Experiment 4: Imaging cells with Nikon Microscopes and

Micro-Manager

(a youtube video demonstrating the software can be found at http://www.youtube.com/watch?v=AB0L9qmx-Qg)



Step 1- Set up a dish of cells and put the dish on the microscope stage


1.Take a 60 mm Petri dish and add about 5x105 cells in 3.5 ml of HL5 (think about what this means in terms of concentration!  What is it approximately?).


2.Bring the dish to your microscope.


3.Each workstation has a microscope (Nikon or Zeiss) with a video camera and computer that controls the camera and acquisition of images.  The microscope is completely manual. 


4.Each microscope has 4x, 10x, 20x, 40x and 100x objectives.  All except the 100x have phase contrast capability. .


5.On both sides of the microscopes are large black focus knobs to move the objectives up and down.  The small knob is fine focus and the large is coarse focus.  Always watch the objectives as you move up and down so you don’t smash the objective into the dish holder or the sample.


6.The 10x objective should already be in position.  Drop the objective to the lowest position with the course focus knob.


7.Adjust the sample holder sliders to a separation that will hold your dish. Loosen the two small screws on the sample support arms and move the two arms that hold the specimen to a distance that will support the dish.  DO NOT OVERTIGHTEN THESE SCREWS.  Gently turning them a half turn each way is sufficient to tighten or loosed them!  If you guessed wrong on the spacing, change the spacing and then try again. Put your sample on the stage. 


8.On the right side of each microscope closest to you is another large black knob that controls the light path.  It has two positions labeled Eye and Side.  In the eye position, the light goes to your eyepieces.  In the Side position, the light goes to the side port where the camera is attached.  Put it in the Eye position.


9.Each microscope has 3 LED illuminators that can be controlled manually or using the computer. They provide light; White, Blue (470nm) and Green (530nm) illumination.  Blue and green are for fluorescence illumination which we will use later.  Today we will only use White which is for transmitted light illumination (the source for white light is located on top of the microscope).  There are three small cubes mounted on a plate next to the microscope. These control the 3 LED’s and are labeled white, blue, green.  Each has a switch on top labeled On/Off/Pulse.  When switched to ON, the LED is on all the time.  When Off, it is always off. In Pulse mode, the LED is switched off and on by Micro-manager.  You should leave the system in Pulse mode at all times so that you can’t accidentally leave the LED on for days.


10. ImageJ is an image analysis program.  Micro-manager is a plug-in to ImageJ that allows control of a microscope, camera or other equipment.  We are using it to control the light sources and the camera that are attached to your microscopes.  We also have a program called Fiji


which is a version of ImageJ that is configured for easy set-up.  You will use Micro-manager to acquire data from the microscope.  You should save that data and then open it in Fiji for analyses and manipulation of the data.  I recommend you download Fiji to your personal computer so you can use it outside of the lab. It is freeware and has a huge development and support community to which anyone can subscribe.  On the two sites below is also documentation of each of the programs if you need help.  These are also bookmarked on the lab computers.


    1. Fiji: http://pacific.mpi-cbg.de/wiki/index.php/Main_Page


Step 2:  Start Micromanager Software


1.Start Micro-manager by clicking once on the icon in the dock. When the program opens you will see the ImageJ toolbar and the Micro-manager control window (see images)


  1. 2. In the Micro-manager window- click LIVE and the light should come on.  The knob on the top of the LED controller allows you to control brightness.  Make sure there is light coming through to your dish



                                                                                                                                   

                                                                                                                           

                                       





















Step 3: Focus on the Specimen


1.Now look through the eyepieces and bring the objective up slowly with the Course Focus Knob until your cells come into focus.  You can use the stage control knobs to move the dish to a field of view you like.  Now use the Fine focus knob to find the focal plane you want to image (usually the lowest in focus plane near the dish surface).

a.IMPORTANT Notes:


A.When using an upright microscope it is a good idea to focus away from the specimen. For instance, if you are not sure where the sample will be in focus, bring the lens close to your sample and then move it away from the sample as you look through the eyepieces to find the correct focal plane.  This protects you from smashing the objective into the sample and breaking your slide or the objective.


B.For our inverted microscopes, because the sample is not held down, overfocusing will simply lift the slide or dish off the surface.  Lens elements are recessed, so it is hard to damage the objective this way.  Therefore, it is okay in that case to slowly and carefully focus toward the specimen.  Keep an eye on the objective as you do this to try not to hit the specimen.


C.It is a good idea to look at the spacing for each objective when in focus so you can estimate the distance where correct focus should be.  As the magnification increases, the working distance decreases, so the 100X oil immersion objective must be very close to the sample whi


le the 10X air objective will be further from your sample when in focus.


2.Now you will align the microscope so it is set up optimally for imaging.  You can do this with teh camera or by eye, but it is easiest to learn while looking through the eyepieces so start with that.  Eventually you will capture images of what you see and post them to your web site.



Step 4: Setting up Köhler Illumination


Adjusting the Condenser


1.Make sure the light is on and the sample is in focus.


2.Just like the objective needing to be brought into focus to see the specimen, the condenser needs to be brought to the correct focal plane to illuminate the specimen.  That is why it is on a carrier that can be moved up and down using the condenser focus knob.


3.Open the aperture diaphragm completely (see diagram on lecture pdf).  Do this by rotating it in both directions.  In one direction no light will be coming through to the objective and in the other lots of light will come through.


4.While looking through the eyepiece, partly close the field diaphragm (at the top of the tilt back arm), so that only about one-quarter of the field is illuminated. 


5.Using the condenser focus knob, move the condenser up and down.  You will see the edges of the dark zone in the center become fuzzy or sharp as you move.  At the correct position, the edge is sharp.  Moving the condenser up or down from that position will make it look larger and fuzzier. 


