The overall goal of this procedure is to use two dimensional mixed magnetic detection scans to analyze thin biological samples containing nanomagnetic particles. This method can help answer key questions in the field of biochemistry and medical diagnosis such as the analysis of tissue sections employing nanomagnetic particles as a leveling compound. The main advantage of this technique is that it allows a meeting of the nanomagnetic particle distribution.
Demonstrating of the procedure will be Eul-Gyoon Lim, Jae-chan Jeong and Jiho Chang, three researcher from my laboratory. The p-FMMD measurement head should be designed in accordance with the text protocols. Details are given on all the wiring and coiling specifications.
Assembly and setup are detailed in the text protocol. This includes adjustment of the high frequency balance and the induced voltage. Next, the measurement electronics are set up, which includes the excitation section, the low and high frequency driver sections and the detection section of the FMMD.
Following this, the preamplifier, first demodulator, intermediate amplifier with filtering, second demodulator, and final amplifier with filtering are all set up. Finally, the 2D scanner is mounted and interfaced with a computer control. For this procedure, have magnetite particles with diameters of 50 nanometers and 100 nanometers and maghemite particles of 1 micron diameter.
Wash the particle stock solutions in water and collect the particles using a magnet. Discard the water and wash them each two more times. Then, dilute the particles into 25 milligrams per milliliter solutions using distilled water.
From the 100 nanometer particle solution, make a five-fold dilution series for concentrations of five, one, 0.2 and 0.04 milligrams per milliliter. Next, punch out pieces of absorptive blotting paper using a biopsy punch. Then, soak the paper punches in the different 100 nanometer particle solutions for 30 seconds.
After the soak, let the paper punches air dry. Next, prepare cut-out pieces of nitrocellulose that are two by 18 millimeters. Soak one piece of nitrocellulose in undiluted one micron diameter particle solution for 10 to 15 seconds, and blow dry using unheated air.
Soak the other piece of nitrocellulose in two solutions of different concentrations to make a concentration gradient, and dry it like the other. Lastly, using capillary action, load a capillary tube with 30 microliters of undiluted 50 nanometer diameter particle solution. Then, load a second capillary with 10 microliters of a 20 times dilution of the same particles.
The scanning direction should be the shorter of the two planar dimensions. Set the starting point and scan length using the ruler marks on the palette. Enter these values in the scanning software, then set the scan offset to be a little smaller than the achievable spatial resolution.
Next, set the scanning speed with consideration of the signal reduction that occurs due to low pass filtering. Use a value between one and seven millimeters per second. Now, set the stepping distance.
The total scanning time is calculated using a formula that is provided in the text protocol. Before scanning, secure the sample with adhesive tape. For the scan, generate an NVD file for the motion control program.
Open the PMC motion control program and load the NVD file. Press the Home button to set the mechanical origin points. Close the motion control program and return to the scanner program.
Then, execute the scans. For these scans, signal intensity was analyzed as a function of the concentration of magnetic beads and the scanning speed was 10 millimeters per minute. A strong correlation was found between the bead concentration and the signal.
The relationship between the speed of the scanning stage and the signal intensity was checked using paper pellets soaked with magnetic beads. Higher signals were obtained at lower scanning speeds. Comparing the p-FMMD scan with an optical image of nitrocellulose membrane sample clearly showed the utility of p-FMMD as an MPI scanner.
The broadness of the scan is due mainly to the sensitivity profile of the measurement head. Similarly, two capillaries filled with different magnetic particle concentrations were photographed and scanned by p-FMMD. Clearly, concentrations differing by a factor of 20 are easily distinguished.
After watching this video, you should have a good understanding of how to analyze 10 samples containing nanomagnetic particles with the FMMD technique. Once mastered, this technique can be done in about one hour if it is performed properly. After its development, this technique paved way for the researcher in the field of biochemistry and medical diagnostics to explore the distribution of the nanomagnetic particles that rather cite specific antibodies in organ system.
