Simultaneous, submicron IR and Raman microscopy O-PTIR and Raman microscope combined
- Simultaneous IR and Raman spectroscopy
- Same spot, same time, same resolution
- Complementary and confirmatory IR/Raman spectroscopy
- Non-contact reflection-based IR measurement with FTIR transmission-like spectral quality
- No IR spectral artifacts like Mie/diffuse scattering or specular reflection
- Wide range of accessories for application flexibility
The world’s first simultaneous IR+Raman microscopy system is a unique dual modality platform with all the advantages of an O-PTIR and Raman microscope combined. O-PTIR, optical photothermal microscopy, using a visible probe laser, is combined with Raman for applications in polymers, defect analysis, microplastic detection and red blood cells.
Spectra, line scans and 2D maps can now be collected from the same spot at the same time, opening up new research opportunities and a more thorough characterization of your sample.
No fluorescence, poor sensitivity or phototoxicity
Simultaneous IR and Raman microscopy
Simultaneous IR and Raman spectral searching, complementary and confirmatory
Traditionally IR and Raman spectral searching has been done separately and the data has been analyzed separately. Now, with Wiley’s recent release of the KnowItAll™ multi-technique of simultaneous searching, the hit list information is displayed in a graphical 2D scatter plot. This multi-dimensional approach plots the HQI of the unknown IR spectrum against the HQI of the unknown Raman spectrum dramatically speeding up data analysis. This greatly simplified display of spectral matches in graphical rather than tabular form greatly improves the accuracy of the identification. IR search results can confirm Raman search results and vice-versa, all simultaneously.
IR+Raman analysis of
red blood cells
Left: Optical image with selected 70 x 70 µm area for subsequent Raman imaging (middle). Right: IR+Raman spectra collected off of a selected red blood cell (~500 nm resolution).
O-PTIR-IR+Raman instrument for microplastic detection
Click to view this video demonstrating the use of the mIRage microscope for O-PTIR plus Raman analysis of microplastics. Dr. Christoph Krafft offers insights into research at Leibniz IPHT, and his research.
Life science – cells
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
Targeted imaging mode (chemically specific imaging) Intra-cellular imaging, off glass slide, at 100nm step sizes
2856 (CH2)/ 1658 (Protein)
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)
Using fluorescence to localize O-PTIR measurements
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
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: bacteria
Single bacterial cell O-PTIR microscopy with deuterium labelled E. coli
Single bacterial cell simultaneous submicron IR+Raman microscopy
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.
Fluorescence imaging + O-PTIR of microplastics
Fluorescence tagging of polymeric beads can help to isolate the polymer particles from other particles for measurement with O-PTIR, thus dramatically speeding up analysis
Sub-micron IR+Raman microplastics
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!
O-PTIR – polymer (PLA-ACM)
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
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
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)
Simultaneous submicron IR and Raman microscopy A world first
Optical photothermal infrared and Raman combined surpasses accepted limitations of IR microscopy, ushering in a new era of analytical capabilities and problem solving. Coupled with simultaneous Raman microscopy, IR+Raman is now finally possible! This webinar discusses O-PTIR and its major benefits across a range of application areas, including defect and failure analysis, polymers, materials, life science, pharmaceuticals and forensics.