Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Our research focuses on developing high-throughput, image-guided, single-cell MALDI mass-spectrometry workflows to reveal cellular heterogeneity and to link the molecular profiles to cell identity, function, and response in complex systems.

Advanced instrumentation, such as MALDI-2 and high spatial resolution mass spectrometers, enable targeted, spatially resolved analysis of single cells, enhancing the sensitivity and expanding the scope of molecular profiling in complex tissues. Current challenges include limited sensitivity, reproducibility across samples, and complex data analysis. MALDI data are often sparse and large datasets with thousands of cells and hundreds of molecules demand robust computational tools.

This protocol addresses the gap in high-throughput methods for analyzing individual cells and even organelles, enabling very detailed studies of heterogeneity and gaining insights into the molecular and cellular biology and function.

This protocol allows rapid targeted analysis of cells scattered across the slide, eliminating the need for cell manipulation or full-area scanning and significantly increasing throughput.

[Narrator] To begin, rinse each slide with two to three milliliters of 150 millimolar ammonium acetate to remove glycerol and salt crystals that can interfere with microscopy and MALDI matrix application, then dry the slides under a gentle stream of nitrogen or allow them to air dry completely. Load the dried slide into the microscope stage. Focus the microscope using at least 10 support points distributed evenly across the entire slide. Using filters suitable for DAPI and bright-field imaging, acquire a tiled fluorescence image of the entire slide at 5x to 10x magnification, ensuring that the fiducial markers on the microscopy slides are clearly captured in the image. Stitch the tiled images using microscopy software, such as ZEN ZEISS. Verify that the vertically adjacent tiles are not offset. Process and export each stitched image as a BigTIFF file using the microscopy software or as a standard TIFF if the final image size is less than two gigabytes. Dissolve 20 milligrams of 2,5-dihydroxyacetophenone in 1.5 milliliters of acetone. Place the slides into the holder of the sublimation chamber, then place the holder into the sublimation apparatus. Pipette the dissolved matrix solution onto the ceramic wafer and allow the acetone to evaporate completely, then close the sublimation chamber to seal it. Fill the coolant chamber with an ice water slush and place it securely on top of the sublimation chamber. Turn on the vacuum pump and allow the system to equilibrate for five minutes. Begin the sublimation by heating the chamber to 200 degrees Celsius for five minutes. After five minutes, remove the ice water bath from the chamber. Turn the temperature to 25 degrees Celsius and place a heat sink on top of the chamber. Slowly vent the sublimation chamber to release pressure. Open the chamber and carefully remove the slides. Open MicroMS and decimate the BigTIFF microscopy images using the image group option to accommodate both bright-field and fluorescence channels. Navigate to the Blob Options tool and adjust the maximum and minimum blob size to define the acceptable blob size range. Set the threshold of the fluorescence channel for blob detection. Specify the circularity value to define how circular the identified cells need to be for consideration, and choose the color. Use the Blob Find option to detect blobs. When prompted, save the blob list under a desired name. Use the Distance Filter tool to set the minimum distance between each cell. Test the error via test points to accurately determine the offset error. Load the slides into the instrument using the MTP Slide Adapter II. After returning to the computer with MicroMS, access the mass spectrometer computer via a remote desktop application. If using the instrument for the first time, verify its position by navigating to Tools, followed by Instrument Settings. In the pop-up window, view the set of coordinates with their X and Y positions and select each of these specific points on the Slide Adapter II geometry. Update the X and Y positions in the MicroMS window. Using the mass spectrometer's camera and stage controls, navigate to an easily identifiable location on the slide and copy the instrument coordinates. In MicroMS, locate the same position in the microscope image, right click, and input the coordinates into the pop-up window. Round the coordinates to the nearest integers and separate them by a space. After three registration points have been added, one of the circles will turn red, indicating that it is the most off position from the registration. Delete the registration point using Control + right click and try again. Under File, go to Save and then Registration to save the registration file. With the blobs visible on the slide, go to File, Save, and then instrument positions to save the instrument position file, then using remote desktop software, transfer this file to the instrument computer. Open the custom XEO file and copy its contents into the MTP Slide Adapter II .XEO file on the instrument computer. Save the file to update this geometry file with the cells' locations. Click on the Automation tab and select New to create a new automatic run. Drag across the displayed sample region to select the cells and right click to add them to the analysis list. Save the automatic run and click Start Automatic Run to begin acquisition. This figure illustrates how single-cell MALDI-2 mass spectrometry profiling reveals lipid-based cellular heterogeneity across and within distinct brain regions. Uniform manifold approximation and projection, or UMAP analysis, separated cells by brain region, forming distinct clusters for striatum, hippocampus, and cortex. Leiden clustering revealed four lipid-based cell subpopulations. Additionally, cluster-specific lipid signatures were observed, with distinct intensity profiles across annotated lipids. Mass spectra from six cortical cells also showed consistent lipid detection across slides. Furthermore, cortical cells from different slides showed overlapping UMAP distributions, indicating minimal batch effects.