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Microplastic Detection Without Nile Red Staining or Fluorescence

ColSpec™ is a rapid, inexpensive, instrumental method to detect microplastics

ColSpec Microplastic detection system: front and side views

ColSpec™ MK2

ColSpec™ A Unique Optical Approach To Microplastic Detection 

A non-destructive test: Detects and reveals microplastics as small as 2-micron.

Detects transparent or colored plastics: Visualize transparent or colored microplastics in high contrast*.

Reveals microplastics: A 40,000 lumen light source in critical-angle darkfield light illumination “CADFLI”. Visit the image gallery.

Eliminates staining and fluorescence: A non-destructive, budget friendly, instrumental alternative to nile red staining or spectroscopy.

Fast acquisition time: One field of view typically takes less than 100-milliseconds.

Minimal sample preparation:  In many cases, no sample prep is required.

High sample throughput: Average 50-80 per hour manually for a series of similar samples.

Morphological characteristics of plastic fragments: High-resolution imaging reveals plastic “shreds” or “chips”.   See the gallery images for examples of each.

Detects cosmetic “glitter”:  Cosmetic glitter often made with PET contaminates oceans, waterways, and landfills.

ColSpec hardware:  Includes: high numerical-aperture, long working distance 12:1 zoom magnification with critical angle illumination, and xyz sample translation,

ColSpec software: Written in Python, provides classification and recognition in color-spectral space, object sizing, areas occupied, and abundance.  See here.

How ColSpec™ with Critical Angle Darkfield Illumination (CADFLI) works

Microplastics: are typically plastic particles less than 5-mm in size.  However, microplastic particles in the 1-micron to 1-mm range are of growing environmental interest. ColSpec detects particles in this size range, then classifies them, determines size ranges and the area they occupy – irrespective of particle shape.

ColSpec overview:  ColSpec is a portable optical instrument that non-destructively detects microplastic candidates from ~2-micron with minimal sample preparation.

  • Designed for both the laboratory and the field, ColSpec reduces the number of samples that require time-consuming, expensive, analytical polymer identification.
  • The secret is in the illumination: when illuminated with high-intensity grazing incidence illumination, microplastic particles appear as bright, high contrast objects on a dark or black background. We refer to this reflection illumination technique as CADFLI “Critical Angle Darkfield Illumination.”

Object illumination at grazing angles: ColSpec works with illumination angles greater than 75-degrees to preferentially reveal microplastics as bright, high contrast, structurally detailed objects.  At the same time, materials such as sediment and most organics fade into the background.

ColSpec quantifies the light scattered by each particle in terms of its Hue, Saturation, and Brightness or HSV (where “Brightness” = Value) to take advantage of the brightness component missing in RGB.

 Technical overview of HSV color-space: Quantified color space was formalized in 1931 by the “Commission Internationale de l’éclairage” (CIE), known in English as the International Commission on Illumination.  Wikipedia describes CIE color space like this:

The CIE 1931 color spaces are the first defined quantitative links between distributions of wavelengths in the electromagnetic visible spectrum, and physiologically perceived colors in human color vision…” (https://en.wikipedia.org/wiki/CIE_1931_color_space).

A simple internet search provides comprehensive technical information describing quantitative color measurement.

Creates HSV libraries with supervised learning: ColSpec training uses supervised learning based on the HSV values of known colored and “relatively transparent” polymers.  Each HSV reference library signature becomes a “class” that correlates with particular plastic material.

 Commonly used color spaces: RGB: or tristimulus, is probably the best-known color space routinely used by color cameras and printed materials.  However, RGB does not provide brightness measurements required by ColSpec.

 HSV: ColSpec and CADFLI utilize HSV color space in darkfield scattered light. 

Both RGB and HSV, as well as alternate color-space models, fall under the CIE umbrella.

Observed colors of objects illuminated by the sun, fluorescent lights, or LEDs depend on the angle of incidence of the illuminant to the FOV.

HSV color values measured at normal incidence (0-degrees or straight-on) differ from illumination angles greater than 0-degrees. At non-zero angles of incidence, reflectivity and surface texture impact HSV values and often reveals additional structural detail of an object. ColSpec utilizes this information to deliver highly sensitive microplastic classification and characterization.

 Plastics detected by ColSpec: ColSpec detects PET (1), HDPE (2), PVC (3), LDPE (4), PP (5) PS (6), and others (7), where the numbers in parentheses are recycling codes. Note: ColSpec detects but does not identify the polymers present in microplastic particles.

 Sample mounting:  Most commonly, up to 47-mm filter membranes are adhered to microscope slides.  ColSpec allows X, Y, and Z sample translation with reference scales.

Speed of analysis: Typically 100-msec per FOV with a sample throughput between 50 to 80 samples per hour. Each FOV can contain an unlimited number of particles.

 Basic software features: ColSpec software determines the particle classes present, their abundance, measures the area they occupy and plots their distribution. The software also detects objects that may be of interest that are not in a library.

 Applications that use color space: Color measurement technology quantifies, verifies, and confirms the colors present in forensics samples, coatings, paints, pharmaceuticals, medical diagnostics, remote sensing, and agriculture.  Paint color swatches and paint color matching illustrates an everyday use of color measurement technology.

 Related technology – darkfield microscopy: Darkfield imaging in transmission microscopy was first formally reviewed over one hundred years ago in the “Transactions of the American Microscopical Society Vol. 39, No. 2, Apr. 1920” For a detailed description of darkfield microscopy, see https://micro.magnet.fsu.edu/primer/techniques/darkfield.html hosted by Florida State University.

ColSpec applies the same principles of microscope-based darkfield transmission into darkfield reflection.

 Pricing and usage: ColSpec is a portable, modular system right-priced for tight budgets.

Bottom line: CADFLI measures the color and brightness of objects in HSV color coordinates.  Illuminating the FOV at angles of incidence greater than 75-degrees reveals target microplastics in scattered light on a dark or black background.

How The ColSpec™ Microplastic Detection System Works With Critical Angle Darkfield Illumination (“CADFLI”)

(A) PET in Critical Angle Darkfield illumination ("CADFLI")

Polyethylene terephthalate (PET, PETE): Only materials that have texture or edges scatter light and are recorded by the ColSpec camera.  Sub-millimeter PET, like almost all domestic plastics, stands out with high-contrast in CADFLI illumination. 

(B) PET in Brightfield illumination

The same PET sample, as above (A), taken in brightfield illumination.  All objects in the FOV reflect light, significantly reducing contrast.

MicroPlastic detection: Sub-millimeter blue polypropylene (PET) bottle cap fragments.  280-micron glitter added as a size reference

Microplastic detection: Sub-millimeter mixed plastic fragments.

How ColSpec Works Video

How to Detect MicroPlastics With ColSpec Software

* ColSpec does NOT replace Raman or IR:  ColSpec does not report the polymer present in objects.

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