Chapter 8 Live Cell Imaging
8.1 Introduction
Live-cell imaging is done under specific incubation conditions, with control of CO\(_2\), temperature, and humidity, and does NOT refer to general imaging of living samples. The Tumor Cartography Core offers a separate training for live-cell imaging with the Zeiss LSM 980. If you have not already completed this training, in addition to the overview LSM 980 training, it is required to book a free training session on Research Cores and to complete the training before use.
8.2 Pre-Experiment Set-Up
The set-up for incubation conditions takes approximately 45 minutes to an hour. It is important to change the stage to the incubation chamber box, as the cover will not fit on the regular stage. Additionally, the condenser head may need to be raised in order to not hit the incubation chamber. When the proper stage is placed, it should be covered and brought to incubation conditions. Note that CO\(_2\) does not need to be turned on at this point, and can be turned on right before the sample is placed in the incubation chamber. The incubation chamber adjusts temperature, humidity, and CO\(_2\) on a feedback loop until it reaches a stable asymptote with the desired conditions (\(37^{\circ}\)C, \(14-15\)psi for the CO\(_2\)).
8.3 Experiment Set-Up and Considerations
High resolution imaging can be difficult when using a multi-well plate and an immersion objective. For resolution purposes, a plate with an image-grade glass bottom is essential, and the \(20\)x objective should be used if possible. Additionally, if not using an immersion objective, Definite Focus is a useful tool to monitor the \(z-\)position of your sample. Under the Definite Focus tab on the software, you can use the “find surface” button, which will find the bottom of the well-plate.”Lock focus” then fixes the working distance as you move the sample in the \(xy-\)plane.
8.4 Focus Strategy
Implementing an effective focus strategy is the most challenging part of
live-cell imaging. The following section was adapted from Nikon MicroscopyU (Joel S. Silfies, n.d.) for our set-up. Because
live-cell imaging experiments usually occur over a longer period of
time, one of the main sources of difficulty in implementing an effective
focus strategy is focal drift. There are a variety of factors that can
cause focal drift, as will be outlined below, that you should consider
when conducting your experiment.
Thermal Drift can affect both the sample and the apparatus, and lead
to focus drift. Fluctuations in thermal energy occur due to a wide
variety of factors, such as conditions in the room or energy introduced
via the light source for the experiment. These fluctuations can cause
unwanted motion (drift) of the sample, changing the focus. Additionally,
the materials of the microscope may contract or expand in changing
temperatures, which can alter the alignment and consequently the focus.
Heterogeneity in Chamber Heating can occur, particularly with high
numerical aperture objectives that require an immersion media. The
reason that heterogeneity in heating is more of an issue with immersion
objectives is that heat is conducted via the metal objective, which
causes a thermal gradient. Additionally, heat from the incubation
chamber can cause evaporation and degradation of the immersion media,
changing its refractive index.
Sample Drift occurs when the sample itself moves, such as a cell
undergoing mitosis, and is no longer in the correct focal plane. While
there are not a lot of ways to mitigate sample drift, there are some
strategies that can be employed to maintain focus as the sample moves.
The Zeiss software provides two main methods of maintaining focus
throughout a live-cell imaging experiment: Definite Focus and AutoFocus.
While these options sound similar, they use different
strategies. Definite focus keeps a consistent focal plane by using an
LED light source to measure the distance to the coverslip. Definite
focus is a great option for samples that may be more delicate and prone
to UV damage or photobleaching. Auto focus actually utilizes feedback
from thesn detector to keep samples in focus, which provides a
relatively precise way of keeping the sample in focus. However, because
auto focus utilizes the laser light, it can be more damaging to samples
in longer experiments.
8.5 Timing Considerations
We ask that you are considerate of the time of other users and do not book more than three peak hours (9am-5pm) for imaging. If your experiment is going to take longer than three hours, please book the remainder of the time during off-peak hours. The updated system will calculate how many cycles you need to image for a given period of time, and you can adjust the lag-time in the experiment set-up.