Sample control:
D.1 Set-up preparation
3. Adjusting time for the CO 2 gas incubation system:
At least half an hour before the set-up should be used, one needs to switch on the gas incubation system with active gas mixer. Usually, the following parameters were used:
• 5 % CO2
• 3 liters gas per hour
• 75 % humidity
To help the System stabilize, keep petri dishes in the chamber to avoid large air / temper-ature / humidity fluctuations due to airflow in and out of the chamber. These petri-dishes should not be the ones one wants to use for the experiments but dummies.
D.1. Set-up preparation 155
Adjustments for room F.03.143:
1. Room temperature:
Imaging in room F.03.143 is quite tricky. In case one doesn’t take care of a stable room temperature, one will end up with massive z-drift problems. The cold air flow is directed almost directly to the live-cell imaging set-up and in case someone is using the other imaging set-up in the room, which causes a temperature increase by 5◦C, it will cool down the live-cell imaging as much as possible. This won’t solve the heat issue but instead cause Z-drift issues on the live-cell imaging set-up.
• The temperature sensor is directly at the door, so it needs to be kept closed all the time during the experiment and - if possible - the night before.
• Furthermore the ventilation system and the temperature control should both be switched on. The temperature control to maximum, while for the ventilation system "on" plus twice as much air-flow is sufficient to avoid heating/cooling problems with the neighbouring set-up. Switch on both the night before, so the room temperature is stable when one starts imaging.
2. Windows-update related saving issues:
Every third Wednesday to Thursday night is Windows - update night. Although there won’t be any updates at the computer connected to the microscope set-up, the connection between the "AG-Schmidt" - server and the computer will be interrup-ted at some point, which will result in a memory error (can’t save data on the server).
To make a long story short:
In case it is the third Wednesday of a month and one wants to start imaging, save the images on an external harddrive.
D.2 Petri dish preparation
Materials needed:
• prepared gel(s) or cover glass(es)
• lint-free tissues
• Isopropanol
• bottomless petri dish(es), 35 mm Ø
• petri dish lid(s)
• UV-curing glue
• tweezer with curved edge
• UV-lamp
• PBS, autoclaved See also 3.9
Procedure:
· Wet a lint-free tissue with isopropanol, clean petri dish and lid with it
· Let dry on a lint-free tissue
· Distribute the glue in a line (not too thick!) along the inner border of the petri dish bottom
· Dry the cover glass (with or without gel) at the bottom side (not the gel side) gently with a lint-free tissue
· Carefully drop the cover glass at the petri dish → bottom side facing the glue
· Make sure all glass edges are touching the glue! If not, the bottom will not be liquid stable and ruin your experiment. To do so, take a tweezer and gently push the glass edges towards the glue. In case you are working with a gel on a cover glass, be careful not to damage the gel.
· Put petri dish and lid on a lint-free tissue and (very close) underneath an UV-lamp and cure for 60 - 90 seconds
· Turn the petri dish upside-down and let cure for another 60 - 90 seconds
D.2. Petri dish preparation 157
· Now the petri dish is liquid stable → fill with 2 ml PBS and put under UV for another 60 minutes
· Put under cell culture hood
· Wash twice with sterile PBS
· Fill with 2 ml cell medium, keep in incubator until cells are seeded (should be within hours afterwards)
D.3 Imaging
The cells need to adhere to the substrate before one can start imaging, so it is necessary to let them sit in the climatic chamber for 45 minutes up to one hour.
After everything is set, imaging can be started. The free and open source software called Micro-Manger [218, 219] is used to automatically obtain images in intervals of 10 minutes between two subsequent images.
The general procedure to start a live-cell movie is the following:
· The climatic chamber as well as the whole set-up is switched on (microscope, lamps, camera).
· Focus on the cells, then optimize the phase contrast by using the "Köhler"-technique.
· Switch the light path from the ocular to the camera
· Start the computer, wait until windows is ready to be used
· Check with D if you need an external hard drive to save the data
· Start Micro-Manager and choose the latest startup configuration (should look like figure D.1a). If loaded successfully, it should look like figure D.1b
D.3. Imaging 159
(a) Micro-Manager startup window
(b) Successful start of Micro-Manager configuration
· Now one needs to open the "Multi-dimensional Acquisition" tool (see figure D.1c). It has several features:
◦ Time points
Here one can choose time intervals between two subsequent images (starting from milliseconds to hours) and the number of time-points which translates to the overall imaging time.
◦ Multiple Positions
To image not only one but several cells at a time, one has the option to save several positions in x-, y- and z-direction in a list. Can be saved separately.
◦ Z-stacks
It might be of interest to image a cell at different z-positions at each time-point.
This feature enables to take several images in a chosen distance, for example every 0.5µm from a chosen start to a chosen ending point.
◦ Channels
In case the sample is fluorescently labeled, it might be useful to chose more than one channel / wavelength during imaging.
◦ Save images
A folder to save the images as well as a name prefix for newly created saving folder can be set here.
◦ Aquisition comments
Anything that might be of interest concerning this particular measurement can be noted here.
D.3. Imaging 161
(c) Multi-dimensional acquisition tool
(d) Choose path to save images
· The used parameters were:
◦ 144 time-points in intervals of 10 minutes
◦ No z-stacks
◦ Channels:
Phase contrast [3.5 Volts and usually 30 ms exposure time]
RFP [300 - 400 ms exposure time]
◦ Mainly saved in the "Carina" folder on the "AG-Schmidt"-server, on a hard drive (FR.01 - property of Florian Rehfeldt) otherwise
◦ Since the settings are more or less the same and can be read out of the data-files, there are almost no comments.
· After adjusting the general parameters, one has to choose the cell positions. Clicking on "Edit position list" will lead to the position list window.
· Choosing a channel and an exposure time in the main Micro-Manager window and clicking "Snap" will give a first view on the focused sample. In the "Live" - mode, the focus can be readjusted and by switching between phase contrast and fluorescence channels fluorescent cells can be found. Once a cell of interest is in focus, one tries to locate it in the center of the field of view. This will allow the cell to move around and still remain on the cameras field of view. If that is done, one click on the "position list" - window on "mark" will save the position in x-, y- and z-direction (see figure D.1e).
· It is useful to save this list every once in a while (every 10-20 positions) to ensure one won’t be searching the cells one more time. In principal Micro-Manager is a very stable program, but in case it crashes it is better to be safe than sorry.
· After a satisfying number of cells has been chosen and the positions have been saved on the list, the only thing left to do is to click "Acquire!" (see figure D.1f).
D.3. Imaging 163
(e) Edit and save position list
(f) Image acquistion in progress