Techniques Available

Transmission measurements are recommended where possible and are predominantly used for solid samples. If you’re interested in liquid or gaseous samples, please contact the IRM team. Transmission requires that samples be very thin (between 5-10 µm), flat and highly polished where applicable. Solid samples should be microtomed to an appropriate thickness (sample dependent), either freestanding or deposited onto an IR transmitting window such as CaF₂, BaF₂, ZnSe (preferably 0.5 mm in thickness) depending on the window properties and your required wavenumber coverage. Less frequently, Si, silicon nitride (Si₃N₄) or silicon carbide (SiC) may be used as substrates for transmission measurements.

A micro-compression cell with diamond or CaF₂ windows, to sandwich your sample in place under the beam, is also available for use. 

Examples of samples for transmission:

• Biological tissues – microtomed and mounted onto an IR transmitting window

• Cultured cells – grown on a substrate such as CaF₂ or Si₃N₄ windows, and freeze-dried or fixed and air dried

• Polymer multilayers – microtomed and placed in a compression cell between two windows of diamond or CaF₂

• Environmental samples - e.g. volcanic olivine crystals set in resin and microtomed; soil micro-aggregates microtomed and mounted onto an IR transmitting window

Reflection studies measure the specular reflectance or transflectance from the surface of a sample. For samples that can reflect IR directly (specular reflectance), it is vital that they be optically polished (to a roughness of 1 µm or less) to reduce scattering of the reflected beam and other interference features.

Transflectance studies are more commonly conducted at the beamline and describe the reflection observed when a sample is deposited as a relatively thick layer (1 - 20 µm) on a non-IR absorbing, reflecting surface e.g. a highly polished metal slide (Au or Al). Experiments on layers thinner than this typically required use of our Grazing Angle Objective (GAO) (see below in Grazing Incidence). Transflectance effectively produces a double-pass transmission spectrum as the beam passes through the sample twice upon reflection, however scattering effects from the surface can impact the data and transmission measurements are preferred where possible.

Examples of samples for reflection:

• Minerals - finely polished with alumina or diamond to a surface roughness of 1 µm or less

• Biological tissues or cultured cells mounted or grown on reflective surfaces, such as Mirr-IR microscope slides (Kevley Technologies, Ohio) or gold coated glass

ATR relies on a form of internal reflection to enable the analysis of more difficult samples with IR spectroscopy, e.g. samples that cannot be microtomed for transmission measurements or that do not adequately reflect IR for reflection. Compared to transmission and reflectance FTIR, ATR offers the advantage of enhanced spatial resolution (where a crystal with a high refractive index is used such as Ge).  Furthermore, the total internal reflectance phenomenon that ATR relies on (occurs when IR radiation travels through a high refractive index crystal a onto a low refractive index sample surface), results in an evanescent wave that penetrates only the surface of the sample (<1 micron), rather than the bulk.

There are 3 types of ATR techniques available at IRM:

Micro-ATR

• A Bruker micro-ATR accessory with a Germanium crystal tip (diameter=100 microns, refractive index=4), where the ATR crystal and the objective (20x) are one unit and the sample stage is independent.

• The main benefit for this ATR method is the ability to raster map over large areas (in comparison to our macro-ATR accessory)

• Disadvantages: potential for sample damage and contamination. Slower measurement as the stage moves in x, y, and z coordinates.

Open

Left: micro-ATR in position in the microscope. Right: Ge crystal - objective unit.

Open

Open

Open

Soft-contact piezo-controlled macro-ATR configured for electrochemical experiments

The IRM beamline has a Bruker Vis/IR Grazing Angle Objective (GAO, 15×) for grazing incidence analyses. This objective is specifically designed to analyse thin layers or films <1 µm thick that are deposited onto non-IR absorbing, reflecting surfaces e.g. highly polished metal slides (Au or Al) or indium tin oxide (ITO) coated glass, for example. GAOs increase the optical path length of a sample by essentially skimming the beam across the surface using high angles of incidence. This objective uses an incident angle of ~84° and shows excellent sensitivity down to monolayer thicknesses, particularly when used with polarised light, and good spatial resolution. The Bruker design relies on transflectance from the sample surface, effectively increasing the sample path length to again assist sensitivity, and uses a polariser in the IR beam; p-polarised light is polarised perpendicular to the surface of the sample and shows enhanced IR absorption when large angles of incidence are used.

Open

Examples of samples grazing angle experiments:

• Thin (sub micron) coating on metallic or other highly IR-reflective surfaces

• Corrosion products on metallic surfaces

Time resolved spectroscopy (TRS) using a FTIR spectrometer offers the advantage of being able to monitor rapidly changing chemical or physical properties of a system in high temporal resolution (up to 65 spectra/sec using a 160 kHz scan velocity @ 16 cm^-1). It allows for the simultaneous observation of the progression and/or decay of various chemical species during an event.

The rapid scan experiments can be initiated by an external trigger, such as a change in voltage, which can be programmed into the rapid-scan method editor in OPUS, making it ideal for the observation of kinetic chemical changed induced by an electrochemical potential event, to provide an example, More information on rapid scan FTIR spectroscopy can be found on the Bruker website.

A fluorescent probe can be used to help locate regions of interest for infrared mapping. This technique is most commonly used for biological samples such as tissue sections or cell cultures.

Called epifluorescence because the incoming light source travels the same path to the sample as the outgoing light (i.e. like reflectance).

Open

Image from: https://www.thermofisher.com/au/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/imaging-basics/fundamentals-of-fluorescence-microscopy/epifluorescence-microscope-basics.html

The Bruker Hyperion 3000 microscope at the IRM beamline uses the LED visible illumination as the light source, with an excitation filter centered at 455 nm (420 - 490 nm), a dichroic beamsplitter of 505 nm, and an emission filter of 520 - 1100 nm.

So far, Calcein (for calcium), BODIPY (for lipids and membranes), and Nile Red (for lipids) fluorescence probes have been successfully used to find regions of interest for IR mapping.