The overall goal of this procedure is to determine the location and structure of calcification event by performing a combination of optical and electron microscopy methods. This is accomplished by monitoring the living larval tube worm during metamorphosis, the life stage responsible for calcification that can be detected using fluorescent indicators sensitive to intracellular pH and calcium signals. Intracellular pH imaging allows us to study the concentration of proton into and out of the cytoplasm in the process of calcification.
To begin, intracellular pH imaging in the marine tube worm larvae, the competent swimming larvae are stained with the intracellular pH indicator dye, SNARF-1 AM.The larvae are then transferred to a dish of IBMX containing seawater with a thin glass observing window and placed on a fluorescent microscope, where they are hit with light at 488 nanometers in wavelength, which is absorbed by the dye. 580 nanometer and 640 nanometer emissions from the dye are collected. The values of intracellular pH can be calculated from the ratios of these values.
Optical microscopy allows us to identify time and tissue region associated with a higher pH level. This is the first step to determine the time points of interest for further analysis. In the second step, preserve the tube worm with fixatives at the relevant time points for electron microscopy.
A newly mineralizing life stage is identified using SEM-EDS mapping for calcium signal. Then the regions with a high calcium signal are screened using SEM/EBSD, identifying the presence of crystalline mineral. Finally, using a focused ion beam the mineral is cut out, a thin section is prepared for TEM observation.
This section is used to obtain a selective area diffraction pattern. The main advantage of this technique or a conventional test methods like bulk X-ray diffraction spectroscopy is that it provides direct observation of specific structures that have been observed by optical and electron microscopy methods, hence, discrete calcification events can be studied directly at both temporal and spatial levels. When calcification is studied with calcium and intracellular pH indicators we can identify the timescale of these relevant physiological events.
Preserving specimen at the right time is critical for a fruitful SEM analysis. When looking for the first event of biological calcification SEM/EDX can be used to screen for the elemental distribution for calcium. A location that has more calcium may indicate the presence of calcium carbonate, however, only the presence of an X-ray diffraction pattern can confirm that there is a crystalline structure.
Such information can be obtained using EBSD and TEM. Electron backscatter diffraction is a crystallographic characterization technique to identify crystalline or polycrystalline material, as long as microstructure and electron REM angle satisfy the Bragg diffraction condition. Typically, backscattered electrons are collected at a tilt of 70 degree angle on a polished specimen with grain or crystal orientation maps.
For an unpolished sample generating such a map from these uneven surfaces is hard, because not all service series satisfies EBSD angle requirements. However, in the form of point IG a Cacucci diffraction pattern can still be collected from these uneven surfaces as long as the EBSD angle requirement is satisfied. The Cacucci diffraction pattern provides unambiguous confirmation that the crystalline mineral is present.
Our method is direct and quick, and can be implied to other mineralizing tissues. Here, a focused ion beam is used to fabricate a microsample, which is also a very direct method of preparing a TEM section. To examine the metamorphosis process, tube worms were cultured in the laboratory for about five days.
The competent larvae will swim in a forward direction instead of a circular motion. This means they are ready for metamorphosis. Incubate competent larvae in the fluorescent solution in seawater, cover the containers with foil to prevent photobleaching and incubate the animals overnight.
The tube worm larvae are washed against the nylon mesh that will retain the larvae but allow solution to pass through. Wash the larvae with filtered seawater two times to remove excess dye solution. Press the strainer to the Petri dish to create a tight seal before releasing the seal to discard the wash solution.
Take care not to dry out the larvae. Then place each of the 10 to 20 larvae in a thin, glass bottom dish with IBMX and cover the dish until imaging. Next, insert the suitable filter cubes with the appropriate dichroic mirrors to detect the fluorescent signals.
Optimize the shutter time to be fast enough to capture images for a living organism. Next, select the optical sectioning parameters on the Z-Stack option, move the microscopic stage up and down to define the start and stop position of image acquisition. Set a one micrometer distance between layers.
After imaging, export all data as grayscale TIF files. This can be performed by selecting File, Export. Make sure the file types appear as TIF images, then click Start.
The grayscale image in each channel, in each layer of Z-Stack will be in the same image folder ready for image analysis. Finally, generate a composite image for each of the fluorescent channels, using ImageJ. The following JoVE videos demonstrate how to perform calibration and measurements for intracellular pH.
Preserve the tube worms undergoing metamorphosis using a crosslinking fixative, using a solution of 4%paraformaldehyde in seawater. Simply mix one part of 16%paraformaldehyde into three parts of seawater and allow fixation to take place overnight. Dispose the primary fixative and refix the specimen for 30 minutes in aqueous solution of 1%osmium tetraoxide to further stabilize the tissue structures.
Be sure to work in a fume hood and have separate waste bottles that are clearly labeled, prepare formaldehydes and osmium tetraoxide. Next, dehydrate the samples using a graded ethanol series, allowing five minutes between changes. The next step is to dehydrate the samples with a 1:1 solution of ethanol and HMDS, just enough to cover the specimen.
