Applications

The advancement of analytical methods and systems are paving the way for innovation in academic, industrial and government sectors. As a result of the fast-paced progress, more research questions are asked that surpass the limits of these technologies. The mIRage IR Microscope is now answering these questions using Optical Photothermal IR (O-PTIR) spectroscopy, as well as IR+Raman – simultaneous submicron IR and Raman microscopy. With more chemical characterization requirements being met, mIRage is ushering in the next generation of applications and technology developments in numerous industries.

Contamination

Successful identification of contamination is a critical step in ensuring product or process quality is maintained. With stricter control standards and the decreasing size of high-tech products, confidently identifying smaller features is becoming increasingly important. With submicron spatial resolution using a non-contact reflection mode, O-PTIR easily resolves the most challenging of contamination issues.

Failure analysis of high technology components

Data illustration showing failure analysis of high technology components
Left; visible image showing location of 6 µm defect, Upper Right; Comparison of unknown O-PTIR spectrum to nearest library match, Lower Right: Comparison of unknown Raman spectrum to nearest library match
Data illustration with failure analysis of high technology components
Upper Left; Schematic representation of sample and measurement, Lower Left; Visible camera image of defect, Right; O-PTIR spectra from on and off the defect. Colors correspond to markers on visible image

Submicron line scan of polystyrene beads embedded in epoxy

Data images showing submicron line scan of polystyrene beads embedded in epoxy
Left: Demonstration of ~400nm spatial resolution as determined from a line scan (at 100nm steps) across 1 µm diameter polystyrene beads embedded in epoxy and sectioned to ~300nm thick. Right: A sharp boundary of only ~400nm is observed on both sides of the polystyrene beads as the IR spectral features transition between the two components.

Film defect identification

Data image showing film defect material identification
Left: Optical image of defect in a 240 µm thick two layer film. Markers on image represent the location of subsequent O-PTIR spectral collection. Right: Spectra collected in the defect-free (red) and defect (blue) region of the sample. The spectra display peaks indicative of isotactic polypropylene (998 cm-1). Insert: In the plot of the varying intensities for the isotactic polypropylene peak, both on the defect and off, The film region shows consistent signal intensity, while the defect region shows significant variability.

Polymers

Polymers are present in virtually all products we interact with daily. With increasing environmental awareness, polymer science is looking at more novel and complex solutions to improve functionality and reduce environmental impact. These requirements often exceed the limits of traditional IR microscopy, especially when it comes to spatial resolution. The mIRage IR Microscope, with its unique submicron spatial resolution using a non-contact reflection mode technique, is able to meet even the most demanding of analytical and sample characterization needs.

Application note: Submicron resolution IR spectroscopy and imaging of multilayer films for food packaging

Submicron IR+Raman Microplastics

mIRage locates PS (0.9 µm, 2.0 µm, 4.5 µm and 10 µm) and PMMA beads (3.0 µm) in salt crystal mixture in hi-res IR images at key absorption bands. Distortion free spectra, even amongst salt crystals at hotspots, confirm the identity of the microplastics and readily searched against IR database. Importantly, and unlike traditional FTIR/QCL systems, spectra are consistent, regardless of particle shape or size when measured in reflection mode – no dispersive scatter artefacts.

O-PTIR – polymer (PLA-ACM) phase dispersions

Data illustrating O-PTIR – polymer (PLA-ACM) phase dispersions
High quality spectra were collected in seconds, with high spatial resolution images collected in minutes. Image on the right shows image resolution of an inclusion of ACM as small as 249nm! Clear spectral differences attributable to the expected chemical domains of PLA and ACM were observed.
IR image: 20x20um, 100nm step size, ~3min/image
Sample courtesy of Dr Rudiger Berger, Max Planck Inst Polymer Research, Mainz, Germany

Polymer laminates analysis with O-PTIR

  • Key peaks at 1642 cm-1 (Nylon) and 1142cm-1 are used for single frequency imaging
  • Image collected at 100nm steps (~3mins per image)
  • Central EVOH layer of 1.6microns clearly visible!
Data image of polymer laminates analysis with O-PTIR

IR spectroscopy for direct fiber characterization

O-PTIR spectra of PP-based nanofibers with 800 nm diameter

Imaging and spectroscopy of bioplastic laminates

Composite (red/green) single frequency images

O-PTIR Scan of Bioplastic Laminate

Linear sampling scan spanning 8.0 µm measured every 100 nm apart (plotted only every 200 nm and across 2 µm for clarity) across the boundary of the bioplastic laminate, moving from the pure PHBHx layer to the pure PLA layer.

Gradual spectral changes over the space much greater than the optical resolution suggest the mixed distribution of PLA and PHBHx without any sharp boundary.

No clear isosbestic point indicates that the system is not a simple binary mixture.

