Imaging particle analysis: Difference between revisions

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{{Short description|Microscopy technique}}

'''Imaging particle analysis''' is a technique for making particle measurements using [[digital imaging]], one of the techniques defined by the broader term [[particle size analysis]]. The measurements that can be made include [[particle size]], particle shape (morphology or [[shape analysis (digital geometry)|shape analysis]] and [[grayscale]] or [[RGB color model#Numeric representations|color]], as well as distributions (graphs) of [[Population (statistics)|statistical population]] measurements.

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Imaging particle analysis uses the techniques common to [[image analysis]] or [[image processing]] for the analysis of particles. Particles are defined here per [[particle size analysis]] as particulate solids, and thereby not including atomic or sub-atomic particles. Furthermore, this article is limited to [[real image]]s (optically formed), as opposed to "synthetic" (computed) images ([[computed tomography]], [[confocal microscopy]], SIM and other [[super resolution microscopy]] techniques, etc.).

Given the above, the primary method for imaging particle analysis is using optical microscopy. While [[optical microscope]]s have been around and used for particle analysis since the 1600s,<ref name="Hogg1887">{{cite book|author=[[Jabez Hogg]]|title=The Microscope: Its History, Construction, and Application: Being a Familiar Introduction to the Use of the Instrument, and the Study of Microscopical Science|url=https://books.google.com/books?id=wzM5AAAAMAAJ&pg=PA8|year=1887|publisher=G. Routledge and Sons|pages=8|edition=12th}}</ref> the "analysis" in the past has been accomplished by humans using the human [[visual system]]. As such, much of this analysis is subjective, or qualitative in nature. Even when some sort of qualitative tools are available, such as a measuring [[reticle]] in the microscope, it has still required a human to determine and record those measurements.

Beginning in the late 1800s<ref name="Tissandier1877">{{cite book|author=Gaston Tissandier|title=A History and Handbook of Photography|url=https://books.google.com/books?id=lWo9AAAAYAAJ&pg=PA1|year=1877|publisher=Sampson, Low, Marston, Low, & Searle|pages=1}}</ref> with the availability of [[photographic plate]]s, it became possible to capture microscope images permanently on film or paper, making measurements easier to acquire by simply using a scaled ruler on the hard copy image. While this significantly speeded up the acquisition of particle measurements, it was still a tedious, labor-intensive process, which not only made it difficult to measure statistically significant particle populations, but also still introduced some degree of human error to the process.

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# A digital camera captures an image of the [[field of view]] in the optical system.

# A gray scale [[Thresholding (image processing)|thresholding]] process is used to perform [[Image segmentation#Thresholding|image segmentation]], segregating out the particles from the background, creating a [[binary image]] of each particle.<ref name=Gonzalez>{{cite book|last=Gonzalez|first=Rafael C.|title=Digital Image Processing|year=2002|publisher=Pearson Education|isbn=978-8178086293|pages=595–611|author2=Woods, Richard E.}}</ref><ref name="Sankur2004">{{cite journal|last1=Sankur|first1=Bulent|title=Survey over image thresholding techniques and quantitative performance evaluation|journal=Journal of Electronic Imaging|volume=13|issue=1|year=2004|pages=146|issn=1017-9909|doi=10.1117/1.1631315|bibcode = 2004JEI....13..146S }}</ref><ref>{{cite journal|title=A Threshold Selection Method from Gray-Level Histograms|journal=IEEE Transactions on Systems, Man, and Cybernetics|volume=9|issue=1|year=1979|pages=62–66|issn=0018-9472|doi=10.1109/TSMC.1979.4310076|last1=Otsu|first1=Nobuyuki}}</ref>

# [[Digital image processing]] techniques are used to perform [[image analysis]] operations, resulting in morphological and grey-scale measurements to be stored for each particle.<ref name="CarterYan2005">{{cite journal|last1=Carter|first1=R M|last2=Yan|first2=Y|title=Measurement of particle shape using digital imaging techniques|journal=Journal of Physics: Conference Series|volume=15|issue=1|year=2005|pages=177–182|issn=1742-6588|doi=10.1088/1742-6596/15/1/030|bibcode = 2005JPhCS..15..177C |doi-access=free}}</ref>

