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Picture perfect

30th May 2014

Posted By Paul Boughton


Unwanted artefacts. Movement artefacts such as striping and colour ghosting hamper live imaging quality on cameras. Striping (1) High quality live image (2); Colour ghosting (3) High quality live image (4)
Sensitivity is important for fluorescence applications. The benefits of using a dedicated monochrome camera are clear, with large pixels and active cooling for high sensitivity
Stereo microscopy image of a GFP-expressing Drosophila sample, overlaying brightfield and fluorescence images. DP80 pixel-precise centring mode and HDR processing of fluorescence image were used
High resolution is crucial for documentation purposes. At low magnification, retrospective analysis is only possible with high resolution. Low resolution (left); High resolution (right)

Flavio Giacobone presents a guide to picking the best digital camera for microscopy applications.

As the 'eyes' of the digital microscope, the camera determines the quality of the digitally acquired image - a key factor in both analysis and documentation. Selecting the most suitable camera depends on the desired application - such as colour imaging, on-screen viewing, fluorescence imaging and digital archiving.

Awareness of the different parameters dictating camera performance therefore determines the ideal camera for each application, enabling users to reach the full potential of their microscope system.

Faithful colour reproduction is essential when considering how the variety of hues and intensities allow different structures in the sample to be distinguished. If the colour is imposed on the sample by specific staining, for example, it can also lead to diagnostic indications and it is vital to reproduce the exact same colour profile on the monitor as viewed through the eyepieces.

Colour cameras are highly specialised for colour imaging, measuring each pixel hue through an RGB (Red, Green, Blue) filter in front of the sensor, and yet accurate colour reproduction has remained a challenge for some time, prior to recent developments in colour profiling technologies. Such technologies avoid distracting artefacts such as excessive saturation and colour mixing, ensuring faithful colour reproduction to the extent allowed by the monitor. Some models now even feature native support of the Adobe RGB colour space, which allows the camera to interpret a greater range of colours.

Speed is an important parameter when it comes to on-screen live image quality, enabling the user to quickly find and focus the region of interest. However, situations can also arise where the camera's main purpose is not grabbing a snapshot but instead displaying a real-time image.

In these situations, not only the speed but also the quality of the live image becomes important as cameras can exhibit unnatural moving images, with problematic striping and colour-ghosting (Fig.1). 'Progressive readout' technology achieves a fast and artefact-free image, reducing user stress during extended sessions and dramatically improving audience understanding during presentations and discussion sessions.

The number one priority for low light and fluorescence applications is high sensitivity. Colour is not required for these techniques, meaning that superior sensitivity is achieved by first removing the RGB colour filters covering the chip. Furthermore, a dedicated monochrome chip will have a higher sensitivity by simply having larger pixels, enabling the capture of a greater number of photons and thus producing a clear and noise-free image (Fig.2). In highly sensitive chips, however, the image noise can be more evident, since the measured signal is so low. The most notorious contributor of noise is thermally derived, and so it is important to actively cool the camera chip, either with a Peltier-effect plate or with forced air or water flow.

Olympus DP80

In the past it seemed that a choice must always be made between having a colour or a monochrome camera, but this is no longer the case with dual-chip cameras. For example, the Olympus DP80 camera houses both a dedicated colour and a monochrome chip, with automatic switching depending on the type of snapshot needed. This versatility is valuable and it also presents opportunities for overlaying colour and fluorescent images with single pixel precision (Fig.3).

Digital technologies yield many advantages for documentation and archiving, making images highly accessible and easily shared across the globe. The transition towards digital sample libraries is facilitated by high resolution imaging, allowing the sample to be analysed retrospectively with the digital zoom (Fig.4). Defined by pixel size (smaller pixels capture finer details), resolution is often indicated by the total number of pixels, measured in mega-pixels (MP).

High resolution is, however, only effective when working at low magnifications. For example, when using a 100X objective a resolution of just 1 MP will be generally sufficient: any additional resolution will merely increase data volume without providing any extra information. An image instead captured at 4X magnification might require more than 10MP to capture all the details from the large field of view. Currently, 'pixel shifting' technology allows the greatest number of pixels to be rendered by a camera.

The finer pixel size necessary to obtain higher resolution will decrease the camera's overall sensitivity. In this case, 'pixel binning' provides the option to group smaller pixels into bigger 'virtual pixels', increasing sensitivity when the application needs it, albeit at the expense of the overall resolution.

Which camera?

The camera sits at the very core of ocular-free microscopy, and each application requires specific features:

* Colour imaging - colour profiling technologies;

* On-screen viewing - progressive readout;

* Fluorescence imaging - monochrome chip with low noise;

* Documentation & digital archiving - high resolution.

For more information at www.scientistlive.com/eurolab

Flavio Giacobone is with the Micro-Imaging Solutions Division, Olympus Europa SE & Co KG, Germany.





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