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What is Flow Imaging Particle Analysis?

FlowCam Flow Imaging Particle Analyzer

A flow imaging particle analyzer, also known as a flow imaging microscope, performs three functions all in one instrument. It examines a fluid under a microscope, takes images of the magnified particles within that fluid, and then characterizes the particles using a variety of measurements.

There are many applications that have benefited from this technology, including the study of microbial life in the world’s oceans to understand key processes driving this ecosystem, analysis of biopharmaceuticals and parenteral drugs to evaluate the stability of the formulation, and quality control of food ingredients where particle shape can affect taste and texture, just to name a few.

Flow Imaging - A Novel Concept

The first imaging flow cytometer for particle analysis, the FlowCam, was developed at Bigelow Laboratory for Ocean Sciences in Boothbay Harbor, Maine.  The research at Bigelow included identifying, counting, and tracking changes in plankton populations across the globe. The available tools – a microscope for plankton identification and a flow cytometer for counting – were time-consuming and laborious, so the scientists at Bigelow sought to develop a better method.

The FlowCam was designed to combine the benefits of a flow cytometer and a microscope in a single instrument. A sample of ocean water could be introduced into the system, particles were magnified and photographed, and then measurements of the imaged particles were taken. This information was presented in a spreadsheet-like fashion, so the user could easily sort, filter, count, and characterize the data.

How Does Flow Imaging Particle Analysis Work?

Flow imaging microscopes and particle analyzers use digital images of particles to measure the particle’s size and shape information. Essentially, the operator in classical microscopy is replaced by a computer to extract the information from the images. The sample containing the particles streams by the microscope optics, and thousands of particle images are captured per second. In order to freeze the moving particles in space, a strobed illumination source is combined synchronously with a very short shutter speed in the camera. As each frame of the field of view is captured, the software, in real time, extracts the particle images from the background and stores them.

Concurrently, a row in a spreadsheet for each particle image is populated with both spatial and gray-scale measurements.  Measurements range from basic shape measurements, such as equivalent spherical diameter (ESD), aspect ratio, and volume, to advanced morphology measurements, like circularity, elongation, and perimeter. Flow rate varies depending on volume of sample, size of flow cell, and magnification level, but rates can reach up to 50,000 particles/minute, with no operator intervention required. This means that you can quickly gather statistically significant populations of particle data.

Direct Particle Measurements

In an imaging-based system, particle measurements are made directly from an image of the particle. Since the optics of the system are fixed, distance measurements on the image can be directly converted to real distance measurements on the object because the overall system magnification is known. Imaging systems do not have to make any assumptions about a particle’s shape because they measure directly from the image. This means that multiple measurements can be made on each particle. Gray-scale measurements can also be made such as intensity, transparency, and color information (when a color camera is used).

Many other automated particle analysis systems, such as laser diffraction, light obscuration and light scattering, take indirect measurements. They do not measure physical dimensions, rather, they measure a signal that is proportional to a physical dimension, typically volume. The volume is then converted to an ESD by assuming that all particles are spherical in shape. These volumetric systems are able to process statistically significant numbers of particles in a short period of time and then present a particle size distribution (PSD) showing particle size versus either frequency or volume.

Ultimately, the benefit of an imaging system is that you get a concrete record of each particle in the form of its image, which you can view directly to better understand the data and to ensure that the data is being properly interpreted. Conversely, other systems can only produce distributions where the only way to validate the answers is to manually check using a microscope.


 

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Topics: FlowCam Technology