Microscopy101
Stereo pairs . Producing stereo pairs requires taking paired photos in which the sample has been tilted. We use a JEOL 1400 TEM with a side-entry goniometer; the specimen height (Z height; eucentric height [2]) is adjusted by selecting the standard focus button and using the Z controls and the X or Y wobble keys and the focus knob to bring the image into fi ne focus. We generally tilt a total of twelve degrees; 6 degrees in the
X direction; then 6 degrees in the Y direction at a magnifi cation of 40,000. Depending on the magnifi cation, the sample can be tilted from 4 to 20 degrees total. Because distortion might be present at the edges of the fi eld at higher magnifi cations, the second photo should be in exactly the same orientation and cover the same fi eld as the fi rst so that the photos will be in full alignment. If the pairs are not in alignment, some of the periphery of the image may be distorted when the pairs are assembled, rendering them unusable. T e problem of achieving good alignment expeditiously is even more challenging when more than one stereo pair is needed of the same cell. To minimize the laborious tracking of a sample, we use a cell phone camera to record the precise orientation of each photographic fi eld. Because each fi eld is accurately recorded, we can save a great deal of time between series of stereo pairs. We begin by fi nding the desired growth cone at a low magnifi cation and photographing it for reference ( Figure 4A ). Keeping the growth cone in view, we tilt the stage -6°. We raise the magnifi cation, again keeping the area of interest in view, and take a whole series of photos along the growth cone’s perimeter and interior, all taken at a stage tilt of -6°. For each
TEM image taken, the operator takes a cell phone photo of the display screen. Once the entire growth cone has been documented, the stage is tilted +6°. Using the cell phone photos as references for framing the fi elds of view, the second photo of each stereo pair is taken. Stereo pairs can be used to prepare an anaglyph [ 3 ] to demonstrate, for example, the three-dimensional distribution of localized protein using antibody-coated gold beads at the edge of the growth cone ( Figure 4B ). In summary, in our experience the small diff erence in price of the center-marked grids (which can be cleaned and reused) and in the time taken to scan the grids using light microscopy is more than compensated by the time and money saved during the TEM session, making time on the instrument less tedious and more productive. Once we adopted this method for photographing stereo pairs, our sessions on the microscope decreased from an hour to about fi ſt een minutes for each growth cone. Using a cell phone camera to assure that stereo pairs align suitably saves a great deal of time and prevents the frustration of having taken stereo pairs and later fi nding that they fail to over- lap properly, necessitating another session on the microscope.
References [1] Kent Makes Formvar Coated Grids:
www.youtube.com/ watch?v=u2auPtTtroM .
[2] J Rodenburg , “Learn to use the TEM; T e Specimen Height and Eucentric Height,”
http://www.rodenburg.org/guide/ t800.html .
[3] F Korobova and T Svitkina , Mol Biol Cell 19 ( 4 ) ( 2008 ) 1561 – 74 . Vibrations Bad for Without Minus K® With Minus K®
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