Life Science

Life science applications of sub-micron, multimodal IR (O-PTIR) microscopy and spectroscopy with co-located fluorescence imaging

Why O-PTIR for life science research?

The mIRage-LS platform is revolutionizing life science research by uniquely enabling sub-micron, sub-cellular label-free chemical imaging via the breakthrough technique of multimodal O-PTIR with simultaneous Raman and co-located fluorescence microscopy to achieve unique scientific insights into the study of tissues, cells, proteins, lipids and many others molecular structures and macromolecules

Highest optical resolution IR spectroscopy achieving <500 nm resolution on biomolecular structures providing unique insights of molecular chemical structures
Co-located with fluorescence microscopy to quickly identify biomolecular structures of interest and subsequent IR resolution microscopy and spectroscopy
Investigate a wide range of life science samples, from single bacterial cells, tissues (hard and soft) to eukaryotic cells and their sub-cellular features, in both dried and hydrated states. Multimodal O-PTIR provides for unparalleled insights into the biochemical distribution of cells to support new scientific discoveries.

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High resolution chemical cell imaging

Cell imaging with co-located O-PTIR and fluorescence microscopy

Neuroglioma cells were stained with G3BP1 for protein stress granules, DAPI for nucleus and BODIPY for lipids.

The top left image is an 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 top right image is the brightfield image. Square markers show locations of O-PTIR spectral collection.

On the bottom is the O-PTIR spectra that 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 from the other locations.

Tissue imaging and protein aggregation

Initial brightfield (BF) and Autofluorescence (AutoFL) images are shown on the left hand side. All images, BF, AutoFL and IR chemical images are inherently perfectly spatially registered as there are no optics (objective) or sample movements between these images.

In red, cell nuclei are clearly highlighted in the image by the 1080/1140cm-1 ratio. In green, creatine is highlighted by the 1040/1655cm-1 ratio and lipid is highlighted by the 1738/1665cm-1 ratio.

Representative single point spectra (~400nm spot size) from each of these chemical images is shown in the bottom pane with location marked by white circles.

Asterisks on spectra indicate the image ratio wavenumber positions described above.

Single bacterial cell simultaneous submicron IR+Raman Microscopy

Upper Left: Visible image of bacterial cells. Orange box indicate region of IR imaging.

Bottom Left: O-PTIR infrared image at 1655 cm^-1, with 50 nm step size.

Collection time ~1 min.

Right: Simultaneous, submicron IR and Raman spectra collected from the indicated spot on the single bacterial cell in the IR image. Spectra are normalized to the most intense band spectra and are ~20 sec accumulations. Raman spectra are baseline corrected.

Here, the clear complementarities of IR and Raman spectroscopy are evident, with the strong C-H stretching bands in the Raman (~2900 cm^-1) being characteristically strong, whilst the IR spectra are very strong with their protein (~1600 cm^-1) and other fingerprint range (1800-800 cm^-1) signatures

The inherent sensitivity advantage of IR is also clear, with the O-PTIR spectrum demonstrating many times better sensitivity (SNR) over the Raman spectrum despite both measurements being the same time, ~20 sec.

Insights into protein aggregation

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 left is shown a brightfield image.

To the 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.

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 = 1 µm

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

Application note:

Sub-500nm IR microscopy and
spectroscopy with co-located fluorescence imaging for life science applications

Webinar:

Live cell imaging by sub-micron IR microscopy: Tracking de novo lipogenesis in single cells with stable isotope probes

Publication:

Fluorescence guided OPTIR for protein specific bioimaging and subcellular level

Webinars

Life Science

Alzheimer’s Disease Research with Sub-micron Simultaneous IR+Raman: Co-localization of Beta-Sheets and Carotenoids in Aggregates

Two-part webinar to learn how submicron IR (O-PTIR) and Raman has been applied to characterize the co-localization of amyloid β aggregates with carotenoids in human brain tissues to help better understand Alzheimer’s Disease progression.

Life Science

Life science applications using novel submicron simultaneous IR and Raman microscopy – A new paradigm in vibrational spectroscopy

Professor Ji-Xin Cheng of Boston University Photonics Center, author of over 230 peer-reviewed papers, co-inventor of CARS microscopy and pioneer in the field of O-PTIR applied to life science systems, in conjunction with Dr Mustafa Kansiz, Director of Product Management at PSC are proud to present this webinar.

Life Science

Amyloid aggregates in neurons – Life science applications using submicron simultaneous IR and Raman microscopy

Dr Oxana Klementieva and co-workers identifying amyloid-beta aggregates directly in neurons (neurites an dendritic spines) at the subcellular (submicron) level using Optical Photothermal Infrared (O-PTIR) imaging (mIRage submicron Infrared Microscope).

Life Science

Collagen orientation, fiber to submicron fibril life science applications of O-PTIR, from tissues to single cells and bacteria

In this webinar, we will show how, submicron simultaneous Optical-Photothermal IR (O-PTIR) spectroscopy and Raman microscopy (IR+Raman) is being used and published in life science applications, from tissues, to cells and even single bacterial cells.

Life Science

Single and intra-cell bacterial IR spectroscopy

Life science applications of O-PTIR, from tissues to live cells and single cell bacteria. In this webinar, we will show how submicron simultaneous Optical-Photothermal IR (O-PTIR) spectroscopy and Raman microscopy (IR+Raman) is being used and published in life science applications, from tissues, to cells and even single bacterial cells.

Life Science

Live single cell analysis with simultaneous submicron IR+Raman spectroscopy

In this webinar, Prof Peter Gardner (Manchester University, UK) will present results from their recent and first published study on live (and fixed) cell analysis with simultaneous submicron IR+Raman microscopy with wide spectral coverage using two pump lasers (QCL and OPO).

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