IR+Raman, O-PTIR plus Raman diagram

Spatial resolution
breakthrough

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Applications

New Dual Range
(C-H/FP) QCL

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Better than a synchrotron

submicron IR microspectroscopy in your lab!

Synchrotron

IR spatial resolution:
5-15 microns (wavelength dependent)
Spectral SNR:
Good
Measurement modes:
Typically transmission or ATR

Sample preparation:
Complex, requires thin sections (1-10 microns) for transmission. Very flat for ATR
Substrates:
Typically IR transparent (eg CaF2 or BaF2) or reflective
Artefacts:
Cell, tissue interfaces and particulates can generate significant dispersive scatter artefacts – Mie scattering

Simultaneous IR+Raman:
No

O-PTIR

IR spatial resolution:
~500nm (wavelength independent)
Spectral SNR:
Excellent
Measurement modes:
Typically reflection (non-contact far field) delivering comparable to FTIR transmission. Transmission mode also available
Sample preparation:
Simple, measure samples from 100nm to >10mm thickness with no saturation issues.
Substrates:
Typically IR transparent, reflective or even glass (with no spectral range limits)
Artefacts:
No dispersive scattering type artefacts, even off cells, tissue interfaces and particulates


Simultaneous IR+Raman:
Yes, simultaneous, submicron IR+Raman

The advantages of O-PTIR

Commonly, FTIR microspectroscopy is performed at IR beamlines at synchrotrons, in an attempt to get the best spatial resolution with also good spectral sensitivity. Even then, spatial resolution is still diffraction limited to ~5-15 microns and acquisition times per point can take 10’s of secs or even minutes.
The recent advent of Optical Photothermal Infrared (O-PTIR) microscopy enables for the first time, truly submicron IR spectroscopy generating high quality information rich FTIR transmission mode-like IR spectra, in both reflection and transmission modes. It can also be coupled with Raman, to provide for a world first, “IR+Raman microscopy” – same spot, same time, same resolution.

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Advantages over
synchrotron IR microscopy;

    • Submicron spatial resolution, typically ~500nm or even less (wavenumber independent)
    • Excellent spectral sensitivity in seconds
    • Rapid targetted imaging via discrete frequency imaging (based on QCL technology)
    • Reflection mode (non-contact, far-field) and/or tranmission without any dispersive scatter artefacts (eg Mie Scattering)
    • Live cell imaging in water
    • Use of glass substrates, with little to no spectral range restriction
    • Less sensitive to water vapour fluctuations
    • Can be coupled to Raman (on same platform) for simultaneous submicron IR+Raman microscopy.
    • Room temperature visible detectors, mean no more liquid N2 handling (for MCTs) and measurement time limits
    • All of the above, in your own lab without having to apply for synchrotron time and travel.
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Applications

Life sciences
Cells
Life sciences
Bacteria
Life sciences
Tissues
Microplastics
Polymers

Life science – cells

Submicron amyloid aggregate
imaging in neurons

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

Data image of submicron amyloid aggregate imaging in neurons

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

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)

Top Left: Lipid Chain length image (2856cm-1 (CH2)/ 2874cm-1 (CH3). Top Right: : Lipid relative to protein image (2856cm-1) (CH2)/ 1658cm-1). Both IR images collected at 100nm pixel size. ~5 mins per image. Bottom Right: O-PTIR Spectra from markers in images (spectra are single scans, ~1sec measurement time, no processing. Bottom Left: 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)

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Submicron O-PTIR imaging
of live cells in water

RGB overlay image

Data illustrations showing submicron O-PTIR imaging of live cells in water

Left: Composite RBG IR chemical image of epithelial cheek cell in water, showing overlay of protein (1551, Blue), nucleic acid (1085, Green) and Lipids (1740, Red) showing key macromolecules are easily spatially located, label-free. Some lipid inclusions seen are <1 micron. Image was collected at 500nm pixel size. Middle: Single spectra (~500nm spot size) from different cellular regions. Note, spectra are not corrected for water and therefore inclusive of water absorbances. Right: Schematic of collection setup.

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)
IR+Raman analysis of 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).

Life science: tissue

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

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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)

Life science

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
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.

Polymers

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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
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. Insert on RHS 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
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

Microplastics

Submicron IR+Raman microplastics
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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.

Spatial resolution breakthrough of O-PTIR

Theoretical spatial resolution comparisons
(FTIR, QCL and O-PTIR microscopes)
Chart showing theoretical spatial resolution comparisons of FTIR, QCL and O-PTIR microscopes
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New “dual range (C-H/FP)” QCL

A new “dual range (C-H/FP)” QCL option is available that now covers, in a single unit, the C-H stretch and fingerprint ranges (3000-2700, 1800-950cm-1)

Spectra showing new “Dual Range (C-H/FP)” QCL

Library (Wiley KnowItAll) search results delivered >95% match

O-PTIR spectra collected in reflection mode. Displayed spectra are raw and unprocessed (<5sec collection time, ~500nm spot size)

O-PTIR spectra are measured off thick polymers (mm’s), vs library FTIR references data off thin films (~10microns)

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