6.If the circle of light is not in the center of the field, use the the two centering screws to bring the image of the field diaphragm (circle of light) into the middle of the field of view.  If it is in the center, play with the screws anyway to see how they work. 


7.Open the field diaphragm so that the lighted portion is just outside the field of view.


8.You are done!  You have set the microscope for correct Koehler illumination.


9. You should check Koehler each time you go to use the microscope.  The centering should not need readjustment.




Step 5. Adjusting the Eyepiece


1.Using the binocular or slider adjustment on the eyepieces, adjust the interpupillary distance (distance between the eyepieces) so that you see one clear, round field of view.

2.One of the eyepieces on the microscope has a scale and can be rotated to adjust the focus.  The other is fixed.


3.Close the eye that looks through the non-focusing eyepiece and bring the specimen into focus. Now look through the focusing eyepiece with the other eye and without touching the focus knob, rotate the eyepiece until the specimen is in focus.  Now open both eyes and both should be in focus.  See if you can find a position that works OK for both partners.  


NOTE:  Some microscopes have one focusing eyepieces, some have two and some have none.  Check your microscope.   

# You would normally put the non-focusing eyepiece on your dominant eye.  To determine which is your dominant eye, focus on a target 20-30 feet away through a cardboard tube.  Without moving the cardboard tube, close one eye.  If the tube is in front of the open eye, that is the dominant eye.  If the tube is in front of the other eye, then the closed eye is your dominant eye.




Step 6: Acquire Still Images for Notebook/Web site


1.  Now switch the knob on the microscope to Side (camera port).


2.  The image should now appear in a window in the software.  If the image is all white, it means the light is too bright. You can either reduce the light power using the knob on the top of the White control box or reduce the exposure time in the Micromanager window.  If it is all black, it means no light is getting to the camera.  Either the light is not on (check by looking at the sample and you should see light shining on it) or the camera/eye knob on the microscope is set to eye (if so you should see light when you look through the eyepieces).  For transmitted white light imaging, usually the best setup is to keep the LED dial at its minimum setting.


3.The camera is usually not perfectly positioned relative to the eyepieces, so you will have to refocus the image while looking at the screen to get it in focus. 


4.Click the Snap button to acquire an image.  You should now save that image to an appropriate place on your computer.  The normal format to use is TIFF.


5.Snap images to show the various stages of alignment that you carried out above.  You want to collect images that show the correct settings vs. what it looks like when incorrectly set.  with the condenser in the correct position and also with it far enough out of correct position so that you can see what it looks like when it is wrong


a.Close the field diaphragm to show what that diapragm does

b.Open the field diaphram too far and snap and image.  You should see less contrast due to flare from light outside of the expected light path

c.Raise and lower the condenser to incorrect positions and snap images to show what it looks like when the condenser is in the wrong place.

d. Close the aperture diaphragm to various positions and show the effect on the image

e. Move the phase ring selector to the wrong position and snap images to show the effect on the image.

f.The A position has no phase ring.  Use that position and close the aperture diaphragm and note the effect. (on the Zeiss, move the condenser turret to one of the DIC positions that has an aperture).

g.You can sometimes get interesting effects if things are not lines up correctly.  Try slightly moving the phase rings from their normal position and notice the effect.

h.Collect images at different exposures times or light intensities

i.As you decrease the exposure, the images will get dark.  Click on the ImageJ toolbar to bring up the ImageJ tools.  Select an image and use Analyze/Histogram to see the distribution of intensities in the image.  You can save that histogram and put it on your web site

j.Now go to Image/Adjust/Brightness/Contrast

k.Adjust the sliders to make the image more appealing then Apply the Lookup Table.  Save that image and measure the Histogram as before.  Note how the values have changed.  Note the extent to which you can “improve” what an image looks like with these adjustments.  You can do the same with some of the images that already looked OK to make them even better.


Step 7: Capture a time lapse image series.


1.Click on the Multi D Acq. Button to bring up the settings for time lapse acquisition

1.


In the upper left, make sure the Time Points box is checked.  Set the time interval between images.  A good starting point would be about 30 second interval

2.Set the Number- this is the total number of images you will acquire at a 30 second interval

3.Make sure Channels box is clicked and click on Channel group and choose channel

4.Set the channel to White and the exposure you found from the Live window

5.Click the box to Save Images.  In the Directory choose a location to save and a name to use.

6.Click Acquire and the computer will collect an image every 30 seconds and you will see them accumulating in the new window. You can scroll through the images as the data is being acquired

a.You can enter information in the comment box that will be saved with the data.


b.Watch how the cells are changing from frame to frame and adjust your time interval and total time (frame number) accordingly.  There is no point in collecting huge amounts of data if the cells are not changing significantly from frame to frame. You can adjust the frame rate of playback using the box at the bottom of the acquisition window.

c.You can try different settings and collect some data until you are happy with the interval. 

d.When the acquisition is done your data is saved, but you should also save the data as a Movie.  Click File/Save As/ Save as/Save as Quicktime Movie.  Choose MPEG as the format and then save.  You should watch your movie (double click on the file to open Quicktime Player) and make sure you are satisfied with it and then upload to iWeb.


2.Watch the movie to get a descriptive view of cell behavior. Do all the cells move? Do they all move at the same rate? Did any divide? What can you say about the mechanism of movement from watching?


Goal- You will be repeating this experiment on Thursday with wild-type and myosin mutants, so this is a trial to work out the software and parameters you will use for that experiment.  You need to figure out what density of cells to plate, how frequently to take images, what magnification to use, how long to image each cell type, etc. 

 
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