![]() ![]() Use "Analyze" and "Set Scale", set the Known Distance and Unit (in this case it's 1.00 and mm), select "Global". ![]() Select the line tool, zoom in (press '+' and hold the space bar down to move the image with your cursor) and carefully trace a known distance in the image, e.g. Download the free image analysis program "ImageJ" from the NIH website ( ).(Include a photo with a hemacytometer or ruler for image scaling.) Images should save automatically to the same folder. Your image will appear at the bottom of the window, and can be renamed. To capture an image, click the "Auto" button in NIS Elements.Turn on the flexible lights and microscope base lights to illuminate the stage as needed. Select the "scale" tab, which will be needed for scaling the image for analysis in ImageJ. Under "camera settings" set "Fast Focus" to 640x480 to increase camera speed when focusing, and set "Quality (capture)" to 2560x1920 to maximize the quality of the captured image. Use a camera-mounted microscope (Nikon Digital Sight) connected to a computer with NIS Elements.Place the plate on the dissecting microscope stage. Pipette DI water over the root until it is covered, to suspend the root hairs. Using forceps, immerse the root in the blue dye for approximately 5 seconds. Fill one Petri dish with DI water, and a second dish with 0.05% Toluidine blue dye.I can count the bright peaks to find that there are 5.5 striae in 5 µm, or 11 striae in 10 µm. The plot window shows the pattern of light and dark that my yellow 5 µm line crosses. Now, go to the menu and select Analyze > Plot Profile. It is important to measure striae in this location! Also note that I carefully drew a line that was 5 µm long. First, note that I drew a line starting at the central area, parallel to the apical axis. You can measure the number of stria density of your diatom. Now you can measure the length and width of your diatom using the line tool. If you check the box for "Global", the calibration will apply to all your images. I entered the known distance as "10" and the units as "micro" (I meant to write micron, oops). For this image, there are 244 pixels for 10 µm. Note that the length of the scale bar (in pixels) will appear. Select Analyze > Set Scale from the menu. (The thin yellow line is to the left of the scale bar). Use the line tool to measure the length of the embedded scale bar. We usually use 10 µm scale bars for LM images. Find out if you get the same results when you measure it in ImageJ. Download this image by dragging it to your desktop. ![]() ![]() (The thin yellow line is to the left of the scale bar in the image to the right). At least one of your digital images needs to have an embedded scale bar. It has a handy tool for measuring diatoms from light micrographs or scanning electron micrographs. ImageJ is a free, powerful software program. ![]()
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