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    • CommentAuthorjuruble
    • CommentTimeJun 22nd 2007 edited
     

    This protocol explains how to: use Olympus fluorescent microscopes to image in vitro experiment slides.

    Protocol revised: 3/15/2007

    Protocol written by: Julie Ruble

    1. Turn on green burner power button first, and then the microscope and then the camera.

    2. Start viewing in fluorescence (change filters using the filter wheel until you reach the green filter). Begin at the corner of the slide. Use the 40X objective lens.

    3. When looking for neurons to image, scan through the slide methodically so as not to miss any:
      slide example

    4. When you find a neuron, position it with the cell body in the field of vision but off to the side (so you'll be able to get more neurite in the picture and therefore take fewer pictures). NOTE: When you are not looking at or imaging the neuron, close the shutter on the right side of the microscope so as not to bleach the neuron.

    5. To take a picture, open SPOT (the program may have a shortcut on the desktop, but you may need to find it in the C folder and create one). Then, to take a fluorescent image:

    6. -Click "manual" on the image settings box.
      -Adjust the "gain" to ~16 and the exposure time to ~9-10 seconds (can increase exposure time if picture is too dim, or decrease if it's too bright).
      -Click "More" and make sure it is set to "green."
      -Open shutter, divert light to camera by pushing in slider on right of microscope, and click the camera icon.
      -Close the shutter when picture is finished exposing!

    7. To take phase images:
      Without moving the stage, turn the filter wheel to phase and turn up the lighting (dimmer switch on right of microscope). Focus on neurites. If you cannot see neurites, you may have to fiddle with light levels, filter on bottom of microscope, focus, etc. Make sure phase ring is set to Ph2 (phase 2).

      To take pictures:
      -Click "auto" on image settings box in SPOT.
      -On the "more" dialog box, make sure it's set to "RGB" instead of "green."
      -Direct light to camera and click camera icon.

    8. Since neurons are larger than camera view, you will have to take several frames to get the full view. While you take them, "build" them with a drawing in your notebook to make it simpler to photoshop them together later. Frame "A" should always contain the cell body. For instance:
      frames

    9. Saving images:
    10. In this experiment, images are saved by experiment number, slide letter, neuron number, frame of neuron letter, and then a lowercase "p" for phase images or "g" for green fluorescent images. For instance, a picture might be saved as: 4G1Bg.jpg. This indicates that the image is part of experiment 4, slide G, is the first neuron imaged on the slide, is the second frame of the neuron, and is the green fluorescent version.

    11. You can stop in the middle of a slide if you need to -- just refresh yourself on your pictures when you return so you don't re-photograph the same neurons (also, you place a piece of tape light on your slide to mark the "row" you were on when you stopped last).

    12. When you turn off the microscope, turn off the camera and turn off the burner last. Record your name, time finished, and bulb hours (found on the burner) on the log. Cover microscope. Do not turn microscope and burner back on within 15 minutes of turning it off -- it needs 15 minutes to cool.

    13. Put slide back in slide box in fix fridge. Fill out chart on front of slidebox with the number of neuron images you obtained from the slide and your initials.

    Examples of images:

    fluorescent example

    40X fluorescent image -- 4G4Ag.jpg

     

    phase example

    40X phase image -- 4G4Ap.jpg

    • CommentAuthorredsox07
    • CommentTimeAug 5th 2007 edited
     

    If you are staining with phalloidin, you will need to take a picture using red fluorescence as well as green fluorescence.  All that is different is that you need to rotate the wheel above the objective lenses one click to the left from the green fluorescent view (which is actually the setting to the right of the green) and open the shutter and you will be able to take a picture in red fluorescence. 

    In my experience, I found that the red fluorescent pictures showed up much fainter than the green fluorescent pictures, making it hard to focus on the neuron and also making you question the purpose of taking pictures using red fluorescence.  However, the pictures come out much better than they appear when you first take them and can provide a pretty neat view when they are screened on top of the green in Adobe Photoshop.  And I found that leaving the focus settings the same for red as I did for green produced pictures that were in focus. 

