4D Video Microscopy

4D Microscopy Using the Zeiss Axioplan 2

and Axiovision 4.5 Software

Perform the following steps in order. Have all the materials ready and on hand and try to be quick as possible.

 

Before you start dissecting out the embryos make sure that you log on to the microscope computer and that the microscope and camera are on.  The Axiovision parameters should be roughly setup (see below) or load you “4D program” from the Experiment section of Multidimension Acquisition.

 

Preparing the embryos and slide:

 

  1. Prepare 50-100ml of 3.5% agarose (the one used for DNA electrophoresis) in distilled water. This concentration works the best for me. Other labs use agar. Dissolve the agarose by heating 2-3 times for at most 30 sec in the microwave. Don’t allow it to overflow. Distribute about 2-5 ml in 10 ml test tubes. Seal the tubes with parafilm and keep them in the fridge until use.
  2. Melt the agarose by heating it directly in the flame, and try not to burn it. You can keep the agarose fluid in the heating block set up at about 50-60oC for 24h.
  3. Fill a well of a the spot plate (made of glass) with about 100 ul M9 and place about 10 gravid hermaphrodites in it.
  4. Prepare the pad: Stick a 0.1mm thick tape (paper tape) along a slide. Make one more and use them as spacers. Place a third slide in parallel between the spacer slides. With a Pasteur pipette spot a drop of agarose in the middle of the third plate (the one without the tape). Flatten the agarose by dropping immediately a fourth slide perpendicularly over the other 3 slides. Do not press. Wait 15-30 seconds to cast and gently slid out the underneath slide with the pad. The pad should be smooth. This step is also described in Ledwich D et al., Methods in Enzymology, vol. 322, 76-88. From this step you should be relatively quick. Don’t allow the pad to dry out.
  5. With a thin needle (27G1/2) cut the worms in the middle of the body under dissecting scope trying to avoid the zones of the gonad with 1-2 cell embryos. The embryos will pop out from the mother. Using a mouth pipette (Sima-Aldrich Cat# A5177 Aspirator tube assemblies for calibrated microcapillary pipettes) and a drawn out microcapillary suck the embryos together with a little M9 and place them on the agarose pad. Remove most of the excess M9 by sucking with the mouth pipette, and cover the pad with a 22x22 coverslip. However, the M9 should cover the whole pad.  Remove the remaining excess M9 with a paper towel inserted between the coverslip and the slide up to the pad. Try to remove all excess M9 or the recording embryo will become out of focus in time.
  6. Seal with immersion oil. Put 1-2 drops of immersion oil at the edge of the coverslip. It will diffuse under the coverslip and completely surround the pad. If it is necessary add one more drop of oil. Use of immersion oil instead of vaseline for sealing has the advantage that it does not mess up your optics. I use cheaper immersion oil for sealing and keep the Zeiss oil only for immersion optics.  
  7. Mount the slide under the microscope. With 10x optics find a 1-2 cell stage embryo, and then switch to 60x. Take 10-12 focal planes at 2-2.2mm distance, for 8h (see below)

 

Axiovison 4.5:

Start Axiovision Rev 4.5 (make sure the camera and microscope are turned on). You want to setup the Axiovison parameters before putting the embryo on the slide.

 

Microscope settings:

Objective 10X or 20X (before oil)

Transmitted light filters should be set at 4 and 2 for low neutral setting (not much light comes through). If you choose low neutral for 4D in channels settings for during acquisition these filter setting will be set for you automatically. 

Camera Settings:

You want to frame your image as 1 embryo will not take up all the camera capture area. This will also reduce the size of your movie file.

See boxed area below. You also want to choose 650 X 514 bin 2X2 mono in the camera mode.  If you use standard mode 1300X1030 your file will be greater than 2GB after 8 hours of time lapsed capture.

 

Multidimensional Acquisition

You can load a saved experiment or setup a new one with the following properties: Experiment: make sure that the Z stack and Time lapse boxes are checked

 

 You can give the experiment a name (and save the parameters for future use) and you can also give the Image a name.

Channel Assignments (C):

During Acquisition choose DIC  (low neutral setting for 4D) You can take a measurement but it will change at 40X or 63X oil. You want to make sure that the exposure time is less than 100ms at 63X objective lens. If the embryo looks too dark do not worry about this as we can increase brightness and contrast after we captured the recordings. You don’t want too much light and too much exposure as the embryo could heat up and die.

After Acquisition setting choose: nothing- i.e. leave this blank (This is very important!)  we used to have the shutter closed here but this results in the shutter being close between each Z-section. Instead close the transmitted light shutter after the Z-stack. See below.

10Xà 40X oil -> 63X oil  (Make sure you have the DIC prism matched with the objective lens i.e DIC prism III with  40X and 63X and DIC II with 20X)

 

 

 Z stack:

While your are in 40X or 63X oil objectives focus in the middle of the embryo.

The Z stack box should be checked and you want the Start/Stop mode (not the Center Mode). 

You want to choose a Z stack of about 20 microns (10 slices at 2 microns each).  To do this first focus to the bottom of the embryo by turning the focusing knob on the right counter clockwise (i.e. your right thumb moves downward).  Focus until you just reach out of focus.  Press the “Start” button . Now move up in the focus (right thumb move up) so you get about 10 slices. If you need to move up more to get out of focus then put in more slices. If you want to add more slices add to the top of the embryo (i.e  focus with right thumb moving up and then press “stop”

The start and stop buttons sets the interval.

If you are making short movies (less than 2 hours) then you may take sections at 1mm and take more sections to get greater details.

Important! Make sure to choose after Z-stack... "transmitted shutter close" This way the light will be off the embryo between time points.

Time points (T):

Choose interval as 1 minute and the duration should be about 8 hours.

 

Once all parameters are set you can hit “start” at the bottom right hand of the work area.  Also when you start the capture, make sure to pull the right knob out all the way and the left one pushed in to allow all the light to go to the camera port (See green arrows below).

 

 

 

After the recording is done:

1) Save your file. Note it may seem that the program has "crashed" as it sometimes takes several minutes. Even if in the task manager it says "application not responding" wait for at least 10 min before rebooting.

2) If the program crashed you can recover your file from the cache. Do a search for files  >100,000kb (100Mb) and then sort by most  recent files. The default is usually in "my Pictures/Zeiss" folder.

3) Make sure to return the objective lens to 10X with the stage up and clean off any oil from the objectives.  Close Axiovision and turn off the microscope and the camera (The camera is turned on/off by the power bar). Don't forget to cover the microscope.