitation at 488 nanometer light using a Leica SPE confocal microscope. Next, prepare a 100 micromolar solution of doxorubicin in saline. Perform spectral imaging of the 100 micromolar doxorubicin solution.
To obtain the pure spectral signature of doxorubicin fluorescence. Acquire the autofluorescence spectral signature from the image cube by imaging untreated tumors. Once all of the data has been collected, generate reference spectral signatures, perform spectral classification of the images and perform linear spectral unmixing of those images to obtain a quantitative assessment of her doc's Accumulation in tumors.
Confocal fluorescence images were obtained at different focal depths in situ, followed by maximum intensity Z projection of the images and acquisition of the mean fluorescence intensity of the projected image. The high tumor fluorescence shown in this image is typical after systemic delivery of her docs and is indicative of its tumor preferential targeting. The fluorescence detected in the skeletal muscle is atypical and may result from anomalies in the injection procedure.
Qualitatively, this image has low background. For quantitative analysis, a background correction is performed using an empty dish as shown here. The resulting background corrected image has a higher background signal at the edge of the image compared to the original because the background image has lower intensities at the edge of the image.
The stacked images shown at the left are representative of the confocal scans of tumor tumor tissue obtained at sequential one micron depths. The image at the right shows the compilation of the stacked images on the left. The compilation image contains the HeroX accumulation information over the indicated Z depths at every pixel, and thus the average of the fluorescence intensities reflects overall herd doc's accumulations in tumors at different depths quantitatively.
To distinguish herd doc's fluorescence from autofluorescence in tumors and quantitatively discriminate the two with high specificity ratio metrics. Spectral imaging and analysis is performed. This image shows the pure spectral signatures for doxorubicin.
The fluorescence intensity of the delineated dotted area at the indicated emission wavelengths produces a histogram showing the profile of doxorubicin over the 510 to 650 nanometer wavelength spectrum. Here, the pure spectral signatures for autofluorescence is shown. The fluorescence emitted from an untreated tumor at the indicated emission wavelengths produces a slope that represents the autofluorescence emission profile.Reference.
Spectral signatures were also created by mixing different ratios of the pure spectral signatures. Using the program, we developed each reference. Spectral signature here represents relative fluorescence of her docks accumulated in tumors with respect to autofluorescence.
After obtaining the spectral signatures of docks and autofluorescence, the dock signals are separated by using linear spectral unmixing. The figure shows the autofluorescence and her docs mixed image before spectral unmixing and the separated her docs image after the spectral unmixing. After watching this video, you should have a good understanding of how to combine fluorescence intensity, comfort car, and spectral imaging modalities to enhance analysis and quantification of tumor delivery.
Don't forget that working with doxorubicin can be hazardous, and precautions such as double gloves and frequent glove changing should always be taken while performing this procedure.