PLA and PHBHx contributions are overlapped and mingled in the fingerprint region

Little to no sample preparation of a multilayer film

Left: A multilayer packaging film block face sample with manually selected markers for subsequent O-PTIR spectra collection. Right: The spectra easily show difference sin composition of each layer.

Submicron spatial resolution between film layers

Left: An optical image of a food multilayer film sample. Right: Corresponding O-PTIR spectra spaced 500 nm apart, with clear spectral distinction.

Life science

From plant biology to medical research, life science is an ever expanding research field that has impact in numerous industries. Providing submicron spatially resolved chemical analysis on biological samples, in a label free and objective approach, has proven itself to be a difficult result to obtain. The mIRage IR Microscope has accomplished this with its non-contact reflection mode O-PTIR technique, and is unlocking numerous applications capabilities.

Single bacterial cell O-PTIR microscopy with deuterium labelled E. coli

Single bacterial cell O-PTIR microscopy with deuterium labelled E. coli

A: O-PTIR image at 1655cm-1 (protein) at 200nm step size. B: O-PTIR image at 2195cm-1 (C-D stretch) at 200nm step size. Both images took 3 min to acquire each. C: Single E. Coli cell (2.6×1.3 microns) imaged at 1655cm-1 with 50nm steps. Image time, ~1 min. D: Four submicron (~500nm spot) O-PTIR spectra were acquired from the single bacterial cell image above (Upper Right), with corresponding colors. Spectra are normalized to 1655cm-1. Intracellular differences are apparent with the Amide I band position and shape indicating intracellular chemical (protein secondary structural) differences being detected. Each spectrum is 10 averages (~15 secs). You can see the C-D absorbances at around 2195cm-1 and 2100cm-1.

Single bacterial cell simultaneous submicron IR+Raman microscopy

Single bacterial cell simultaneous submicron IR+Raman microscopy

A: Visible image of bacterial cells. Orange box indicate region of IR imaging. B: O-PTIR infrared image at 1655cm-1, with 50nm step size. Collection time ~1 min. C: Simultaneous, submicron IR and Raman spectra collected from the indicated spot on the single bacterial cell. Spectra are normalized to the most intense band spectra are ~20sec accumulations. O-PTIR spectra are collected with a Dual Range (C-H/FP) QCL, covering 3000-2700, 1800-950cm-1 in a single unit. O-PTIR spectra are raw (no processing). Raman spectra are baseline corrected.
SNR of the OPTIR (~500nm spot) is ~4000:1 (RMS, taking amide band intensity as the peak and the baseline noise at the amide I position measured on a CaF2 blank) with ~20 sec accumulations.

Targeted imaging mode (chemically specific imaging) Intra-cellular imaging, off glass slide, at 100nm step sizes

Lipid relative to protein
2856 (CH2)/ 1658 (Protein)
Lipid chain length image
2856 (CH2)/2874 (CH3)
Data illustration showing targeted imaging mode (chemically specific imaging) Intra-cellular imaging, off glass slide, at 100nm step sizes

A: Lipid Chain length image (2856cm-1 (CH2)/ 2874cm-1 (CH3). B: : Lipid relative to protein image (2856cm-1) (CH2)/ 1658cm-1). Both IR images collected at 100nm pixel size. ~5 mins per image. D: O-PTIR Spectra from markers in images (spectra are single scans, ~1sec measurement time, no processing. C: Optical image.
Data collected using the new “Dual range (C-H/FP)” QCL, with spectral range coverage of 3000-2700, 1800-950cm-1.
Sample courtesy of Prof Jose Sule-Suso, Keele University, UK.
Publication in preparation (Dec, 2020)

IR Polarized O-PTIR to study collagen orientation in individual fibrils and tendon

Data images showing IR Polarized O-PTIR to study collagen orientation in individual fibrils and tendon

A: Spectra obtained with O-PTIR from control tendon fibrils on CaF2 window. B: Single frequency image at right recorded at 1655 cm-1 in perpendicular orientation. markers denote locations at which spectra were acquired. Scale bar = 1µm
C and D: Optical photothermal IR (O-PTIR) spectra from intact tendon, from ~500 nm measurement spots. (B) Individual spectra obtained from the two orientations of a section mounted on a CaF2 window, relative to the linearly polarized QCL. Inserted visual image shows the 6 locations, all of which lie within the region imaged with FTIR FPA; scale bar = 70 μm.
Colored markers (+) correspond to spectral colors. (C) Comparison of spectra obtained from CaF2 (top) and glass (bottom) substrates in parallel and perpendicular orientations to linearly polarized QCL.
Published: Gorker Bakir et al., “Orientation Matters: Polarization Dependent IR Spectroscopy of Collagen from Intact Tendon Down to the Single Fibril Level”, Molecules 2020, 25, 4295   https://www.mdpi.com/1420-3049/25/18/4295

Breast tissue calcification – Demonstration of <1 micron spatial resolution with O-PTIR

Data images of breast tissue calcification, demonstration of sub micron spatial resolution with O-PTIR

