Microscopy101
Figure 2 : (A) A processed, formvar-coated grid (note the “ L ” in the center) with neurons and glial cells, as viewed with phase optics. (B) The insert in 2A is shown here at a higher magnifi cation that allows neuronal growth cones to be easily visualized. Note the brown “burned in” spot (arrow) where a growth cone has been viewed with TEM.
Figure 4 : (A) Low magnifi cation of a growth cone with region of interest (B) indicated. (B) Stereo pairs of the growth cone in (A) were taken at a magnifi cation of 40,000 at +6° and -6° angles by tilting the goniometer. The resulting stereo pairs were used to produce an anaglyph by placing one image in the red channel and the other in the blue channel using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA) according to [ 3 ]. View the anaglyph image in 3D using red (left lens)/cyan (right lens) glasses. Scale bars are 2 µm (A) and 0.2 µm (B).
Figure 3 : A portion of a grid “map” in which the center “ L ” shape has been recorded. All cells of interest are indicated, as well as notes on experimental details and the sample condition.
56
immuno-TEM. T e grids are fi led in numbered slots in a grid box labeled with the date of the experiment. Map making . To map the location of cells of interest, we view each grid with light microscopy and enter the specifi c cell positions on a “grid map” that is preprinted with a pictorial representation of a 50 mesh grid, repeated in a column of six per page. Each grid is placed on a clean slide and viewed using phase optics with an inverted microscope ( Figure 2 ). T is examination reveals information that is useful for prioritizing samples, for example, sample quality and holes or wrinkles in the formvar. On each grid map, we record the grid identity, the orientation of the grid’s L shape, the location of growth cones of interest relative to the L , and notes on sample quality ( Figure 3 ). When viewing the grid in TEM, the map is turned to refl ect the orientation of the grid as seen on the screen. We can then quickly toggle over to any cells we wish to view on that grid.
www.microscopy-today.com • 2015 July
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84