The overall goal of this protocol is to describe the major steps necessary for IR-MALDESI mass spectrometry imaging of whole body tissue sections. This method can effectively answer key questions in the field of mass spectrometry imaging, such as discerning the spatial distribution of endogenous and exogenous analytes in multiple organs simultaneously. The main advantage of this technique is that it operates at atmospheric pressure and the exogenous ice matrix helps account for variations in water content in different organs.
Although this protocol provides insight into a targeted whole body imaging. IR-MALDESI mass spectrometry imaging can also be applied to targeted and quantitative drug distribution analysis. The following protocol describes how to perform IR-MALDESI MSI experiments.
In depth details about the optimized geometry of the IR-MALDESI source and it's synchronization with the laser, stage, and mass spectrometer can be found elsewhere. To prepare the specimen, wear protective gloves, and apply a layer of OCT mounting medium onto a 40 millimeter cryostat specimen disc. Then, gently place a frozen whole mouse specimen onto the OCT coated holder and orient it for sectioning.
OCT is used solely to adhere the tissue to the specimen disc. Do not completely embed the tissue in the OCT. Place the specimen disc on the Peltier sample holder in the cryostat.
Turn on the Peltier and wait 10 minutes for the sample to equilibrate. Then, place the specimen disc inside the disc holder, orient it to slice to the desired plane. Section the specimen at 20 degrees Celsius at, for instance, 25 micorons for whole body analysis.
Make use of the anti-roll glass plate and use the cryostat vacuum and brushes to remove unwanted tissue debris. After the tissue is sectioned, cover the blade with a blade guard. Now, orient the frozen section on the specimen plate and thaw mount them onto pre-cleaned glass microscope slides.
Bring the slides as close as possible to the sections without touching. Keep the mounted slides inside the cryostat until the IR-MALDESI calibration is complete to maintain the tissues cryopreservation. To begin, turn on the mid-IR laser and launch the laser control application.
Then, choose the desired wavelength. IR-MALDESI experiments are typically performed at 2940 nanometers. Next, turn on the stage controller and launch the control program.
Using the program, calibrate the stage position using the home button. Next, fill a one milliliter syringe with electrospray solvent and flush the silica capillary. Then, align the ESI emitter with the MS inlet using the source parameters, such as ESI-spot distance, spot-inlet distance, sample height, and solvent flow rate.
All of which have been optimized using statistical DOE for analysis in tissue sections. Now start the electrospray and evaluate it's stability for at least 10 minutes. Monitor the total ion current, which at this point, consists solely of ambient compounds.
Variation under 10%over a 10-15 minute period indicates the system is stable. After turning off the electrospray and ensuring it is off, transfer a specimen slide from the cryostat to the IR-MALDESI sample plate. Then, stand back from the ESI emitter and restart the electrospray.
Close the access door of the IR-MALDESI source and purge the enclosure with dry nitrogen to prevent condensation of water on the tissue. Once the relative humidity inside the enclosure is less than 3%switch on the Peltier plate's DC power supply to about 12 volts to cool the sample plate to 9 degrees Celsius. After about 5-10 minutes, stop the nitrogen purge and open the source access door to expose the tissue to the ambient humidity.
A thin layer of ice will form on the stage and mounted tissue section. If the air is dry, place a beaker of warm water inside the enclosure. Now, close the door and restart the nitrogen flow to reduce the humidity to a around 10%Adjust the nitrogen flow as needed to maintain a stable layer of ice on the sample during the experiment.
Using the Rastier GUI, move the stage to the analysis position by clicking the LASER Position button. Use the adjustment diode of the laser to ensure an off tissue region will be abladed. Then, fire a test shot to calibrate the laser's offset with respect to the camera view.
Click the Laser Test Fire button to update the laser position. Now, return to camera position and update the laser position in Rastier by placing the laser alignment reticle on the laser ablation spot. Then, click the ROI button and select the region of interest.
Include some off tissue area to serve as control. Use this off tissue section for peak picking. The spot size of the laser on tissue is about 150 microns.
A smaller diameter is possible with over sampling. Now, input the appropriate experimental parameters and name the mass spectrometer imaging file in the project name box. Then, select the appropriate laser frequency from the drop down box.
Typically, use two laser pulses per voxel at 20 Hertz to completely desorb the material. Also, set the delay time to 10 milliseconds so the ions have time to reach the mass analyzer. The repetition rate should be set to the laser's maximum.
Now, choose mass spectrometer parameters, such as ionization mode, electrospray voltage, solvent flow rate, capillary temperature, and the injection time in the mass spectrometer software. Once all parameters have been set, place the mass spectrometer in handshake mode for synchronization and start the mass spectrometer acquisition. Using the Rastier imaging software, verify that all steps in the checklist have been completed and then load the program.
Once the RUN button becomes available, start the MSI signal acquisition. Upon completion of the imaging experiment, stop mass spectrometer acquisition and place the instrument in standby mode. Turn off the nitrogen purge to the enclosure.
Turn off the stage controller. Turn off the power supply to the Peltier plate and turn off the laser. Then, open the access door.
Remove the eletrospray emitter tip from the inside of the source enclosure and wearing protective gloves, remove the very hot extended metal mass spectrometer inlet. Wearing gloves and safety goggles, clean the inlet using an ultrasonic bath. First, use 15%nitric acid for 10 minutes.
Then, use HPLC grade water for 10 minutes and finally, use HPLC grade methanol for 10 minutes. Dry the metal capillary inlet under a stream of nitrogen and reinsert it into the mass spectrometer for future analysis. Spatial distribution of metabolites in different organs of whole body tissue sections were analyzed using the described method.
Cholesterol was observed in all tissues because of it's structural role in cell membranes. Other lipid metabolites exhibited distinct distributions within specific organs or tissue compartments. For example, some lipids, such as punitively assigned desmosterol were observed to have a specific localization in the brain.
Whereas other lipids exhibited a higher distribution in the spinal cord. And other lipids were uniforming distributed across all organs except for the brain. IR-MALDESI mass spectrometry imaging is a disruptive technology that allows for the relative and absolute quantification of analytes in tissues with minimal sample preparation at atmospheric pressure.
Once mastered, all of the sample preparation steps can be done in approximately 30 minutes. A key step in sample preparation is the formation of the ice matrix, which facilitates efficient desorption of tissue related material. The ice matrix also helps account for variations in water content in different organs.
In tandem with this protocol, supplementary analysis, such as histological and immunohistochemical staining of the serial section can be performed.