    You may need to expose the neurons to the fluorescent light longer in red fluorescence than you did with the green, which can contribute to bleaching the neurons if done to the extreme, but I still found taking the red fluorescent pictures worthwhile when it came time for measuring neurons in Image-Pro.

    Mike Neri, summer 2007

  1.  
    If you have a hard time finding neurons in phase and you're worried you might be in the wrong focal plane, just quickly check to see if the edge of the coverslip is in focus (or at least nearly in focus). You will find neurons in the focal plane immediately above the coverslip (if in culture dish) or just below (if culture is mounted on slide).
    • CommentAuthorjoiordanou
    • CommentTimeNov 10th 2007
     
    General tips- If you are having trouble finding neurons make sure your phase rings match the objective you are using and try using only the fine focus, it might take a little more time but you can easily skip over the neurons focal plane if you use the coarse focus.
    • CommentAuthorkilang
    • CommentTimeNov 11th 2007 edited
     
    Tips:
    -If you feel that you need it, there is a 'White Balance' button on the top toolbar (it looks like a red green and blue arrow pointing downwards. It's the 5th button from the left).

    -If are having trouble focusing, try clicking the Focus button (it is the second from the left and has a picture of a hand and a camera). It will let you select a region of your image and watch the effects as you play with the focus knob on the microscope.

    -Turning the fine focus knob very slightly back and forth while scanning can help you to see neurites; they may be faint in one focus plane but become much clearer when the focus shifts a tiny bit.
    • CommentAuthorkacole
    • CommentTimeDec 15th 2007
     
    If the contrast seems off, try cleaning the objective. Carefully remove the objective, then use a lens cleaning cloth or q-tip (NEVER USE A KIM WIPE) dampened with window cleaner or water to remove any debris. This usually improves image clarity and restores contrast.
    • CommentAuthordoippolito
    • CommentTimeDec 20th 2007
     
    You'll find that imaging entire slides can be quite time consuming. Here are a couple quick things you can do with the camera software to shave some time off.

    1. Speed over quality in fluorescence:
    Remember that the primary purpose of the fluorescent imaging is to verify that a given neuron is an RGC. Since you really only need to verify the presence of fluorescence in your neurites, there is no need to wait any more than a few seconds to get your image. One of the easiest ways to do this is to go to your 'Image Settings' display and slide the bar from 'Quality' all the way over to 'Speed.' Make sure that you adjust brightness and gamma so you can see each neurite, but sacrificing some image quality for speed can be a big time-saver, especially if your immunostaining is dim.

    2. Get into a pattern with your imaging:
    In my experience, constantly having to alter the image settings between fluorescence and phase was not only time consuming, but also frustrating in that I had a hard time keeping any momentum progressing through the slide (I found the preset options to be finicky as well, so I just stopped trying to use them). I was able to get a much better flow going by following an alternating pattern between image types from neuron to neuron. Since that probably is vague, here’s a basic outline of what I found helpful:

    a. Find a neuron
    b. Set Spot32 camera settings for phase
    c. Take phase image
    d. Set Spot32 camera settings for fluorescence
    e. Take fluorescent image
    f. Find next neuron
    g. Take fluorescent image
    h. Set camera settings for phase
    i. Take phase image
    j. Find new neuron
    k. Take phase image
    l. Set camera settings for fluorescence….and so on and so forth, until you’re done
    (Just something to try if you get frustrated with your pace.)

    Oh, and don't forget to wipe your slide down before you get started!
    • CommentAuthorjuruble
    • CommentTimeJan 4th 2008
     

    There are two versions of the SPOT program on the computer, Basic and Advanced.  If you can't seem to find something, you may be using the wrong version.

    • CommentAuthoraldeal
    • CommentTimeDec 15th 2010
     

    If images appear consistently out of focus, it may be that the camera is out of focus. You can adjust the focus of the camera by turning the "Camera Focus Adjustment Ring." (see below)