# The measurements saved for each particle are then used to generate image population statistics,<ref>{{cite web|last=Pouli|first=T.|title=Image Statistics and their Applications in Computer Graphics (2010)|url=http://www.cs.bris.ac.uk/~reinhard/papers/eg2010_tania.pdf|archive-url=https://wayback.archive-it.org/all/20110401055809/http://www.cs.bris.ac.uk/~reinhard/papers/eg2010_tania.pdf|url-status=dead|archive-date=1 April 2011|publisher=Eurographics, State of the Art|accessdate=2 January 2014|author2=Cunningham, D|author3=Reinhard, E.}}{{Dead link|date=January 2020 |bot=InternetArchiveBot |fix-attempted=yes }}</ref> or as inputs to algorithms for filtering and sorting the particles into groups of similar types. In some systems, sophisticated [[pattern recognition]] techniques<ref name="Rosenfeld1981">{{cite journal|last1=Rosenfeld|first1=A.|title=Image pattern recognition|journal=Proceedings of the IEEE|volume=69|issue=5|year=1981|pages=596–605|issn=0018-9219|doi=10.1109/PROC.1981.12027|s2cid=13410801}}</ref><ref>{{cite book|last=Young|first=T. Y.|title=Handbook of Pattern Recognition and Image Processing|year=1986|publisher=Academic Press|isbn=978-0127745602|url-access=registration|url=https://archive.org/details/handbookofpatter0000unse}}</ref> may also be employed in order to separate different particle types contained in a heterogeneous sample.

Imaging particle analyzers can be subdivided into two distinct types, static and dynamic, based upon the image acquisition methods. While the basic principles are the same, the methods of image acquisition are different in nature, and each has advantages and disadvantages.

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Static image acquisition is the most common form. Almost all microscopes can be easily adapted to accept a digital camera via a [[C mount]] adaptor. This type of set-up is often referred to as a [[digital microscope]], although many systems using that name are used only for displaying an image on a [[Video monitor#video display|monitor]].

The sample is prepared on a microscope slide which is placed on the [[Optical microscope#Stage|microscope stage]]. Once the sample has been focused on, then an image can be acquired and displayed on the monitor. If it is a [[digital camera]] or a [[frame grabber]] is present, the image can now be saved in digital format, and image processing algorithms can be used to isolate particles in the field of view and measure them.<ref>{{cite book|last=Russ|first=J.C.|title=Computer-Assisted Microscopy: The Measurement and Analysis of Images|year=1990|publisher=Springer US|isbn=978-1-4612-7868-9}}</ref><ref>{{cite book|author1=Hazelwood, Kristin L.|author2=Olenych, Scott G.|author3=Griffin, John D.|author4=Cathcart, Judith A.|author5=Davidson, Michael W.|editor1=Shorte, Spencer L.|editor2=Frischknecht, Friedrich|title=Imaging Cellular and Molecular Biological Functions|url=https://archive.org/details/imagingcellularm00shor|url-access=limited|year=2007|publisher=Springer|isbn=978-3-540-71330-2|pages=3–43[https://archive.org/details/imagingcellularm00shor/page/n21 3]–43|chapter=Entering the Portal: Understanding the Digital Image Recorded Through a Microscope}}</ref>

In static image acquisition only one field of view image is captured at a time. If the user wishes to image other portions of the same sample on the slide, they can use the X-Y positioning hardware (typically composed of two [[linear stage]]s on the microscope to move to a different area of the slide. Care must be taken to insure that two images do not overlap so as not to count and measure the same particles more than once.

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[[File:Basic flow through diag on white.png|thumb|Diagram showing flow-through architecture for dynamic imaging particle analysis.]]In Dynamic image acquisition, large amounts of sample are imaged by moving the sample past the microscope optics and using [[flash (photography)#High speed flash|high speed flash]] illumination to effectively "freeze" the motion of the sample. The flash is [[synchronization|synchronized]] with a high [[shutter speed]] in the camera to further prevent motion blur. In a dry particle system, the particles are dispensed from a shaker table and fall by gravity past the optical system. In fluid imaging particle analysis systems, the liquid is passed across the optical axis by use of a narrow flow cell as shown at right.

[[File:Flow cell Cross Section.png|thumb|Flow cell Cross Section|Diagram showing the flow cell cross-section perpendicular to the optical axis in a dynamic imaging particle analyzer. Credit: Fluid Imaging Technologies, Inc.]]The flow cell is characterized by its depth perpendicular to the optical axis, as shown in the second diagram on right. In order to keep the particles in focus, the flow depth is restricted so that the particles remain in a plane of best focus perpendicular to the optical axis. This is similar in concept to the effect of the microscope slide plus cover slip in a static imaging system. Since depth of field decreases exponentially with increasing magnification, the depth of the flow cell must be narrowed significantly with higher magnifications.

The major drawback to dynamic image acquisition is that the flow cell depth must be limited as described above. This means that, in general, particles larger in size than the flow cell depth can not be allowed in the sample being processed, because they will probably clog the system. So the sample will typically have to be filtered to remove particles larger than the flow cell depth prior to being evaluated. If it is desired to look at a very wide range of particle size, this may mean that the sample would have to be fractionated into smaller size range components, and run with different magnification/flow cell combinations.<ref name=Brown />

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[[Category:Laboratory equipment]]

[[Category:Counting instruments]]

[[Category:Microscopy]]