Pollen grains are the natural source of some of the most frequent inhalant allergens. Many different varieties of pollen source material are used in allergy research as well as in the manufacture of diagnostic or immunotherapy products. The composition of the active allergen source material selected for use in the production of allergy diagnostic products influences their clinical efficacy. Hence, pollen source material characterization is an essential step in the production of allergy diagnostic products.
Manual microscope techniques have been traditionally used for product purity control and determination of foreign particles like foreign pollens and spores. The Morphologi® G3 automated particle characterization system performs rapid analysis and delivers microscope quality images and statistically significant data. This powerful analytical tool analyzes thousands of particles with minimum or no operator interference, thus saving labor and time significantly when compared to manual microscope techniques. A wide range of pollens can be analyzed using the system.
This experiment studied three types of pollen, one of which consisted of a mixture of two species (Taraxacum Officinale and Chrysanthemum Leucanthemum (TF&CL)). The remaining types were single species (Parietaria Judaica (PJ) and Avena Fatua (AF)).
The integrated Sample Dispersion Unit (SDU) of the Morphologi G3SE was used to dry-disperse the three samples, which were then measured using Standard Operating Procedures (SOPs), describing all of the hardware and software settings of the instrument. Filters were then used on the analyzes for removal of images with a low pixel area and so limited shape information, and images of touching particles from the final results.
Each sample was subjected to three repeat measurements. Figure 1 illustrates the field of view images of the samples captured using the manual microscope facility of the Morphologi G3. An image of each particle examined during the measurement is retained. Figure 2 shows illustrative images for each of the pollen samples.
Figure 1. Example field of view images of the three dispersed pollen samples taken with the 10X objective.
Figure 2. a) Example particle images for PJ pollen. b) Example particle images for AF pollen. c) Example particle images of TO and CL pollen.
The comparison of the analysis results clearly shows the variations in the size and shape of the pollen samples analyzed. Figure 3 presents the resulting size distributions for the first measurement of the samples in terms of circular equivalent (CE) diameter on a number basis (3a), and on a volume basis (3b).
Figure 3. a) CE diameter distribution for the three pollen samples on a number basis. b) CE diameter distribution for the three pollen samples on a volume basis.
The shape distributions for the pollen sample types in terms of high sensitivity circularity, convexity and elongation are presented in Figures 4, 5, and 6, respectively. Convexity distribution describes the surface roughness and elongation distribution describes the overall form or how 'needle-like' the particles are.
Figure 4. HS Circularity distribution for the three samples.
Figure 5. Convexity distribution for the three samples.
Figure 6. Elongation distribution for the three samples.
The CE diameter of the sample AF is the largest and that of the sample PJ is the smallest. It is bi-modal in the case of the mixed sample. The circularity of the sample AF is higher than other samples and that of the mixed sample is much lower when compared to other samples. In terms of the distribution, the mixed sample is bi-modal.
The convexity of samples AV and PJ is very similar, showing higher convexity with comparatively smoother edges than the mixed sample. The lower convexity of the mixed sample indicates the presence of rough edges.
The highest elongation is observed for the sample PJ, whereas the elongation of the mixed sample is the lowest, revealing the higher similarity of the length and width of the particles in the mixed sample when compared to other samples.
A scatter plot for the results obtained from the three repeat measurements for each of the samples is depicted in Figure 7, showing how measurements performed on each type of sample cluster together. When the results of an unknown pollen sample were added to the plot, its type can be determined from its position on the plot corresponding to the cluster.
Figure 7. Scatter plot of mean CED vs mean HS circularity which shows how measurements made on each type of sample cluster together.
The proportions of the different types of particles present in the mixed sample were determined by applying post measurement classifications. For this analysis, particle images were defined as either spherical or spiky. The classification results for the first measurement, in terms of the proportions of each type of particle detected, in conjunction with illustrative images of the spherical and spiky particles, are shown in Figure 8.
Figure 8. Results of the classification of the mixed samples into ‘spiky’ and ‘spherical’ classes along with example particle images.
The Morphologi G3 can perform automated characterization of pollen samples in terms of size and shape. Results help with differentiating the various types of pollen, and in combination with the particle images, enable analysis of samples consisting of the desired types of pollen. It is possible to characterize the mixed samples to identify the ratio of each type of pollen present in the samples.
This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.
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