Setup of a 4D live cell imaging system at an LSM 410

the demands for cell viability and for microscopy have to be met. The cells should be kept on the microscope stage under sterile conditions at a temperature of 37°C and at a pH of 7.4. They have to be supplied with fresh cell culture medium. The microscope should allow to automatically record time series of 3D image stacks with stable imaging conditions.

Figure 3.29: Experimental setup (A) and scheme (B) of the live cell imaging system.

The system consists of a Bioptechs FCS2 live cell chamber (1), temperature control units for chamber and objective-heater (1b), flexible tube pump (2) connected to the control PC via a solid state relay (SSR), syringe for supply of fresh medium (3), disposable syringe filter (4), waste container (5).

A system, which meets these conditions, was established at the Zeiss LSM 410 confocal microscope in Munich (Figure 3.29). The cells were kept in a temperature controlled closed live cell chamber (Bioptechs FCS2), and were imaged with a 63x/1.4 Plan-Apochromat oil immersion objective. As the immersion oil has considerable heat conductivity, the objective acts as a heat sink, if not warmed to 37°C. Hence, the objective was operated with an objective heater, which kept the objective collar at 37°C. The chamber was perfused with medium through a metal

tube on one side of the chamber, and waste medium was disposed through an identical tube on the opposite side. To keep the pH stable the cells were kept in medium with carbonate buffer and HEPES buffer. The carbonate buffer needs an atmosphere with 5% CO2. As this could not be supplied on the microscope stage, the

medium was CO2-adapted in a gas permeable container in a cell culture incubator for

at least half an hour. The medium was then filled into a syringe, which was connected to the chamber by plastic tubes via a flexible tube pump and a sterile filter. On the other side of the chamber a plastic tube led the waste medium to a waste container. The whole assembly was airtight to prevent the loss of CO2 from the

medium. The flexible tube pump was switched on and off by a solid-state relay, which was connected to the parallel port of the PC. The relay could be switched on or off by setting bit 0 of the register at the base address of the parallel port (0x378) to either 1 or 0 with the “OutPort” command of the LSM 410 macro language. The pump was run at minimal speed for 1 minute each hour. At this setting approximately 1 ml medium was perfused every three hours.

Figure 3.30: Macro for live cell confocal microscopy at the LSM 410.

The 4D-imaging macro records an overview image of the whole field of view and high-resolution z- stacks of selected ROIs at regular time intervals. Imaging parameters can be adjusted for each ROI individually. Bars: 10 µm

The control software of the LSM 410 allows recording focus series (“3D image stacks”) and time series of light optical sections. Its capabilities were extended for live cell confocal microscopy by a macro written in the proprietary macro language of the LSM 410 control software. The macro can record time series of 3D image stacks in multiple regions of interest (ROIs) at high zoom within a common field of view (Figure 3.30). It compensates for z-axis drift, a common problem in live cell microscopy, by imaging the interface between the coverslip and the cell culture

medium in reflection contrast before each image stack is recorded. The reflected light intensity has a sharp maximum at one z-position, which is used as reference position. The macro also switches the perfusion pump. To avoid distortions in the imaging process induced by vibrations of the pump or the pressure of the pumped medium, the pump is switched on in the interval between two successive time points of imaging for a selectable duration. For the various demands of the users of this microscope versions of the macro have been created, which record image stacks for one, two and three fluorescence channels. For this work, only one fluorescence channel was needed. This version of the macro (“TimePr1”) also records a transmission image produced by the transmitted excitation light at every z-position simultaneously with the fluorescence image. For the fast and easy visualization of the recorded time series a macro (“Dispt”) for the dummy version of the LSM 410 software was created, which allows to scroll through the z-slices and time points of the 4D series. With this live cell observation system cells could be followed for several days on the microscope stage until they grew confluent.

To perform laser-photobleaching of GFP with this system, the Ar+ laser of the LSM 410 was used (488 nm, 15 mW, operated at 30% power with an attenuation filter). An image of the cell was recorded at high resolution (512x512 pixels, Zoom 8). For bleaching of stripes a line in this image was selected with the “Line Scan” function, and 2 - 5 line scans were performed without attenuation filter. For bleaching of larger areas a rectangular region of interest (ROI) was defined, and 2 - 4 images of this ROI were recorded without attenuation filter. The ROI-function of the LSM 410 only displays pixels within the selected ROI, but scans the laser along the fast scanning axis over the full width of the image. Therefore the height of the bleached region is defined by the selected ROI, but its width is defined by the image width, i.e. by the zoom factor.