Advances in the automated image analysis of particles and cells

1st April 2013

Particle shape and function are intimately linked. However, because of the lack of a universal shape analysis technique, the exact relationship between the two has been hard to define. As David Higgs explains, new technology is now likely to solve this problem.

Particle shape is an important variable that is known to influence the efficiency of a wide range of industrial processes. In many such processes, particle shape affects powder flowability. It can also have a bearing on the build up of electrostatic charge and may influence the compacting ability of a powder. However, the simple measurement of particle shape has proved to be a challenge and a barrier to further understanding and control. Now, an automated technique based on image analysis is stimulating renewed interest in particle shape measurement.

Traditionally, particle shape measurements have involved onerous and subjective methods of analysis, requiring laborious sample preparation and manual microscopy. In order for particle shape measurement to become a reliable, repeatable and routine operation, there had to be a significant improvement in the methodologies available. The Malvern Instruments FPIA-2100, or flow particle size analyser, uses imaging techniques to deliver the simultaneous and automated analysis of particle size, shape and count.

Comprehensive particle characterisation data are generated in a very short time, typically less than five minutes, and with minimal sample preparation. Extensive information about particle shape is acquired from a large number of particles, and displays of size and shape distributions are supported by images of the particles to provide further visual understanding of the measurement data.

In developing an automated technique for particle shape analysis, it has been necessary to address key problem areas encountered when using manual microscope-based methods. Firstly, there is sample preparation. Achieving a homogeneous distribution on a microscope slide makes sample preparation for traditional image analysis extremely demanding. The particles should be present as a monolayer, since imaging will see overlapping particles as single entities. Then there are imaging limitations. The laws of optics dictate that the greater the magnification used, the smaller the depth of focus. To look at small particles requires the use of high magnification lenses, which introduces imaging limitations. If the sample being analysed contains particles with a wide range of sizes, the operator will find it necessary to adjust the focus according to the size of the particles. Again, this is a complicating factor where image analysis is applied using standard software packages.

Then there is the problem of subjective interpretation at each step. This introduces bias, the level of which varies from analyst to analyst. An automated method must remove operator bias as completely as possible.

The technology employed in the FPIA-2100 has overcome these issues. The system is designed to provide automated particle shape and size measurement using imaging techniques. Fundamental to its successful operation is a patented sheath flow cell, which ensures a wide flat particle stream and appropriate presentation of particles to the camera (Fig. 1).

The sheath flow cell

Particles sampled from a dilute suspension are transferred into a stirred chamber. From here a controlled stream is ejected from a jet nozzle into the sheath flow cell between the strobe light and the CCD camera.

The flow cell is designed to achieve a flat sample flow. As the particle suspension is injected into the cell, it is sandwiched by the sheath flow. The combination of nozzle design, flow cell geometry and hydrodynamic effects transform the particle flow into a flat flow. This has a number of benefits:

€ The particles are presented as a monolayer, with no overlapping, enabling the image analysis software to recognise each particle individually. If particles have agglomerated, the sheath flow will not alter that state, so there is no sample modification.

€ Particles are all at the centre of the sheath flow cell. This means that they will all be in focus, regardless of their size, allowing the simultaneous measurement of particles from one micron to more than 160 microns.

€ The sheath flow cell ensures that all particles present their largest projected area to the camera. This ensures reproducible analysis whatever the shape of the particles.

The particle images (taken every 1/30 second) are processed on a real time basis, achieved through the use of a high-speed image processor. Because the images always have a constant volume, the number of particles per unit volume can be calculated quantitatively by taking a known number of images.

Each image is processed through a series of sophisticated digital imaging stages to isolate and quantify every particle. For each particle image the projected area, circle equivalent diameter and perimeter are directly calculated and the circularity is deduced. The circularity is defined as the ratio between the perimeter of a circle of equivalent area to the particle and the perimeter of the particle itself. Circularity can be viewed as an index of the degree of irregularities on the surface of a particle. So if the circularity was a1' then the shape would be perfectly spherical. As this value decreases, the surface shape becomes more and more complicated.

A selection of images is stored and classified according to size, adding valuable visual information that helps in developing greater understanding of the particles and their properties.

There are numerous applications for automated particle shape and size analysis. In the pharmaceutical industry for example, active drug substances are often produced with different particle sizes and morphologies depending on the batch. Such batch-to-batch variations can lead to differences in the way materials are handled and processed. Monitoring particle shape as well as size, assists the rapid characterisation of micronised drugs in order to achieve greater consistency.

The absence of a universal shape analysis technique has, in the past, created difficulties in associating the function of particles with their shape characteristics. The application of new technology is likely to solve this problem across a wide range of industrial applications.

Dr David Higgs is Product Marketing Manager with Malvern Instruments Ltd, email





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