New Life Science Applications of O-PTIR
O-PTIR (Optical Photothermal IR) is a breakthrough technique that’s opening up new possibilities in life science and biological research. It can be used to image live cells in aqueous conditions, with sub-micron spatially-resolved chemical analysis, in a label-free and objective approach. This article provides several examples of how O-PTIR is being used in life science.
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O-PTIR in Life Science
O-PTIR is a breakthrough technique that provides sub-micron spatially resolved chemical analysis on biological samples, with a label free and objective approach. In addition to this, it’s a non-contact reflection mode technique, which unlocks numerous possibilities in life science and biological applications.
This article provides just a few examples of how it’s being used in life science. Contact us to find out more, and to discuss whether this technique would be suitable for your research.
Single Bacterial Cell
This first example shows O-PTIR microscopy of a single bacterial cell of deuterium labelled E. coli.
The first two images of the protein (A and B) took 3 minutes each to acquire. Image C shows a single E. Coli cell (2.6×1.3 microns) imaged in around one minute.
Below this are four sub-micron (~500nm spot) O-PTIR spectra acquired from the single bacterial cell image (colour-coded). Intracellular variation can be seen, with the Amide I band position and shape indicating intracellular chemical differences, in the protein secondary structure. The spectra are normalised to 1655cm-1; 10 averages each (~15 seconds).
Single Bacterial Cell: IR + Raman
In this example, a bacterial cell was imaged with simultaneous sub-micron IR+Raman microscopy. Infrared and Raman data were collected from the same spot, at the same time and with the same resolution. More about IR+Raman…
The visible image (A) shows the region selected for IR imaging of a single bacterial cell (B).
Sub-micron IR and Raman spectra were collected and normalised to the most intense band. The O-PTIR spectra are raw (no processing), collected with a dual range (C-H/FP) QCL, covering 3000-2700, 1800-950cm-1 in a single unit. The Raman spectra are baseline corrected.
The 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 second accumulations.
Chemically Specific Imaging
Next is an example of targeted imaging mode, which provides chemically specific imaging. Intra-cellular images were acquired off a glass slide, at 100nm step sizes. This is from research by Prof Jose Sule-Suso at Keele University (publication in preparation, December 2020).
First is the lipid chain length image (2856cm-1 (CH2)/ 2874cm-1 (CH3). Next in B, the lipid relative to protein image (2856cm-1) (CH2)/ 1658cm-1). Both IR images were collected at 100nm pixel size, taking around 5 mins each. C is the optical image.
O-PTIR spectra were acquired from the highlighted points. They are single scans, ~1 second measurement time, with no processing.
This data was collected using the new dual range (C-H/FP) QCL, with spectral range coverage of 3000-2700, 1800-950cm-1.
Collagen Orientation in Fibrils / Tendon
In the example below, IR polarised O-PTIR was used to study collagen orientation in individual fibrils and tendon. This research was published by 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.
The first O-PTIR spectra was acquired from control tendon fibrils on a CaF2 window. In B, the single frequency image was recorded at 1655 cm-1 in perpendicular orientation. The markers show where the spectra were acquired. Scale bar = 1µm.
The O-PTIR spectra in C and D are from intact tendon, from ~500 nm measurement spots. Individual spectra were obtained from the two orientations of a section mounted on a CaF2 window, relative to the linearly polarised QCL. The visual image in C shows the 6 locations (colour-coded), all of which are within the region imaged with FTIR FPA.
Breast Tissue Calcification
This example of breast tissue calcification demonstrates O-PTIR’s <1 micron spatial resolution. The sample is courtesy of Prof Nick Stone of Exeter University (publication in preparation Dec 2020).
The mosaic optical image in A shows the location of the IR image (B), which shows the locations where spectra were collected from the calcifications. The IR image is 200×200 microns at 500nm step size, taken in approx. 10mins.
Calcifications are clearly resolved at 1050cm-1. They are only a few microns in size, with many even <1 micron. Such small and localised calcifications had not been seen before, because the spatial resolution of traditional FTIR is much larger than these features (~12microns).
Amyloid Aggregate in Neurons
Here, sub-micron amyloid aggregate was imaged in neurons. This was published Oxana Klementieva et al., “Super-resolution infrared imaging of polymorphic amyloid aggregates directly in neurons” in Adv Sci 2020, 1903004.
The O-PTIR single frequency ratio image (1630/1656cm-1) shows the distribution of beta protein structures with separation of 282nm.
The spectra were taken on (red) and off (black) the beta protein structure. You can see the differences in the amide I band, typical of beta sheet structured proteins – even though the two locations are only 282nm apart. This demonstates the resolution and precision of O-PTIR.
Single Cell Analysis
In this analysis of a single mammalian cell, sub-micron O-PTIR was performed on cells deposited on glass slide with no dispersive scatter artefacts. This is from research by Prof Jose Sule-Suso of Keele University (publication in preparation, Dec, 2020).
The optical image shows where spectra were taken from each cell, in reflection mode from regular glass slides. The spectrum has three averaged cell lines (one normal and two cancerous). In the shaded area, the standard deviation is 1.
Spectra were collected in reflection mode off regular glass slides. Other than averaging each cell line and area normalisation to the amide I and II bands, no other pre-processing was performed (eg baseline correction). Variation in the glass spectral region (1300-900cm-1) is due to differences in cell thickness.
This data was collected using the dual range (C-H/FP) QCL, with spectral range coverage of 3000-2700, 1800-950cm-1.
mIRage IR Microscope
These examples were all acquired using the mIRage from Photothermal Spectroscopy Corp.
- Breaks the diffraction limit of traditional infrared.
- Non-contact – fast and easy-to-use.
- Transmission quality IR spectra in reflection mode
- None of the dispersive artifacts or contact limitations of ATR.
Blue Scientific is the official distributor of the mIRage IR microscope in the UK and Nordic region. We’re available to answer all your questions – just get in touch: