The overall goal of this procedure is to offer a simple and robust method to map and quantify organic and inorganic elements in biological tissue with tabletop instrumentation fully compatible with conventional optical microscopy. This is accomplished by focusing a laser pulse on the sample of interest to initiate the breakdown and spark of the material. The plasma emits radiation that is subsequently analyzed by a spectrometer.
The second step is to select and identify the spectral lines corresponding to the elements of interest and scan the surface of the sample. Next, perform calibration measurements to allow elemental concentrations to be determined. The final step is to construct the elemental mapping of the sample tissue from all the recorded spectra using a relative intensity or a concentration scale.
Ultimately, laser induced breakdown. Spectroscopy is used to show the tissue distribution of various elements without any labeling Compared other existing methods such as micro x-ray fluorescence, or mass spectroscopy based techniques. Laser induced breakdown spectroscopy or lips is simple to implement, runs at atmospheric pressure and is compatible with most of optical microscope systems.
In addition, it allows to reach PPM level in sensitivity with dense of micron in resolution. This method can help answer a key question in nanotechnology applied to biological studies such as the distribution within a tissue sample of nanoparticles containing metallic element without the need of nanoparticle labeling. The technique can extend to pathology due to its low detection limit in detecting abnormal metallic elements such as copper, iron, and aluminum that may exist in different organs of the body, especially the brain Through this method can provide insight into nanoparticle detection and ification.
It can also be applied to other systems like detection of inorganic element into TCU or heterogeneous element distribution. Scientists who will have to conduct such experiments will have to struggle with acquisition settings in order to get as much a signal as possible without damaging the samples too much. Begin the experiment by preparing sample slides to be studied for this protocol.
Obtain organs from tumor bearing mice, injected with gadolinium nanoparticle solution to make slides. Place 100 micrometer thick slices of the organs onto a quartz slide or Petri dish. Always store the slides at negative 80 degrees Celsius.
Next, prepare samples for calibration. Label several vials labeled with concentrations, starting at zero nanomolar and ending at five millimolar. For each vial, prepare 100 microliters of gadolinium in water mixture with the appropriate concentration.
Once this is done, ready a slide. Measure five microliters from the first vial and drop it along the quartz slide or Petri dish. Be certain to identify the drop for later use.
Move three millimeters along the circumference and place five microliters of the next concentration on the slide. Do the same for each of the remaining solutions. Dry the slide contents for 20 minutes at room temperature.
This lip setup uses a nanosecond neodymium atrium, aluminum garnet laser producing a 1064 nanometer wavelength beam. Use part of the beam to synchronize the spectrometer system. Direct the other part through a computer controlled attenuator used to adjust and stabilize the laser pulse energy during the entire experiment.
Next, direct the beam to the sample region there. Focus the beam onto the software controlled translation stage. That will hold the sample mount one CCD camera to monitor the sample surface mount.
Another to monitor the plasma plume. Analyze light from the sample breakdown In a turn turner spectrometer with an ICCD camera held at negative 26 degrees Celsius. For the measurement, start stabilizing the laser and cooling the camera at negative 26 degrees Celsius.
10 minutes before the experiment. When ready, use the attenuator to lock the pulse energy to five millijoules. Next, retrieve a sample slide to study and prepare to mount it on the sample stage.
Secure the sample on the motorized sample holder. Once this is done, return to the computer. To position the sample, move the stage, so part of the sample is in the beam of the laser.
Adjust the laser focus to about 100 micrometers below the sample surface. When this is done, the laser forms craters of 50 micrometers or less. Use the computer to set the spectrometer input slit value to 40 micrometers and the ICCD parameters for the mapping measurement.
Adjust the holder position to target a region of the sample. Take a high resolution panoramic image of the sample with a broad spectrum light source. Then use software and the captured image to define the libs scanning region and resolution.
Here the grid is 100 by 100 with 100 micrometers between points. Start the automated acquisition of the data at each point on the grid. A spectrum is measured and recorded to file.
When the measurements are done in about 30 minutes, take a second image of the sample slice, then consolidate all the spectra into one data file for later use. Next, remove the sample slide and prepare to mount the calibration samples. Secure the quartz slide with the calibration samples on the translation stage back at the computer.
Position the center of the first calibration sample in the laser path. Maintain the experiment parameters and record a spectrum. Do the same for each of the different gadolinium concentration samples.
These are examples of a single shot spectra recorded from different regions of kidney tissue. The blue spectrum was recorded in the central region of the kidney. The red spectrum corresponds to the kidney membrane and the green spectrum is for the peripheral area.
The injected, gadolinium based nanoparticles are composed of a polys laane matrix with gadolinium ion chelated by doca ligand on their surfaces. In the 286 nanometer to 320 nanometer spectral range used gadolinium, calcium, iron, silicon, and aluminum can be detected. Note how the intensities vary in the different regions of the sample, suggesting a large heterogeneity of these element concentrations in the tissue mapping of gadolinium, silicon, and iron in a slice of mouse kidney was done using the data from libs with a resolution of about 100 micrometers.
The scale intensity is expressed in an arbitrary unit. This is a natural light image. For comparison, similar results were obtained with tumor samples.
As with this map of gadolinium and SQ 20 B tumor tissue, a false color scale has been superimposed on the natural light picture. For this experiment, nanoparticles were administered directly into the tumor. After one hour, the tumor was removed and prepared for analysis.
Note that approximately half of the tumor volume contains some particles On masters. This technique can been done in less than one hour for one square centimeter samples with a resolution of 100 micrometer if it is Performed properly. When doing lips, it's important to remember that measurements are performed from laser induced plasma and it is crucial to control all the parameters during the entire experiment Following the procedure.
Other methods can be performed like immunohistochemistry on adjacent slices in order to answer further questions like the type of cell involved in the process of interest After his development. This technique pave the way for researcher in the field of nanotechnology applied to biology and medicine to explore the distribution of compounds containing metallic element in biological tissue. After watching this video, you should have a good understanding on how to use the lips techniques in order to map multi elements in biological tissues.
