This protocol uses Terahertz imaging and spectroscopy to evaluate the surgical margins of breast tumors with the aim of substantially decreasing the need for a second surgery. Terahertz imaging can be used to distinguish between cancerous and non-cancerous tissues based on their optical properties, in a non-ionizing and biologically safe manner processes. This technique was developed for breast cancer margin assessment, but it's applicable to other solid tumors that require surgical excision and margin assessment.
This method allows imaging of the inside of FFPE tissue blocks without the need for physical slicing, facilitating the identification of tumor margins at any depth precise. This protocol requires the performance of critical steps such as tissue handling and stage balancing that can be difficult to understand without visual demonstration. Before handling the tissue cover stainless steel metal tray with a biohazard bag and place all of the appropriate lab materials within easy reach of the covered tray.
Next, transfer the fresh tumor sample into a petri dish on the tray and use gross inspection to select a side of the tumor that is sufficiently flat and has little blood and few blood vessels. Then, place the side to be imaged on grade one filter paper to remove any excess demand medium and to clear the tissue of fluid or secretions, repositioning the tumor to a dry spot as the paper saturates. While keeping the tumor drying for five minutes, place a 1.2 millimetre thick polystyrene plate onto the scanning window within an approximately 37 millimetre diameter and place the scanning window and the polystyrene plate onto the sample stage of the Terahertz imager.
In the main window click the Fixed Point Scan icon to activate the Terahertz antennas and to start sending and receiving the reflected Terahertz signal from a single point on the polystyrene plate. Click the Motor Stage dialog icon. The Motor Control window will open up.
To center the reflected pulse from the polystyrene in the main window, click the optical delay axis arrows to adjust the optical delay axis. Click the Data Acquisition Settings button to open the Data Acquisition Settings Dialogue window and change the optical delay value from five to four volts. Adjust the vertical position of the scanning stage with the micrometre scale, until the minimum of the secondary pulse is the strongest and adjust the optical delay the axis in the Motor Control window to put the primary reflection outside of the range of the reflected signal being measured.
To level the sample stage and record the reference signal, click the motor control dialogue button to open the motor control window and reposition the motor control and main software windows so that the time domain signal is visible while adjusting the motor positions. To level the A axis using the following steps in the motor control window, change the value of the x axis from zero to negative 10 and hit Enter. The stage will move to the minus 10 millimetre position of the A axis and a shift in the signal position will be observed in the main window.
Use the adjustable micrometre scale to move the minimum peak of the signal back to the previous vertical position and change the A axis value to plus 10. Click Enter. The stage will move to the plus 10 millimetre position on the A axis and a shift in the signal will be observed again.
Note that the direction and distance that the signal shifted from its previous position and change the A axis value back to minus 10. The signal will return to the original vertical position. Rotate the leveling screw on the A axis of the scanning stage and shift the signal to double the distance in the same direction, the stage moved from the original position.
Use the micrometer on the scanning stage to shift the signal back to the original position and repeat the adjustment until the signal at plus 10 and minus 10 are equal in the peak for both positions is focused at the original position. Once the leveling of the A axis has been achieved, change the A axis value to zero and repeat the same procedure for the B axis. Once both axes have been leveled, return both the A and B axes to zero millimeters, enclose the motor control window, then verify that the signal is in its original position in case it has shifted a little.
To record this signal as the reference in the Data Acquisition Properties window, change the averaging value to five and leave all of the other parameters at their default settings. Then click new reference. The averaging counter will count from zero to 20.
Once the counter reaches 20, change the averaging value to one and click OK.The reflected signal from the polystyrene will be saved as the reference for any subsequent scans. When all of the parameters have been set, transfer the imaging window from the scanning stage to the tissue handling area and mount the tumor onto the polystyrene plate. Remove any air bubbles within the tumor with tweezers or gently roll the sample onto the polystyrene until the air gaps are minimized.
Place absorptive spacers at regular intervals around the test sample and place another polystyrene plate above the tumor. Gently press the tumor surface as flat as possible and tape down the tumor and polystyrene plate arrangement in the sample window. A flat tissue surface and a good contact with the polystyrene plate are vital as air bubbles, excess fluid and an uneven surface can ruin the tumor tissue imaging.
Flip the sample window and take photos of the tumor to keep a record of its orientation. Return the sample window to the scanning stage and click the image parameter dialogue button to open the image acquisition parameters window. Set the values of axis one minimum, axis one maximum, axis two minimum and axis two maximum to fully enclose the position of the tumor in the imaging window.
Set axis one step and axis two step to 0.2 millimeters for the imaging scan. Then, under the Measure menu, select Flyback 2D scan and create a folder and file name under which to save the scan data in the pop up window. In this analysis, a grade one to two infiltrating ductal carcinoma obtained from a 49 year old woman via left breast lumpectomy surgery procedure was assessed.
Upon correlating the Terahertz image with the pathology image, it was clear that the cancer region exhibited a higher reflection than the fat region. Tomographic imaging revealed that as the frequency increased the calculated absorption coefficient values for the cancer in fat pixels increased with the cancer pixels showing higher values than the fat at both frequencies. In contrast, the refractive index of both tissues decreased as the frequency increased.
Transmission spectroscopy analysis of the same tumor reveals a good agreement for the frequency range of both the extracted absorption coefficient and refractive index for both sections extracted from the tumor. Note that insufficient handling of the tissue can lead to misleading imaging results, resulting for example in suggesting a larger presence of cancer in the tumor due to the presence of excess liquid within the undried tumor sample. Additional characterization can be performed on the Terahertz image data to obtain information about the frequency dependent properties of the tissue and statistical analysis can be performed for automated image segmentation.
This technique has led to the development of imaging and spectroscopy algorithms for investigating the signatures of ductal carcinoma in situ, to differentiate low grade tumors from deadly high grade tumors. Remember, to handle cancerous tissues and the formalin solution with caution and that any system elements or tools that contacts the tumor tissue must be cleaned or discarded appropriately.