Wait for the specimen to evaporate dry in the fume hood, then repeat the procedure with pure HMDS. To create a controlled fracture of the sample run the diamond blade against the dish, enclosing a few individuals to make a triangular cut. With the Petri dish covered, place the culture dish against an aluminum stub, gently press to make a controlled fracture.
Observe using a dissection microscope, carefully pick up a fracture piece that contains a few intact tube worms. With silver paint, stick the sample to the SEM sample holder, paint around the edges with silver paint to reduce charging. The sample is now ready to be imaged for SEM-EDS analysis.
Insert the sample in the holder into the microscope. Orientate the sample and place the sample underneath the electron column. Analyze the sample under VP SEM mode.
Optimize the focus and astigmatism as necessary. Select the magnification such that a single organism fills the entire field of view. Analyze the elemental distribution of calcium on the sample by acquiring a map of the entire field of view, run the map for 15 minutes or longer or until the data shows distinct localization of calcium within the organism.
To obtain point quantification for a region of interest select the Point ID option using the Single Point tool. Click on the area of interest to perform a scan. Record carefully the location by capturing a series of images with decreasing magnifications.
To prepare the samples for EBSD remove the sample from the flat SEM holder and stick it to a 45 degree pre-tilted stub using silver paint. Using the reference SEM images locate the animal of interest and exact region of the animal to be examined. Tilt the stage 25 degrees so that the sample surface is now approximately 70 degrees to the electron beam.
Raise the stage. Select EBSD mode in the AZtec software. Choose aragonite as the phases of interest.
Insert the EBSD camera, set camera conditions to achieve a signal of about 90%Using a fast beam raster, acquire a background, then capture an image in Aztec. Using the Spot Analysis tool click on the location of the sample that has shown a higher calcium signal. Scan to see if Cacucci patterns are detected.
If patterns are present and match any of the selected phases in the database they will automatically be indexed. Protect the prepared SEM specimen with layer of platinum by sputter coating before proceeding to microsample preparation using focused ion beam. Insert the sample into the FIB SEM chamber, position the sample and acquire a live, ion-induced secondary electron image.
Locate the area of interest from the referenced images as previously determined by EDS mapping in EBSD. Rotate the stage as necessary to get the features of interest in line with the frame of the window, adjust magnification to show the area to accommodate the features of interest, then define a box to deposit carbon and tungsten. Capture an FIB snapshot, then draw a pattern of four boxes around the tungsten cap to define the location of microsampling.
Then tilt the stage at 58 degrees and cut through the bottom of the microsample. Tilt the stage back to zero, insert the tungsten microsampling probe, then lower it until it contacts the sample. Connect the tungsten probe and the sample by depositing tungsten between the tip of the probe and the tungsten coating, then make the final cut, detach the microsample from the rest of the island and raise the probe with attached microsample.
Place a TEM copper half grid into a side entry holder, lower the tungsten probe until the microsample contacts the TEM grid. The microsample is further milled until it becomes electron transparent. Before TEM analysis, fill both dewars with liquid nitrogen and set the accelerating voltage to 300 kilovolts.
Place the half grid with attached lamellae in the standard TEM holder. Center the beam on the phosphor screen, contract and expand the beam and ensure that all beam movement is concentric. Use the stage movement knobs to navigate and locate the sample, increase magnification on the sample and adjust the Z-Height until the sample is in focus.
Use the optical eyepieces as necessary. Insert the camera and remove the phosphor screen. Expand the beam as necessary to avoid oversaturating the camera.
Adjust focus and the camera parameters to capture an image. To acquire a diffraction pattern, center the feature of interest, remove the objective aperture and insert the diffraction aperture. Change to Diffraction Lens mode.
Insert the blanker to prevent the center of the diffraction pattern from burning the camera or phosphor screen. Capture an image of the pattern for crystallographic analysis. The following are some observations of the calcification process during metamorphosis of the tube worm.
The pH values of the tube worms increased after IBMX stimulation of metamorphosis and intracellular pH started to increase beyond eight at 31 hours. The tissue relevant to calcification is the collar region. A map on the SEM-EDS signals show that the day one tube worm had a homogenous distribution of calcium, indicating the calcification process had not yet begun.
Two days after metamorphosis stimulation some tube worms had already calcified too much and are no longer suitable for observing newly calcified materials. In a tube worm specimen, such as the one used in this example, calcification is still at its early stage, so calcium quantification helped to determine the location of interest for characterizing the presence of minerals. When examined using SEM-EBSD the localized area with the strong calcium signal also had a crystalline structure of aragonite.
Using focused ion beam the specimen was prepared for TEM and the diffraction pattern from the aragonite mineral was analyzed in greater crystallographic detail. After watching this video you should have a good understanding of how to locate a mineralization event in a multicellular organism. When optical and electron microscopy are used together we can acquire a great level of physiological and material understanding about the process of calcification.