A: Optical image (mosaic). Red box indicates IR image measurement area. B: Single frequency image at 1050cm-1 to highlight calcification locations. C: O-PTIR Spectra from colored circle markers in IR image (B).
IR image area 200×200 microns at 500nm step size. Image time, ~10mins.
Calcification IR image at 1050cm-1, clearly resolves calcifications averaging only a few microns in size, many even <1 micron. At 1050cm-1, traditional FTIR has a spatial of ~12microns, which is much larger than the actual features, which is why such small an localized calcifications had not been seen before.
Sample courtesy of Prof Nick Stone, Exeter University, UK. Publication in preparation (Dec, 2020)

Submicron amyloid aggregate
imaging in neurons

O-PTIR image, 1630/1656                       O-PTIR spectra

Left; O-PTIR, single frequency ratio image of 1630/1656cm-1. Shows distribution of beta protein structures with separation of 282nm! Right; O-PTIR spectra from IR image (left) showing spectra on (#1) and off (#2) the beta protein structure. Spectral differences, clearly show the differences in the amide I band, typical of beta sheet structured proteins, despite these two locations only being separated by 282nm!
Published: Oxana Klementieva et al., “Super-resolution infrared imaging of polymorphic amyloid aggregates directly in neurons”, Adv Sci, Adv. Sci. 2020, 1903004 https://doi.org/10.1002/advs.201903004

Single mammalian cell analysis – submicron O-PTIR off glass slide with no dispersive scatter artefacts
Data image showing single mammalian cell analysis – submicron O-PTIR off glass slide with no dispersive scatter artefacts

Left; Optical image of a cells deposited on regular glass slide. Markers show replicate spectra locations per cell.
Right; Average spectrum per three different cell lines (normal and two cancerous). Shaded area are 1 standard deviation from replicate spectra. Spectra are collected in reflection mode off regular glass slides. Other than averaging (per cell line) and area normalization to the amide I and II bands, no other pre-processing (eg baseline corrections etc) were performed. Variability in the glass spectral region from 1300-900cm-1) are due to cell thickness differences.
Data collected using the new “Dual range (C-H/FP)” QCL, with spectral range coverage of 3000-2700, 1800-950cm-1).
Sample courtesy of Prof Jose Sule-Suso, Keele University, UK. Publication in preparation (Dec, 2020)

Submicron O-PTIR imaging of live cells in water

Data illustrations showing submicron O-PTIR imaging of live cells in water
Left: Optical image of hydrated epithelial cheek cells in water. Middle: Key macromolecules are easily spectrally discerned and spatially isolated, with the lipid inclusion as small as 0.5-1 µm being easily resolved. Spectra are not corrected for water and therefore inclusive of water absorbances. Images were collected at 500 nm step size. Right: The measurements
were collected using a 0.5 µm step size in transmission mode.

IR+Raman analysis of red blood cells

Left: Optical image with selected 70 x 70 µm area for subsequent Raman imaging. Middle: Subsequent Raman image at 1583 cm-1. Right: IR+Raman spectra collected off of a selected red blood cell (~500 nm resolution).

Microplastics contamination in oceans and waterways

An emerging environmental concern especially for ocean/aquatic life is microplastics pollution. With research continuing to increase to assess the potential harmful affects to aquatic life and the food chain, accurate characterization and identification of microplastics is becoming critical. Microplastics are small plastic microscopic particles and fibers that range from 1micron to 5mm. Microplastics have been found in virtually all environments, from waterways, to wastewater, to the air we breathe and the food we eat. Owing to their small sizes (1 micron to 5mm) their accurate identification and morphological characterization can be challenging. O-PTIR, with its ability to measure submicron particles and fibers of varied sizes in far field reflection mode to yield FTIR transmission-like spectral quality regardless of particle shape and size, makes it the ideal technique for microplastics/particulate characterization. When coupled with simultaneous Raman (IR+Raman), it becomes an even more powerful tool that brings together the best of IR and Raman into a single measurement, for more thorough and accurate characterization for microplastics contaminants.

O-PTIR image and spectra of PS and PMMA dispersed in saline

mIRage locates PS (0.9 µm, 2.0 µm, 4.5 µm and 10 µm) and PMMA beads (3.0 µm) in salt crystal mixture in hi-res IR images at key absorption bands. Distortion free spectra, even amongst salt crystals at hotspots, confirm the identity of the microplastics and readily searched against IR database. Importantly, and unlike traditional FTIR/QCL systems, spectra are consistent, regardless of particle shape or size when measured in reflection mode – no dispersive scatter artefacts.

Application Notes

Submicron simultaneous IR+Raman spectroscopy for failure analysis of high technology components. Open PDF

Submicron, simultaneous and non-contact IR+Raman spectroscopy for direct fiber characterization. Open PDF

Submicron resolution IR spectroscopy and imaging of
multilayer films for food packaging. Open PDF