The overall goal of the following experiment is to eject the content of a single live cell and ionize the released biomolecules for analysis by a mass spectrometer as atmospheric pressure. This is achieved by selecting a single cell through a long distance microscope and ablating the selected cell by a laser pulse of 2.94 micrometer wavelength delivered through a sharpened optical fiber. The ablation of a single cell will result in formation of a microscopic plume of neutral particulate matter to ionize the ablation plume.
It is intercepted by an electro spray. The charge droplets from the spray coalesce with the particulates of the ablation plume originating from the cell. The resulting droplets are seeded with the molecules from the cell and produce the related ions.
Results are obtained that show a mass spectrum from a single cell. The spectrum contains numerous peaks related to the metabolites and lipids of the cell. Hi, I'm Dr.Bin Drester from the Verese Laboratory in the Department of Chemistry at the George Washington University.
Today we'll show you procedure for direct analysis of single life cells by mass spectrometry at atmospheric pressure. We use this procedure in our laboratory to study metabolites and lipids from single plant and animal cells. So let's get started.
Laser ablation for laser ablation, electro spray ionization, mass spectrometry, or lazy MS is produced by a five nanosecond laser pulse at a 2.94 micrometer wavelength. Tunable optical. Parametric oscillator driven by a neodymium YAG laser is used for ablation in this study.
To begin mount the etched end of an optical fiber on a microm manipulator and bring it to close proximity of the cell surface. For efficient energy deposition, hold the non etched end of an optical fiber by a bare fiber chuck for optical alignment. The fiber chuck is mounted on a five axis translator.
Couple the laser energy into the fiber by focusing the beam by a plano convex calcium, fluoride, or anti reflection coated zinc selenide lens onto its non etched end use protected gold mirrors to steer the mid infrared laser beam onto the focusing lens. To maintain the distance between the sharpened fiber tip and the cell surface, use a long distance video microscope mounted at a 15 to 30 degree angle measured from the surface. This system also consists of a five times infinity corrected objective lens equipped with a CCD camera for monitoring and image capture.
A second video microscope is mounted at a right angle to the sample surface to visualize the cells from the top and select them for analysis. This system provides visual feedback to align the fiber tip over the selected cell for ablation and analysis. It consists of a seven times precision zoom optic fitter with a 10 times infinity corrected long working distance objective lens and a CCD camera.
The electro spray system was constructed using a metal union with conductive per fluoro, elastomer, feral fittings, tubing, sleeve needle port, as well as FU silica tubing. The recommended emitter is made of stainless steel and has a tapered tip with a 50 micrometer in a diameter, prepare a 50%methanol solution with 0.1%acetic acid for the electro spray. Pump the solution at a rate of 200 to 300 nanoliters per minute by a syringe pump.
Using a 500 microliter syringe, a high voltage between 2, 803, 000 bolts will be applied to the metal union by a regulated power supply. To generate a steady electro spray, make sure that all electric connections are secure and shielded with the shielding, properly grounded as direct contact with exposed high voltage can result in electric shock. Obtain the live tissue or cell sample from an appropriate source and keep them as recommended before the analysis.
The allium seper bulbs for this demonstration were purchased from a local store for allium seper bulb sample preparation cutter bulb longitudinally with a surgical scalpel section approximately a two square centimeter segment of the scale from an onion bulb. Then peel off an intact monolayer of the inner epidermal tissue. Use the wet surface of the epidermis to mount the tissue on a pre-cleaned glass microscope.Slide.
Hold the slide by a general purpose plate holder, mounted on a three axis translation stage for positioning. Immediately after mounting the sample, acquire the mass spectra to prevent cell degradation. Begin by turning on the laser, the electro spray and the mass spectrometer.
Optimize the geometry of the lazy ion source by adjusting the position of the ablation spot and electro spray emitter relative to each other and the orifice of the mass spectrometer. To achieve maximum ion intensity, select the cell of interest for analysis by using the top view visualization system. After selection, move the sample stage to bring the selected cell directly below the sharpened tip of the optical fiber.
Using the side view microscope, readjust the tip to cell surface distance to approximately 20 microns. Once in the correct position via laser pulses at the selected cell through the etched fiber tip. This results in the perforation of the cell wall and the ejection of the cytoplasm in the form of particulate matter.
After post ionization by the electro spray, the produced ions are collected by the mass spectrometer and mass spectra recorded. After data requisition, put the laser, the electro spray and the mass spectrometer into standby mode or turn them off. Keep the ablated allium cper tissue sample for optional observation by light microscopy, successful ablation of a single cell results in the bursting of the cell wall and the collection of a lazy mass spectrum.
Here are the results from the lazy MS analysis of a single embedded epidermal cell of an allium SEPA bulb showing a representative optical microscopy image. After the sampling of a cell, the cell wall is perforated and the extent of perforation is limited by the boundaries with the neighboring cells. The adjacent cells appear to be intact.
Here a representative lazy mass spectrum produced from the cell is shown. A wide variety of ions are detected from the allium sea per cell, and based on their accurate masses, isotope distribution patterns and tandem mass spectra ions are attentively assigned to endogenous metabolites. The ions detected from a single cell were similar to the ones observed in the analysis of the same tissue by conventional lazy MS of multiple cells.
We have just shown you how to analyze single life cells by mass spectometry at atmospheric pressure. When doing this procedure, it's important to remember to wear protective eyewear to avoid accidental exposure from class four infrared lasers. So that's it.
Thanks for watching and good luck with your experiments.
