MFI is a flow microscopy technology, where bright field images are captured in successive frames as a continuous sample stream passes through a flow cell centered in the field-of-view of a custom magnification system having a well-characterized and extended depth-of-field. The instrument configuration is shown in Fig. . A standard bench-top MFI configuration uses a simple fluidics system, where sample fluid is drawn either directly from a pipette tip or larger container through the flow cell using a peristaltic pump. The combination of system magnification and flow-cell depth determines the accuracy of concentration measurement. Concentration and parameter measurements are absolute but may be re-verified using particle standards. Typical sample volumes range from <0.25 to tens of milliliters. Frame images displayed during operation provide immediate visual feedback on the nature of the particle population in the sample. The system software extracts particle images by using a sensitive threshold to identify pixel groups which define each particle. Successive frames, each containing many particle images, are analyzed in real time. Maximum instrument sensitivity for detecting near-transparent particles is achieved by automatically optimizing threshold values, using low-noise electronics, implementing noise reduction algorithms, and compensating for all possible non-uniformities in spatial and pulse-to-pulse illumination. Ten-bit grayscale resolution is used to improve threshold accuracy.
Micro-flow imaging instrument configuration
Images are analyzed to compile a database containing count, size, concentration, as well as a range of shape and image contrast parameters. This database is interrogated by the computer’s application software to produce parameter distributions using histograms and scatter plots. The software supports image filtering by calculating a trial filter based on user selected representative particles and then interacting with the user to optimize this filter to extract similar particles from the total population. This feature allows particle sub-populations to be isolated and independently analyzed. Particle images are available for verification, further investigation, and analysis. Once a successful assay has been developed and validated, the resulting protocol, including run parameters, software filters, and report formats, can be saved for future use.
Direct imaging particle measurement technologies have a number of advantages over indirect obscuration or scattering-based measurements. They do not rely on a correlation between particle size and the magnitude of a scattered or obscured optical signal as calibrated using polystyrene reference beads. Provided the contrast in the particle image is sufficient for the pixels to be resolved by the system threshold, the particle will be detected and measured. No calibration by the user is required. The particle images captured by the system also provide qualitative and quantitative information about the target particle population. Qualification studies based on National Institute of Standards and Technology-traceable polystyrene beads have shown that the technology can meet high standards for sizing, concentration accuracy, and repeatability (3
). Representative performance parameters for MFI instrument configurations optimized for applications related to sub-visible particle analysis in protein formulation development are shown in Table .
Typical Micro-Flow Imaging Performance Parameters
Work to be described in the sections below includes comparison of MFI with USP <788> methods, measurement studies on a variety of typical protein formulations, morphological software filter development, assessment of consistency and repeatability of the technology, and optimization of MFI technology for this application.