Co-located fluorescence + sub-micron IR of cells

Neuroglioma cells were stained with G3BP1 for protein stress granules, DAPI for nucleus and BODIPY for lipids.
The left image: RBG overlay widefield epi-fluorescence image with red showing protein stress granules, Blue showing nucleus and Green showing lipids. Square markers show locations of O-PTIR spectral collection.
The center image shows the brightfield image. Square markers show locations of O-PTIR spectral collection.
The right side O-PTIR spectra was collected in seconds from the marker locations shown in the left and middle panes. Clear spectral differences can be observed, consistent with the targeted sub-cellular features. Of particular note, is the subtle shift in the Amide I band of the protein stress granule indicating a likely different protein secondary structure to the other locations.

Cheek cells on glass coverslip (170nm)
Oil immersion objective, 100x, NA 1.30
Spectra collected in seconds
Images in minutes
Images collected in single frequency mode with 50nm step size
Probe beam detected in single frequency mode
Spatial resolution of ~285nm

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

Both IR images at the top were collected at 100nm pixel size. ~5 mins per image. At the bottom right is the spectra from markers (spectra are single scans, ~1sec measurement time, no processing. 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

An Alzheimer’s disease mouse model brain tissue section was stained with Amytracker 630 to highlight amyloid aggregates, AF488 to highlight proteins and DAPI
for the nucleus.
In the figure to the right is shown a brightfield image of the stained sample.
In the top right is the RBG composite fluorescence image, which highlights in red/orange the regions of amyloid aggregation. Note how some amyloid aggregates highlighted in the fluorescence image are not readily distinguishable in the brightfield image.
At the bottom is an averaged O-PTIR spectra, from the line profile indicated in the fluorescence image, with spectra averaged on (in blue) and off (in red) the aggregate. The average spectrum of the aggregate shows distinct spectral differences in the amide I band with a significant spectral feature at 1631cm^-1, typical of protein beta sheet structures. This clearly demonstrates the utility of combining fluorescence imaging to highlight regions of amyloid aggregation, some of which cannot be readily seen in brightfield microscopy, with submicrometer O-PTIR spectroscopy which can then provide the molecular compositional information, in this case, being particularly sensitive to protein secondary structure, a characteristic strength of IR spectroscopy.

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)

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

Single bacterial cell simultaneous submicron IR+Raman microscopy

Sub-micron IR shape and size independent spectra

As a demonstration of O-PTIR capability to measure various particle shapes and sizes with artifact-free data collection in the presence of highly scattering salt crystals, we created a mixed model sample consisting of Polystyrene (PS) beads (900nm, 2μm, 4.5μm and 10μm) and Poly(methyl methacrylate) (PMMA). When using an FT-IR/QCL system, not only would these particles sizes be too small to measure, but the range of different particle sizes and the nearby presence of salt crystals would generate dispersive scatter artifacts which can significantly alter the spectra making accurate identification more difficult. To further demonstrate the relative immunity to scattering artifacts, in the figure to the right is shown these mixed polymer bead samples suspended in salt water and deposited on to a CaF2 substrate, so that the polymer beads would be interspersed with salt crystals.
It is noteworthy that despite the spectra being collected from differently sized beads the spectra all look very consistent with their polymer type (i.e., no dispersive scatter artifacts are present). This ensures that correct identification can be determined, regardless of particle shape and size.

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)
phase dispersions

Imaging and spectroscopy of bioplastic laminates

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

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Life science: tissue

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