The overall goal of this procedure is to use analytical electron microscopy to quantitatively measure the concentration of biologically important elements such as calcium at subcellular resolution in cultured neurons. This is accomplished by first rapidly freezing live cell cultures and preparing ultra thin cryo sections. The second step is to transfer cryo sections into an analytical electron microscope and freeze dry them.
Next images and x-ray spectra are required from selected subcellular regions, including neuronal mitochondria. The final step is to quantitatively analyze the x-ray spectra to determine localized elemental concentrations. Ultimately, this approach known as EPMA is used to reveal changes in calcium concentrations and distribution that underlie intracellular calcium signaling.
The main advantage of this technique over related methods like fluorescence, microscopy, radioisotope uptake, or flame photometry, is single organelle level resolution and sub millimolar sensitivity. Demonstrating the procedure will be Dr.Natalia pva, a senior staff scientist in our laboratory. To begin prepare a freezing bath by condensing gaseous ethane at negative 160 degrees Celsius in the center well of a liquid nitrogen cooled system.
Then blot the edge of a cover slip containing adhered cells to remove all but a thin film of liquid. Take care not to dry out the sample. Next, hold the cover slip by the edge and quickly immerse the sample into the liquid ethane.
After two to three seconds, remove the cover slip from the ethane and place it into a styrofoam bowl of liquid nitrogen Transfer. Frozen cover slips into a cryo microtome pre-cool to negative 135 degrees Celsius. Cut the frozen cover slips into three millimeter by three millimeter squares using a sharp pre-cool scalpel.
Then embed them facing up on a three millimeter diameter aluminum pin in a cryo glue mixture consisting of one part ethanol and six parts.Isopropanol. Solidify the cryo glue by lowering the temperature of the cryo box to negative 160 degrees Celsius. Next, trim the cell rich areas of the cover slip piece to an approximately 250 by 250 micron block face, and 100 micron depth.
Using a diamond trimming tool, dry cut ribbons of thin sections at negative 160 degrees Celsius using a 35 degree diamond cryo knife as described in the accompanying text protocol. Then transfer the sections onto glow discharged carbon coated peola form. Support films cast over 100 mesh folding copper grids sitting in an open indium envelope.
The grid is closed. Then the envelope and the sandwich pressed with a cooled pressing tool prior to the cryo transfer of specimens to the electron microscope. Cool the cryo holder to its minimum temperature of at least negative 160 degrees Celsius while under high vacuum within the microscope stage.
Then tilt the goniometer 45 degrees clockwise and remove the holder. Quickly transfer the cooled cryo holder from the microscope to the cooled cryo workstation and retract the holder's frost shield. Then transfer the specimen to the cryo holder.
Secure the grid with the retaining ring using the spanner tool provided and close the frost shield. Next, quickly remove the cryo holder from the cryo workstation and insert it into the airlock of the microscope. Go through the pumping sequence as quickly as possible with the sample cryo holder in place.
Return the goniometer to a zero degree tilt. Reconnect and reenergize the control box and confirm that the specimen temperature is less than or equal to negative 150 degrees Celsius. Finally, refill the doer of the specimen holder and allow the vacuum and temperature to fully recover.
First, visually evaluate the specimen at 250 times magnification and low illumination. Selected sections should be thin and smooth, not folded or overlapping. They should also be flat well attached to the support film and generally not obscured by grid bars.
Use the digital goniometer stage to store the coordinates of the selected sections. Next, increase the holder temperature to negative 100 degrees Celsius for 30 minutes in order to freeze dry the sections to stabilize them and improve contrast. Then recoil the samples back down to negative 160 degrees Celsius or below.
Before high resolution imaging. Turn off the cryo holder control unit and physically disconnect the controller cable to avoid image drift due to thermal cycling and or vibration pickup. Then activate the electron microscope in high magnification mode.
Survey subcellular regions at around 2000 times magnification that are within selected sections whose locations were previously stored. Record and store TIFF images of promising areas. Then evaluate the images in order to select and annotate regions of interest for x-ray analysis.
Once regions of interest have been selected, configure the electron microscope for x-ray acquisition by inserting the EDX detector into the column, withdrawing the objective aperture and inserting and centering any stray radiation apertures. Tilt the holder 20 degrees towards the detector in spot mode. Increase the beam current to around three nano amps as measured in a 100 nanometer spot.
Then raise the cryo holder temperature to negative 100 degrees Celsius or below to avoid frost contamination under wide field imaging conditions. Choose a mitochondria for analysis. Return to spot mode.
Launch the spectrum acquisition software and begin 102nd acquisitions. Save the recorded spectra in the standard EMSA file format. Sample enough mitochondria in different cells to ensure a statistically significant data set.
In order to qualitatively analyze an EDX spectrum, utilize an available software database and peak matching routine such as DTSA to identify the major elements in the spectra. Identify the elements by the characteristic energy positions of their peaks. Biological specimens usually contains sodium, cassium, phosphorus, sulfur, chlorine, potassium, and calcium.
Pay extra attention to the calcium and potassium peaks as they overlap for quantitative analysis. Start by extracting the integrated area of identified peaks using any one of several standard fitting algorithms such as the simplex method built into DTSA next estimate dry mass by integrating the continuum between 1.45 and 1.61 kilo electron volts. Alternative interference free regions, for example, 4.0 to 5.0 kilo electron volts can be used to avoid extraneous non-biological signals in the primary continuum region.
Finally, calculate peak over continuum ratios and convert to concentrations by comparison to standards. This approach is known as the hall peak over continuum method. Shown here is a micrograph of a cultured hippocampal neuron, rapidly frozen following exposure to an cytotoxic stimulus, which leads to the opening of ion channels and calcium in flux.
Most of the mitochondria shown here appear structurally damaged as they're highly swollen and contain small, dark inclusions. Electron probe Microanalysis was then used to perform elemental analysis within and exclusive of the mitochondrial inclusions. The study revealed that inclusions are largely composed of extremely high amounts of calcium and phosphorus.
This explains their extraordinary calcium buffering capacity and why overwhelming this buffering mechanism leads to calcium overload triggered cell death. The hippocampus, an area of the brain critical for learning and memory is a major site of injury after ischemia. The functionally distinct region named CA three is far less vulnerable to ischemic injury than is the adjacent synaptically connected ca one region electron probe microanalysis following toxic stimuli of these slices showed much larger calcium elevations in vulnerable CA one neurons than in resistant CA three neurons.
Consequently CA one mitochondria exhibit extensive injury and dysfunction indicating that calcium overload induced mitochondrial dysfunction is a determining factor in the selective vulnerability of CA one neurons. After watching this video, you should have a good understanding of how to measure the concentration of total calcium in single mitochondria of cultural neurons using collection probe microanalysis.
