Fast, high-speed cell imaging takes centre stage

As the latest innovations demonstrate, cell imaging continues to play a key role in the development of the life sciences industry. And speed is key, as Sean Ottewell finds out.

Scientists at the Department of Biochemistry, The University of Oxford, rely on a number of powerful imaging systems, several of which are supplied by Preston-based Image Solutions (UK). Their efforts are directed to understand how cells become polarised during embryonic development.

Defects in this polarisation process are known to cause birth defects, and also are similar to the processes that go wrong in Alzheimer's disease and Fragile X Syndrome.

By studying neurons of the fruit fly Drosophila, the scientists are trying to understand how RNA moves and becomes localised during this process.[Page Break]

Cell extremities

Professor Ilan Davis, Welcome Trust Senior Research Fellow with the Department of Biochemistry, explains: "We know that RNA has a role in its own right and one that can be localised in the cytoplasm a long way from the cell nucleus.

"A very good example of this occurs in Drosophila neurons, where the RNA can be localised at the cell extremities, where it is regulated and controlled locally. This local control and regulation is known to be important in human diseases such as Alzheimer's and Fragile X Syndrome."

Professor Davis and his team use three DeltaVision Core systems and an OMX 'super resolution' instrument to study how RNA behaves in real time, in living cells.

The DeltaVision Core is designed as an imaging workhorse to image a large number of probes and samples with great precision.

The OMX is a more specialised and advanced instrument that uses 3D structured illumination technology, developed by a team at the University of California San Francisco (UCSF): this doubles the spatial and axial resolution of a widefield light microscope.

Additionally, this system can deliver high temporal resolution data for the study of fast dynamics in widefield mode.

"Imaging is complex and scientists have many types of conflicting requirements, however here we are looking at very fast moving objects travelling at 1-2 microns/second, which requires wide field microscopy," added Davis.[Page Break]

Upright platform

One DeltaVision Core has been modified to work with an upright Olympus platform microscope, rather than the usual inverted microscope. Samples can therefore be viewed from above.

"This makes a big difference to us because we can image RNA as it moves in axons of motor neurons. We have also made modifications to allow micro-injections from the stage plate in order to carry out neurophysiological experiments," he said.

The DeltaVision OMX, on the other hand, gives the Department the ability to image at speeds of up to 100 frames/second (fps) with excellent spatial resolution.

Real-time molecule movement

Researchers can now watch molecules move in living cells, literally millisecond by millisecond, thanks to a new microscope developed by scientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany.

By combining light-sheet microscopy and single molecule spectroscopy, the new microscope can record the fluorescence of every pixel within view, and take snapshots at intervals of less than one millisecond.

With it, scientists can watch and measure very fast processes, such as the way molecules diffuse, across a whole sample, even one containing several cells (Fig.1). This is a considerable step up from previous techniques, based on confocal microscopy, in which researchers could only observe at most a few isolated spots in a sample at a time.

"It's really visual biochemistry," says Malte Wachsmuth, who developed the microscope at EMBL. "We can follow fluorescently-tagged molecules in whole live cells, in 3D, and see how their biochemical properties, like interaction rates and binding affinities, vary throughout the cell."

Until now, chromatin - the combination of DNA, RNA and proteins that forms chromosomes - had been observed in two states: wound tightly together, with most of its DNA inaccessible to the cell's gene-reading machinery, in which case it is called heterochromatin; or loosely packed and easily readable, called euchromatin. But when they used the new microscope to measure the interaction between chromatin and a protein called HP1-a, the EMBL scientists made an intriguing discovery.

"In some areas that look like euchromatin, HP1-a behaves as it would in the presence of heterochromatin," says Michael Knop, now at the University of Heidelberg, Germany. "This suggests that chromatin may also exist in an intermediate state between hetero- and euchromatin, which was not observable before in living cells."

By providing a tool to watch molecules that move very fast, the scientists believe this new microscope will help to investigate processes ranging from the role of growth hormones in cancer to the regulation of cell division and signalling and the patterning of tissue development in the embryo.[Page Break]

Stereo applications

Meanwhile, with its TL3000 ST (brightfield), TL4000 BFDF (bright-/darkfield), TL4000 RC/TL4000 RCI (Rottermann Contrast technology) and the new flat TL5000 Ergo (automatic, zoom dependent contrasting) transmitted-light bases, Leica Microsystems is introducing a series of bases specially designed for a variety of stereo and macroscope applications from documentation of single cells to screening of whole animals.

The Leica TL3000 ST transmitted-light base is the all-rounder of the line. Its integrated, innovative halogen illumination system guarantees perfect conditions for viewing transmitted-light specimens.

Thanks to its modest power consumption, base and specimen warming has been reduced significantly.

The Leica TL4000 BFDF darkfield base is suitable for observing stained amplitude specimens. With its rapid switching between bright and darkfield as well as its sensitive deflection mirror adjustment, the base is optimal for semi-transparent specimens such as embryos.

With their innovative Rottermann Contrast technology, the Leica TL4000 RC and TL4000 RCI transmitted-light bases permit refractive index variations to be displayed as differences in brightness. This relief effect provides a wealth of variations that allow the maximum amount of information to be obtained from any specimen.

Together with the automated Leica M205 A or M205 FA stereomicroscopes, a computer and the Leica Application Suite (LAS) or the Leica Application Suite Advanced Fluorescence (LAS AF), many steps of the workflow can be automated for major gains in efficiency and time savings.

The new flat LED powered Leica TL5000 Ergo defines new standards in reproducibility and automatic contrasting. The homogenous field of view with its 65 mm diameter is optimised for single cells to whole animal examinations.[Page Break]

Diffuse light

The base has an integrated zoom dependent automatic aperture, which exclude diffuse light in brightfield to increase contrast. By displacing the light source out of the beam paths, perfect oblique illumination in Rottermann contrast appears.

Furthermore, each contrast mode is coded and automatised, resulting in complete reproducibility and full controllability via the Leica LAS or LAS AF software.

The transmitted-light bases truly play out their strengths when combined with their accessories. The Leica mechanical stage, for example, noticeably simplifies day-to-day laboratory work with its low profile and precise X and Y control.

Heating stages, adapters for micromanipulators and the entire Leica Live on Stage product line open a full range of applications that the company says were previously beyond the capabilities of stereomicroscopes.

Finally, the new Leica DFC365 FX digital microscope camera combines exceptional image quality with very high temporal resolution for rapid time-lapse recordings.

The Leica DFC365 FX is setting new standards in its class enabling researchers to work efficiently, even with weakly fluorescing specimens.

Equipped with a highly sensitive CCD sensor (pixel size 6.45 µm) and active Peltier cooling, it is ideal for a wide range of applications - from basic fluorescence documentation to demanding experiments such as total internal reflection fluorescence (TIRF), fluorescence resonance energy transfer (FRET) or structured illumination.

The Leica DFC365 FX achieves acquisition rates of 21 fps at full resolution.[Page Break]

Image acquisition

In 'overlapping mode', an image can be captured while the previous image is still being read out. Data are transferred rapidly to the PC via a FireWire-B interface. Besides high-speed image acquisition at 40 MHz, the pixel clocking rate of the sensor can also be set to 20 MHz or 1.6 MHz as required. According to the company this yields brilliant fluorescence images with a superb signal-to-noise ratio.

The optional NIR (near infra-red) mode extends the operating range of the camera for fluorescence markers emitting in the wavelength range above 700nm, which are difficult to capture with conventional CCD technology.

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