Calcium Imaging

Techniques and approaches in high speed calcium imaging

Introduction of ion sensitive dye into living cells:

There are a number of possibilities to introduce a fluorescent dye into cells:

• Ester loading
• Low pH loading
• Electroporation
• Ionophoretic microinjection
• Pressure microinjection

For further reading please refer to:

A.J. Lacey (ed.): "Light Microscopy in Biology". A Practical Approach. 2nd ed. Oxford University Press, 1989.

And to Molecular probes:
Important considerations for live cell imaging
Fluorescence intensity

The observed fluorescence emission is a relatively weak (factor 10^-4 to 10^-9) compared to the power of the excitation light. Therefore it is very important to achieve efficient labelling of your cells. The fluorescence intensity is directly correlated to the amount of fluorescent dye molecules in a certain volume. In addition it is important to use a powerful and stable light source and to introduce the light into the microscope with high efficiency. In the Agilent system, epifluorescence condensers are specifically designed for different microscope models to ensure efficient and homogeneous illumination of your sample.

Selection of the most suitable dye

Furthermore it is important to choose the correct fluorescent dye in terms of a good quantum yield, photobleaching and quenching. Please also refer to Fluorescent dye parameters. Other factors such as cell permeability and life time in a living cell or toxicity are also important to consider as well as the most suitable dye loading method.

Bleaching

Too much excitation light for fluorescence imaging may cause cell damage and photobleaching of the dye. Photobleaching is a non-linear process and is in addition dependent of the dye used. In order to reduce photobleaching to a minimum you should illuminate your sample only when images are really acquired. This requires a system being able to accurately synchronize the illumination of your sample and the image acquisition with the camera. If this synchronization is inaccurate, you will get artefacts due to a drift between the illumination and image acquisition timing. Most imaging system use the PC for synchronization. However the operating system of a PC will introduce delays of a few milliseconds, which, especially for short integration times, may lead to a drift in timing and cause artefacts.

Use optimal optics

If you use dyes excited in the UV you need to use specific objectives with a high UV transmission. In addition the NA (numerical aperture) of an objective determines the total amount of light passing through the objective. The higher the NA, the larger the cone of light focused. Since the light passes the objective twice, the NA influences the total light output by the factor of NA2. Therefore High NA objectives are always preferable for fluorescence measurements. DIC optics should be removed when measuring fluorescence in order to capture as much emitted light as possible and to keep exposure times low.

Suitable filter set

For each fluorescent dye a suitable filter set should be used. For new dyes it may be useful to run an excitation spectrum to find the best signal to background ratio for a given dichroic mirror. If you use dyes with different excitation wavelengths (e.g. FURA and GFP), you can also use one single filter set with a specific dichroic reflecting two or more wavelengths efficiently. Using the Polychrome 5000 you can quickly switch between different wavelengths and also run an excitation spectrum. If you have a specific configuration and you would like to know which filter set to use, please do not hesitate to contact us to discuss the options you have.

Light Sources

For fluorescence imaging usually Hg and Xe light sources are used. Hg is usually used in standard epifluorescence illumination in most microscopes. It has a high intensity at certain wavelengths. The traditional fluorescent dyes have been designed to match these Hg lines. However with more complex applications new dyes are used which are excited at wavelengths where the Hg lamp does not have any peaks. Compared to the Hg lamp the spectrum of the Xe lamp shows a very even distribution of intensity over the wavelengths. This makes it more suitable for dyes excited at wavelengths between the Hg peaks. With a Xenon lamp in a monochromator any wavelength in the visible spectrum can be chosen and the light output will have a comparable intensity over all wavelengths. The Polychrome 5000 is a monochromator combined with a Xenon light source.

High Speed Imaging

In order to speed up image acquisition to highest speed, the following factors have to be taken into account:

• the time needed to select an excitation wavelength
• the exposure time
• the read out time of the camera system

With the Polychrome 5000, the wavelength can be changed within 2 ms.

The exposure time can be minimized by on-chip binning by the factor of 4 and much more

The readout time of the camera system is dependent on

• Resolution/binning
• Size of the area of interest

The camera driver

In the selection of the excitation wavelength or the camera speed becomes the bottleneck in terms of speed. After image acquisition, the image data has to be read out of the camera chip and saved before the next image can be taken. In the TILLvisION high speed imaging system, one image can be acquired while the previous image is being processed and saved at the same time. This enables a complete overlap of image acquisition and camera read out and makes use of the complete "dead time" which is otherwise lost during camera readout. Therefore if you compare imaging cameras make sure you also compare the exposure time at a given frame rate. For more details on the high speed modus in TILLvisION please click here or contact us.

First measurements with living cells

If you are using new cells (or a new dye) and you don't know the expected response you can first test your cells and your dye with some simple experiments:

• Measure the intensity of your dye at different dye and ion concentrations.
• Test the response of your cells e.g. with ionophores to see the maximum response possible in your specific application.
• Usually it is useful to grow the cells (if possible) on cover slips in order to make them stick to a surface. This coverslip can be easily used for imaging later on.