Imaging and microscopy: Perfusion chambers
Introduction about perfusion chambers for live-cell imaging and microscopy
The first live-cell imaging chambers were designed in the early twentieth century, shortly after mammalian cell culture techniques were developped. A wide range of perfusion chambers is now available, from simple sealed coverslip on a microscope glass to sophisticated perfusion chamber allowing a full control of the cells environment. Recently, the advance of microfluidics has led to the developpement of integrated cell culture system.
Perfusions chambers are a critical aspect of live-cell imaging, and they must fulfilled two equally important requierements: maintain cells in a healthy state and allow the living cells to be observed with the highest possible resolution.
This short review will describe different types of perfusion chambers and their requierements. For more general information about perfusion for live-cell imaging, you can check our review. If you are interested in a perfusion system for live-cell imaging, check our Perfusion Pack.
Closed perfusion chambers
Two main categories of culture chambers are available. The first category is open chambers. They are more basic and very similar to Petri dishes. With open chambers, the medium surrounding the cells slowly approaches the equilibrium with the surrounding atmosphere during the experiment. They allow to easily access the cells, but exhibit a low level of control on the cells environment.
Closed chambers are sealed to avoid evaporation of the culture medium and ensure a large control over the environmental variables, such as temperature, pH or carbon dioxide concentration. Most closed chambers are designed with ports allowing the addition of fresh medium or drug during the experiment. Closed perfusion chambers are thus more suited to long term experiments.
Dimensions of the chamber. The depth of the perfusion chamber should be minimized to obtain the highest possible optical quality for transmitted light. Concerning the coverslip surface area, large viewing areas are more sensitive to leaks and physial damage, but allow the use of high numerical aperture objectives, that often have large diameter barrels.
Flow characteristics. For experiments involving the addition of other agents to the culture medium, such as drugs or growth factors, it is very important to obtain laminar flow, ensuring controlled media exchange. In order to maintain laminar flow inside the culture chamber, the chamber cross section should be very close to that of the inlet tube. Several fluids delivery techniques are available, but the uniformity of the flow should as be taken into account, since hydrodynamic pulses can harm the cells or produce coverslip flex. For more informations, see our review about perfusion systems.
Temperature control. For long term experiments, temperature control of the medium surrounding the cell should be ensured. Many commercial systems include heating elements coupled to the chamber or the mounting stage. However, some parts of the microscope, such as frame and objectives can act as heating sink and disrupt heat stability of the perfusion chamber. Moreover, small changes in ambient temperature can lead to thermal extension or contraction in some part of the microscope, modifying the plane of focus. Systems such as objective heaters or incubater box are available to tighly control the microscope temperature.
Microscope slides for cell culture
For short term experiments, a coverslip can be attached onto a microscope slide using spacers. For longuer experiments, more sophisticated microscope slides can be found. They include inlet and outlet allowing to refresh the culture medium or inject drugs during the experiment. These cell culture slides oftenly include multiple chambers in order to multiplex the experiments.
Microscope slides can also be used with warmers, such as Peltier modules or air blowers, for experiments on longer periods of time.
However, these simple culture chambers are restricted to short term experiments. For more complex experiments, closed perfusion chambers are usually preferable.
Microfluidics is currently a growing and promising field in biology. Microfluidics allows a high degree of control on cells environment, on flow dynamics and is compatible with existing high resolution microscopes. A lot of research is currently undertaken in this field, and simple microfluidic chips are already commercially available.
Due to the versatility of microfabrication processes, numerous microfluidics lab-on-chips have been developped by researchers for a wide range of applications, from simple shear stress exposure experiments to complex organs on chip.
For more information, consult our review about microfluidics and cell biology.
Cultivating cells inside perfusion chambers
Maintaining cells in a healthy state on the microscope stage can be very challenging. Culture techniques depends on the cell line used during the experiment.
One very important requierement is the attachement of the cells to the coverslip, in order to obtain a stable focus plan. Adherent cell lines will be naturally immobilized on the glass covership, whereas non adherent cell lines will have to be stabilized. Several techniques have been used, such as adhesion promoting agents (poly-L-lysin or collagen), or thin agar pad.
Cell culture requiere a carbon dioxide and bicarbonte buffer system in order to regulate pH. In conventionnal cell culture, a controlled atmosphere is created in dedicated incubators. For live-cell imaging, some perfusion chambers are specifically designed to create a regulated atmosphere. However, it is possible to use synthetic biological buffers, such as TRIS or HEPES to control the pH in absence of a controlled atmosphere.
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