New: Widefield Fluorescence detected
sub-500nm infrared multimodal microscopy
New: Widefield Fluorescence detected
sub-500nm infrared multimodal microscopy
Widefield O-PTIR™
Widefield fluorescence-detected sub-500nm infrared multimodal microscopy
A major advantage of widefield O-PTIR is its fast (snapshot) single IR wavenumber and hyperspectral imaging speed. It is up to 50x faster than standard O-PTIR, with typical image acquisition times of <1-10 seconds for single wavelength IR images and minutes for hyperspectral IR images, with measurement speeds of up to 5 FPS for dynamic single frequency chemical IR imaging.
FL-PTIR uses changes in fluorescent emission intensity from a sample to detect localized, chemically specific IR absorption at over
Widefield O-PTIR is powered by fluorescence-detected photothermal infrared technology (FL-PTIR), a patented, novel widefield chemical IR imaging approach based on a combination of Optical Photothermal Infrared (O-PTIR) and fluorescence microscopy.
Widefield fluorescence-detected sub-500nm infrared multimodal microscope
Widefield O-PTIR mode is available on the mIRage-LS product which also supports standard O-PTIR and co-located fluorescence capabilities.
To the right watch this video that takes you through the widefield fluorescence-detected sub-500nm infrared multimodal microscopy process applied to a diatom.
Widefield O-PTIR chemical composite image of a diatom (Diatom courtesy of Prof. K Gough, University of Manitoba. MCR-ALS composite image created in CytoSpec)
Chemical spatial resolution <300nm
MCR-ALS spectral components extract from diatom hyperspectral data stack using CytoSpec
Diatom Image above: Image ratio at 1200/1072cm-1 with a 80cm-1 peak integration width. Green box shows size of 260nm 2×2 pixel ROI used to calculate spectra below. Dashed line shows approximate location of cross-section measurements.
The IR spectra above is from 260nm regions of interest with the positions indicated. Note both the substantial spectral variation over 260nm length scales and spectral similarity in the alternating regions of the periodic structures (striae) in the diatom frustule. Spectra are normalized to 1072cm-1.
Demonstration of sub-500nm spatial resolution. The plot above shows the intensity of 1260cm-1 IR band measured at single pixels along white dashed line.
Fluorescently labeled, autofluorescence, IR tagged, or label-free experiments
The possibilities for life science measurements are endless. mIRage-LS with widefield O-PTIR supports a broad range of experimental set up and sample types. Whether your samples are fluorescently labeled, label-free, auto fluorescent or you are detecting molecules with IR tags, the mIRage-LS now supports virtually all biological image contrast enhancement tools, making the system one of the most flexible vibrational spectroscopy platforms available for a broad range of life science research.
Label free measurement of autoflourescent samples
Autofluorescence is now your friend!
Until now, those practicing vibrational spectroscopy with Raman and fluorescence microscopy were often at the mercy of unwanted sample autofluorescence, which can be a severe interferent. With FL-PTIR, autofluorescence becomes a major enabler and sensitivity enhancer providing a path to label-free chemical imaging for many biological sample types.
Label-free chemical imaging
For experiments where fluorescence labelling is undesirable, or autofluorescence does not occur, one can uniquely perform true label-free chemical imaging with sub-500nm O-PTIR, relying in the inherent chemical contrast through direct detection of macromolecules such as proteins (and their secondary structure), lipids, nucleic acids, carbohydrates and more.
IR Tags for enhanced molecular specificity
Increasingly as researchers are requiring the spatial specificity that labelling provides, the use of IR tags is growing in popularity. IR tags provide the sought after spatial specificity, but being much smaller in molecular weight and size, they provide opportunities to follow and study small molecule metabolism, without perturbations that would not be possible with conventional bulky fluorescent labels.
FL-PTIR measurements on an autofluorescent collagen fibril at different orientations. (A) Autofluorescent emission image of collagen fibril excited at 365 nm. (B-C) FL-PTIR absorption images at 1660 cm<span style=”font-size: 70%; vertical-align: top;”>-1</span>, 1550 cm<span style=”font-size: 70%; vertical-align: top;”>-1</span>, respectively. (D) FL-PTIR spectra extracted from an IR hyperspectral image stack from the indicated regions of interest for IR radiation oriented substantially perpendicular (E) to the fibril. (Sample courtesy of Prof. K Gough, University of Manitoba).
High throughput, automated Infrared spectroscopy with featurefindIR™
The mIRage-LS also provides for high-throughput, automated measurements of a large number of high-speed single point spectra of biological features or cells over the region of interest. Either brightfield (unlabeled), autofluorescence or fluorescence images can be used as inputs for automated feature/cell detection with subsequent measurement of 100’s to 1000’s of individual IR and/or Raman spectra. As a result, featurefindIR significantly improves the productivity of single spectra data collection, providing a basis for population level spectroscopy measurements.
Simultaneous sub-micron IR and Raman spectroscopy
The world’s first simultaneous IR+Raman microscopy system provides unique dual modality with all the advantages of O-PTIR and Raman microscope combined. Spectra, line scans and 2D maps can now be collected from the same spot at the same time, with the same sub-micron chemical spatial resolution enabling a more thorough characterization of your sample and opening new research opportunities.
IR+Raman analysis of red blood cells:
Left: Optical image with selected 70 x 70µm area for subsequent Raman imaging (middle). Right: IR+Raman spectra collected off of a selected red blood cell (~500nm resolution).
Source O-PTIR and Raman for microplastic detection and red blood cells (photothermal.com)
The most comprehensive vibrational spectroscopy platform for life science research
The mIRage-LS offers unique vibrational spectroscopy and microscopy capabilities with O-PTIR, integrated fluorescence microscopy and optional Raman spectroscopy. Both high speed widefield O-PTIR chemical imaging and high throughput single point IR spectroscopy capabilities are enabled in an easy-to-use optical microscope-based multimodal platform.
Interested in sample measurements or talk to technical sales ?
Email us at [email protected] for more information
FL-PTIR is a patented technique
FL-PTIR is protected by issued patents US11,519,861, US11,885,745, US11,561,179B2 and pending patent applications WO2022051636A1 WO2022221290A1 and other international counterparts. For a complete list of patents see www.photothermal.com/patents.
Publications References
The following publications provide more detailed information about the integration of O-PTIR and Fluorescence microscopy for different application types.
Single cell mapping of lipid metabolites using infrared probe in human derived model systems IR Tags / Co-located Fluorescence and O-PTIR. Nat. comms. Bai et al. doi.org/10.1038/s41467-023-44675-0.
Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level. Prater et al. doi:10.1021/acs.jmedchem.2c01359
Label-Free High-Resolution Photothermal Optical Infrared Spectroscopy for Spatiotemporal Chemical Analysis in Fresh, Hydrated Living Tissues and Embryo. Gvazava et al. jacs. doi10.1021/jacs.3c08854
Photothermal Spectroscopy Corp.
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Phone: (805) 845-6568
Email: info [at] photothermal.com