Raman microscopy couples a standard optical microscope with a Raman spectrometer, allowing researchers to obtain “chemical” maps of their samples. The addition of an aperture within the detector path provides the confocal ability of the Raman microscope. This eliminates out-of-focus signal and allows the microscope’s control of the “sampling volume” in the z-axes, allowing the ability to obtain high spatial resolution.

Raman microscopy provides chemical mapping of samples down to near diffraction-limited spatial resolution (for example, using a λ_ex = 514.5 nm laser and 100X (NA = 0.9) objective will can provide theoretical lateral resolution of approximately 300 nm). Raman microscopy does not require any chemical labelling, and the image acquisition speed will be dependent on the sample and preparation. Raman microscopy also allows chemical mapping of materials sub-surface (assuming the sample matrix is transparent to λ_ex).

Figure 1. Confocal Raman microscopy merges a Raman spectrometer, and a confocal optical microscope to provide very high spatial resolution “chemical” mapping for researchers. The laser excitation source is guided towards the sample by mirrors (M). An optical objective focuses the laser onto the sample, and the backscattered light is mainly elastically scattered and rejected with a Rayleigh filter. A small portion of light is inelastically scattered (1 part in 10 million), and the Stokes Raman scattered light (slightly lower energy from laser excitation source) passes through the edge filter. An optical lens (L1) focuses the Stokes Raman photons through an aperture, and rejects out of focus light from optically focused plane. A second lens (L2) expands the light to the grating assembly. The grating will separate the photons to the specific energies (measured in Raman shift), which are then focused onto the CCD detector by a third optical lens (L3).

The SSSC has 2 Renishaw Invia Reflex Raman microscopes. Raman imaging can be easily obtained and the instrument provides a few different options:

• Streamline^TM Imaging– A method unique to Renishaw in which acquires fast image acquisition using the linescan function and collecting spectra from multiple pixels simultaneously. This method can only be run in “static” mode (grating centered at user selected Raman shift).
• Regular Mapping – Traditional raster scanned image acquisition where each image pixel is measured. It is possible to collect full spectra in this mode, but it may be time consuming.
• Line Scan – Measure across a user specified line
• Depth Scan – Measure the depth of a user specified line.

Typical applications include:

• Catalyst distribution on support material
• Carbon nanomaterials
• Tissue mapping

Instrument Specifications

Instrument –Renishaw Invia Reflex Raman Microscope (with FTIR)

The Renishaw Invia Reflex Microscope is equipped with an IlluminatIRII FTIR microscope accessory (Smith’s Detection, Danbury, CT), and it is the only one available in Canada. The microscope is equipped with a MS20 encoded stage that allows precise steps (100 nm) for high resolution mapping capabilities. The Invia Reflex has 514.5 nm and 785 nm laser excitations, along with sophisticated software that allows automatic switching of lasers. This allows multiple excitation wavelength Raman and FTIR spectroscopies to be obtained on the same spot with relative ease for the general user.

Instrument Specifications

Instrument –Renishaw Invia Reflex Raman Microscope (with Fiber Optic Probe)

A recent addition to the SSSC, this Renishaw Invia Reflex Microscope is equipped with an external fiber-optic probe (λ_ex = 514.5 nm) that allows Raman measurements on larger samples (ex. In-situ process monitoring). The microscope is equipped with a MS10 encoded stage that allows precise steps (100 nm) for high resolution mapping capabilities. The Invia Reflex has 514.5 nm and 785 nm laser excitations, along with sophisticated software that allows automatic switching of lasers.

Instrument Specifications

Image Caption: Streamline^TM Raman mapping (λ_ex = 514.5 nm, 60s acquisition time) of a reduced graphene oxide (r-GO) flake deposited on Au-coated Si wafer. Raman measurements were acquired using a 100X (NA = 0.9) objective, and corresponding maps were 0.5µm x 0.5µm pixel size for a total of 2604 spectral acquisitions competed in approximately 2 hrs. Each Raman spectral acquisition was deconvoluted into the corresponding D, G, D’, and 2D curves (collected separately). Raman maps (right) are the deconvoluted peak intensities for the G, D, and 2D curves. A higher intensity would correspond to areas that were multilayered graphene (graphite). The peak intensity ratios can be converted into corresponding images to reveal changes in both the 2D/G and D/G peak intensity ratios. Representative Raman spectra for regions (1) and (2) are also shown. More details on research can be found: Zhou et al. Nano-scale chemical imaging of a single sheet of reduced graphene oxide, J. Mater. Chem., 2011, 21(38), 14622. (https://doi.org/10.1039/C1JM11071C)

Image Caption: Streamline^TM Raman mapping (λ_ex = 514.5 nm, 5s acquisition time) of a graphene oxide (GO flake deposited on Au-coated Si wafer. Raman measurements were acquired using a 50X (NA = 0.75) objective, and corresponding maps were 1.2µm x 1.2µm pixel size for a total of 2112 spectral acquisitions competed in approximately 7 min. Each Raman spectral acquisition was deconvoluted into 5 peaks (D’, G, D1, D3, D4). Raman maps are the deconvoluted peak intensities for the G and D curves. Here, higher peak intensities correspond to the lighter coloured flakes in found in the optical picture. The darker pieces appear to be large graphite pieces that did not break into smaller graphene layers. This mapping procedure shows a fast way to assess GO flakes and their chemical preparation.

Image Caption – Streamline^TM mapping λ_ex = 514.5 nm, 30s acquisition time) of photothrombodic induced stroke brain tissue. (A)  Raman survey maps for total lipid (2830-60 cm^-1 Signal-to-baseline), unsaturated lipids (3000-3030 cm^-1 Signal-to-baseline), protein aromatic side-chains (3035-90 cm^-1 Signal-to-baseline), and sample autofluorescence (2750-90 cm^-1 Signal-to-axis). (B)  Representative Raman spectra from regions of interest. (C) Peak areas from representative areas.  Sample measurements are an average of 500-1000 pixels.  Image scale bar is 100 µm. More details on research can be found: Caine et al., A novel multi-modal platform to image molecular and elemental alterations in ischemic stroke, Neurobiology of Disease, 2016, 91, 132. (https://doi.org/10.1016/j.nbd.2016.03.006)

Transmission electron microscopy (TEM) is an ultra-high resolution imaging technique where a focused electron beam is transmitted through a specimen. TEM imaging is performed under vacuum using a highly focused beam. Because this a transmission mode imaging technique, contrast will also be a function of the specimen thickness. Therefore, specimens are generally ultra-thin (100nm) sections generated from an ultra-microtome, and mounted on conductive TEM grids.

Instrumentation

Hitachi HT7700 (located and operated by our partners at the at the WCVM Imaging Centre)

The Hitachi HT7700 features a dual mode objective lens for superior high-contrast and high-resolution imaging making it an excellent choice for both biological and materials science. Digital imaging and refined automation with intuitive user interface allow for high throughput and advanced imagery. An addition EDX detector is available on this instrument and allows elemental fluorescent emission imaging capabilities.

Instrument Specifications