Product Survey: qLive cell imaging systems
In Pursuit of Happiness
by Harald Zähringer, Labtimes 05/2013
Live cell imaging is a permanent balancing act between image quality and the cells’ well-being.
When asked in a recent Science webinar (http://webinar.sciencemag.org/webinar/archive/live-cell-imaging) for some guidelines on successful live cell imaging experiments, Eduard Campbell from Loyola University in Chicago, gave out very simple advice: “A key to keeping your cells alive during a live cell imaging experiment is making sure that they know that you love them well in advance of that because it’s just a matter of fact that happy cells live longer.” Keeping the cells happy, however, is not only necessary before they are sacrificed for the imaging experiment. It is also the main objective during cell imaging that dictates almost all settings of the microscope as well as the design of the specimen chambers placed on the microscope stage.
Live cell imaging chambers are available in many different formats, ranging from simple coverslip chambers to glass-bottom petri dishes and sophisticated perfusion chambers offering complete control of temperature, humidity, pH and gas composition in the culture media. Coverslip chambers may be easily prepared by installing a coverslip on thin silicon gaskets placed on a microscope slide, to prevent smashing of the cells between the coverslip and the slide. Commercial microslide chambers are usually a bit more elaborate; so called lab-on-a-slide systems drawn with several channels may even allow perfusion of media or other solutions through the chamber. But similar to their homemade cousins, they are basically intended for short-term imaging experiments, usually conducted on an upright microscope.
Petri dishes for live cell imaging slightly differ from typical, disposable standard cell culture dishes. They often come with a circular opening in the centre of the bottom which has been sealed with a borosilicate glass coverslip of high optical quality, to enable high resolution monitoring of the cells with an inverted microscope. Similar to standard dishes, the surface may be coated with lysin or collagen to promote better adhesion of the cells. The dishes are installed in petri dish warmers or incubators mounted on the microscope stage to keep the temperature in a physiological range, which is crucial for live cell imaging over extended time periods.
Commercial perfusion chambers are available in different formats and materials such as glass, stainless steel or silicone. Their main purpose, however, is always the same: continuously supplying the cells with everything they need during prolonged imaging experiments. On top of that, open or closed perfusion chambers enable researchers to add drugs, tracers or whatever substances they want at defined time points without touching the culture chamber or disturbing the view field of the microscope. The fluids are driven into the chambers by peristaltic or syringe pumps; pH and gas composition of perfused solutions may additionally be regulated by humidifier and pH control units.
Petri dishes and perfusion chambers are usually installed on the stages of inverted microscopes. Live cell imaging microscopes are, at first sight, not very different from the standard inverted microscope, e.g. in cell culture, except that a plexiglas housing usually encloses the whole microscope or at least the specimen chamber. Light coming from a source installed above the working stage initially passes through the specimen chamber and the objective. It is guided via an optical system to the eyepieces and several camera ports arranged on different sides of the microscope. In contrast to standard microscopes, however, live cell imaging systems are optimised to get the highest image quality with the lowest levels of light.
High light intensities are not only phototoxic to the cells, they may also trigger unwanted reactions in the sample or lead to photobleaching of fluorophores. Hence, researchers apply every trick they know to enhance the sensitivity of the microscope’s optical system and try to keep the signal-to-noise ratio of the installed cameras as high as possible.
Boosting the numerical aperture (NA), i.e., the refractive index multiplied by the lens acceptance angle of the objectives, to the limits is one way to maximise the image quality. Since the resolution of a microscope is equal to a constant number, multiplied by the wavelength of light and divided by the NA, it is quite obvious that a higher NA leads to a higher resolution. That’s why oil immersion microscopy, conducted with high numerical aperture objectives and high refraction index immersion oils, is almost mandatory for live cell imaging.
Another critical factor that depends on the numeric aperture is the brightness or intensity of the light signal. It varies directly with the fourth power of the NA and inversely to the square of the magnification. Even a small increase in NA improves the intensity of the light signal, while a higher magnification decreases it.
Choosing an appropriate live cell imaging camera is the key to optimising the signal-to-noise ratio of the imaging system. Photomultipliers and charge-coupled devices (CCDs) used for image generation are sources of significant background noises that interfere with the light signal. Standard CCDs, for example, show a dark current arising from thermal energy within the silicon lattice of the camera chip. To reduce the dark noise, CCD-cameras mounted to live cell imaging systems are usually cooled to -30 to -50°C. Photon and read noise are two other typical noises intrinsic to CCD-cameras.
It is impossible to completely eliminate these three noises, neither by camera design, setup or operation. However, they may be kept as low as possible to get an ideal balance between light intensity, resolution, speed of image acquisition and cell viability – which is the main goal in live cell imaging and a constant challenge for the manufacturers of live cell imaging systems.
First published in Labtimes 05/2013. We give no guarantee and assume no liability for article and PDF-download.
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