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Observing processes on a cellular level with minimal disturbance of the cells

Published on
March 19, 2013

High quality 3-dimensional video recordings of the processes inside and interactions between living cells can be made with the new spinning disk confocal microscope that CAT-AgroFood has purchased. The cooled electron-multiplied CCD camera on this machine is so sensitive that even small amounts of fluorophores can be registered to visualise dynamic processes in cells. The camera, in essence an array of many light sensors, allows the simultaneous observation of many points in the sample. This allows image acquisition at much faster rates compared to a traditional confocal microscope that scans a sample using only one light sensor.


Little light

Because very little light is required to make images, the studied cells don’t experience much stress, are able to stay alive for hours, or even days, and behave in a natural manner. “Light destroys fluorophores , a phenomenon known as photobleaching, so it is important to keep light levels low to monitor cells for prolonged times”, explains Prof. Marcel Janson, in charge of Wageningen University’s Laboratory for Cell Biology. “The sensitivity of the spinning disk microscope allows us to do just that.”

5-D images

Spinning disk microscopy is a confocal technique, meaning that it can be used to reconstruct the 3-dimensional geometry of a sample, without out-of-focus blur. When visualising a variety of fluorescent probes in a sample, all separated by their different colours, several processes can be monitored simultaneously and dynamic interactions can be studied, when needed also in 3D. Norbert de Ruijter, a spinning disk confocal microscopy specialist at the Laboratory of Cell Biology and the Wageningen Light Microscopy Centre, therefore speaks of “high resolution multidimensional imaging”. “This machine allows up to 5-dimensional imaging, since depth can be reconstructed from a series of optical image planes in z-direction (3-D), rapid time-series capture the 4th dimension and (multi)colour information is referred to as the 5th dimension. Consequently, it is possible to observe movements and interactions of multiple different fluorescent molecules at the (sub)micron scale.”

Many applications

“Because we can see the dynamics of the processes on the level of a cell, we learn to better understand how cells work”, says Janson. “For example, we can see how a plant cell builds a cell wall, the structure of the cell that determines how strong or flexible a plant is.” That fundamental knowledge of how plant cells get structured, could make it possible to design plants that have better properties for all kinds of applications: wood, paper, cotton, biofuel, et cetera. Viewing the processes inside cells also makes it possible to, for instance, get a better understanding of the interplay between plants and plant pathogens, and the role of pesticides. “In fact, for any study that needs high magnifications to discriminate low fluorescent signals in flat samples the spinning disk microscope outcompetes other systems”, De Ruijter adds.

Temperature controlled chamber

Thanks to a separate piece of equipment purchased by CAT-AgroFood – a chamber that can be placed on the microscope stage to keep the temperature at 37 degrees Celsius and to optimise the carbon dioxide levels – it not only possible to observe plant cells, but also living human or animal cells over a longer period of time. De Ruijter: “We used a similar chamber to study the activation of bitter taste receptors from the human nose and tongue. We exposed cloned bitter receptors to specific bitter substances and studied which receptors were triggered. We got a clear view of the cell activation in space and time. With our software we were able to quantify these signal changes over time and review the cellular activation in 3D.”

Years of experience

De Ruijter and his colleagues have been working with a confocal spinning disk microscope for four years. This newest version is, of course, a little bit more advanced, but that doesn’t make the other one obsolete. “The demand for that machine is so high, that we simply needed an extra one”, he explains. The older machine is mainly being used for truly experimental research. This has been resulting in exciting new insights which could be translated into more applicable, innovative research. The newer machine will be reserved for that second step: finding applications for newly found knowledge.

 

More information about the spinning disk confocal microscope

Come to the workshop about light microscopy on 13 June