Particle Analysis

Summary

This Application Note illustrates the use of a new quantitative phase imaging (QPI) technique, called Spatial Light Interference Microscopy (SLIM) to provide fast and accurate quantitative measurements of protein particle solutions. SLIM (Spatial Light Interference Microscopy) is a Widefield imaging technique that does not require biomarkers. This results in faster, nondestructive assays for protein production, characterization and aggregation monitoring. Segmentation and classification of silicon oil impurities is also possible.

Introductory and Procedure

Spatial Light Interference Microscopy (SLIM)

Phi Optics SLIM QPI module systems plug into existing inverted optical microscopes via an available side C-Mount port. SLIM works with a Phase contrast microscope set up. The CellVista software interfaces directly with the microscope to control all functions when the SLIM QPI mode is turned on, yielding label-free data about assays for protein production, particle characterization and aggregation.

SLIM provides high contrast and quantitative data with single particle, submicron resolution with a range between 200 nm to 100 µm. 3D scanning (XYZ) of glass-bottomed, consumable cell dishes or multi-well cell plates enable high throughput (up to 100 uL/min), without cross-contamination.

SLIM works well with cell and protein assays. SLIM can monitor shape, structure and processes with nanometer sensitivity (along Z-axis), providing quantitative assessment of protein content with femtogram sensitivity. SLIM has no minimum sample size, which enables researchers to work with small batch quantities. Samples can be imaged in consumable glass-bottomed cell dishes or multi-well plates, via direct pipetting from bioreactor, where there is no particle carry-over, no cross-contamination, no washing, and no leaking or clogging.

SLIM can also be integrated in table top-instrument (using disposable flow chamber) for in-line monitoring of bioreactor cultures. A more detailed description of SLIM can be found in the reference pages in the “Technology” page of this website.

Image Acquisition and Software

CellVista Pro software provides programmed acquisition of data for 2D (mosaicking), 3D (z-stack), and 4D imaging (3D + time series), using SLIM and any number of channels present on the microscope (e.g. fluorescence).

The resulting SLIM images can be segmented using any imaging analysis software package. The example below shows the procedure using the freely available ImageJ (NIH, DOI: 10.1038/nmeth.2089).

The measured path length information at each pixel is directly related to the dry mass density at that point, and, thus, by integrating over a Region of Interest (ROI), the total dry mass in the ROI is measured. When translated into dry mass, SLIM’s sensitivity corresponds to changes on the order of femtograms.

Particle counting and dry mass measurement is performed using Phi Optics ImageJ plugin:

1. Load a SLIM image or a time series in ImageJ. The color map is a measurement of the phase shift (optical path length difference) in every pixel. Insert is a detail of the larger SLIM map. Phi Optics plugins are conveniently loaded in ImageJ.

Clipboard01

Load a SLIM image or a time series in ImageJ

2. Segment the image using Threshold and Analyze Particles plugins and add the profile of each particle in the field of view to the ROI manager.

3. Select the Dry Mass plugin in Phi Optics 2D Analysis menu and press OK to measure the Total Dry Mass for each ROI loaded in the ROI manager. See Figure 2.

Clipboard02

Segmentation is done via ImageJ Threshold and Particle Analyzation plugins

4. For each frame, the morphology (area, Feret diameter, height, equivalent spherical diameter – ESD ) and total dry mass for each particle is displayed in a separate column and can be saved into an Excel file for further analysis. See figure 3:

Clipboard03-v2

Protein Analysis reporting based on particle mass, shape and 3D volume are available.

Metrics

SLIM system capabilities include live segmentation, particle counting, 3D volume morphology and tracking, protein content (picograms), and refractive index ID. The reporting is Excel based: Cell density, cell viability, and protein content. The data are repeatable, sample invariant and suitable for machine learning applications.

Quantification reports are available for an individual particle or an entire population (Particle size 0.01 – 100 microns). Instruments are capable of measuring high density with high throughput (Particle count 6 x 10^10 particles/mL.)

Applications

Protein Formulation Analysis

Preformulation