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Infrared microspectroscopy
Australian Synchrotron beamlines

Infrared microspectroscopy


The combination of high brilliance and collimation of the synchrotron beam through a Bruker VERTEX 80v Fourier transform infrared (FTIR) spectrometer and into the Hyperion 3000 IR microscope enables high signal-to-noise ratios at diffraction limited spatial resolutions between 3-8 μm, making the Infrared Microspectroscopy (IRM) beamline ideally suited to the analysis of microscopic samples e.g. small particles, thin films and layers within complex matrices, as well as single cells and complex biological systems. 

Infrared microspectroscopy is a non-destructive method used to gain chemical and structural information for a diverse range of samples from biological, biomedical, materials, forensic and food sciences as well as cultural heritage and geology to name a few.  The benefit of FTIR microspectroscopy compared to traditional FTIR spectroscopy, is that spatially resolved chemical information is acquired, enabling the production of "heat" maps that visually demonstrate the spatial distribution of specific chemical functionalities.  The benefits of the synchrotron radiation sourced infrared light compared to traditional globar source is the smaller focused beam spot enhances the spatial resolution achievable and signal to noise of the resulting spectra.

In addition to the online Hyperion 3000 microscope, which is equipped with a single point MCT detector, an offline Hyperion 3000 microscope attached to a Bruker VERTEX V70 spectrometer (using traditional globar source IR), is also available.  This microscope is equipped with both an MCT and a 64 x 64 focal plane array detector (FPA), enabling the rapid acquisition of high resolution chemical maps.  Users can request access to this microscope during their beamtime, if required.

Both the online and offline microscopes can be operated in the following modes of data collection (see techniques below for more information on each acquisition mode) and can be coupled to a number of different accessories to assist with the wide variety of experiments conducted at the beamline:

  • Transmission
  • macro Attenuated Total Reflectance (ATR)
  • Reflectance
  • Grazing Incidence

In each of the above modes a motorised stage allows the point-by-point collection (raster mapping) of individual spectra from a sample, to create the spatially resolved “spectral maps” (see Techniques Available for more information).

Figure 1. The IR Microspectroscopy cabin
Figure 1. The IR Microspectroscopy cabin


Inside the IR Microscopy hutch housing a Hyperion 3000 IR Microscope and a Bruker V80v FTIR spectrometer
Figure 2. The IR beamline uses a Bruker V80v FTIR spectrometer attached to a Hyperion 3000 IR Microscope (right)

Techniques available

The Hyperion microscope, combined with our range of commercial and in-house accessories, enables FTIR mapping in transmission, reflectance, grazing incidence and ATR modes.  The choice of operational mode will be largely dependant on your samples. Some basic information regarding each mode of operation can be found below. If you're unsure which is most suitable for your samples, please get in touch with one of the infrared microspectroscopy beamline scientists.


Transmission IRM is recommended where possible and can be used for solid, liquid or gaseous samples. Transmission requires that samples be very thin (between 5-10 µm), flat and highly polished where applicable.  Solid samples should be microtomed to the correct thickness ranged for analysis, 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. We also recommend Crystran Ltd,, for further details. Also, please see the FAQs section under the beamline guide tab for further information relating to IR transmissive windows.  Less frequently, Si or Si3N4 may be used as substrates for transmission IR.

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 silicon nitride 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

Combined IR transmission studies & X-ray Microscopy:

Samples can be analysed by both transmission IRM and X-ray fluorescence microscopy at the X-ray Fluorescence Microprobe (XFM) beamline when they are deposited on silicon nitride (Si3N4) windows. NOTE Window thickness is restricted to a maximum of 500 nm and the material shows a lower wavenumber cut-off in the mid-IR at 1100 cm-1.  Please contact a Beamline Scientist for more information.

Attenuated Total Reflection (ATR)

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 the surface of the sample, rather than the bulk

The IRM beamline has a Bruker single-bounce micro-ATR accessory with a Germanium crystal tip (diameter=100 microns, refractive index=4). Traditional microscopic ATR methods often cause sample damage, contamination and re-positioning issues, where the ATR crystal is attached to the objective. However, with the macro-ATR methods developed in-house and described below, these issues are avoided by single contact ATR mapping.

Two models of the macro-ATR are available for use, and are expanded upon below:

"hybrid" macro-ATR
  • The hybrid macro-ATR accessory presents a Germanium crystal tip (refractive index=4) mounted on a macro-ATR cantilever arm (Figure 3).
  •  3 sizes of ATR tip are available. The largest tip, which is 1 mm, works well with softer materials that do not require a high pressure to achieve a good contact. The small tips, which are 250 µm and 100 µm in diameter, can provide higher pressure and allow measurements inside smaller regions with limited access suitable for hard/rough surfaces.
  • The macro-ATR device allows users to manually adjust the pressure between the sample and Ge crystal and only requires a single contact throughout the measurement thereby preventing damage to the sample and increasing the scanning speed compared to micro-ATR technique.

hybrid macro-ATR and Ge crystals
Figure 3. (L) hybrid macro-ATR cantilevers and range of Ge crystals, (R) demonstration of crystal-sample contact positions

 Sample Mounting
  • Samples are mounted on metal discs for hybrid macro-ATR mapping (Figure 4).  The maximum sample-disc height is 11 mm and samples should mounted so that the region of interest is in the central 5 mm2 of the discs.  This is due to the limited travel range of the sample stage
  • A range of discs are available (Figure 5), ranging in height from 2-10 mm, with various engineered reservoirs, such as for resin embedding, and with heating a cooling capabilities to perform temperature controlled studies


sample mounting hybrid macro-ATR
Figure 4. Metal discs used for mounting samples for hybrid macro-ATR mapping

hybrid macro-ATR sample discs
Figure 5. (L) A range of discs are available from 2-10 mm in height and (R) with heating/cooling capabilities

Examples of samples for hybrid macro-ATR:

  • single fibre analysis (carbon fibres, wool)
  • dairy products (cheeses)
  • biological samples such as cells, tissue sections and skin biopsies
  • Insect wings
  • polymeric materials
  • Eucalyptus leaves


Model 2 is the "soft-contact" piezo-controlled macro-ATR, please extend the tab below for more information. 

"soft-contact" piezo controlled macro-ATR

•For this model, we use ZnSe or Ge hemispheres, which has 1 mm diameter flat sensing surface at the bottom. The hemispherical crystal is then held inside a magnetic mount, which provides precise positioning of the crystals when changing the sample.

•The outstanding feature of this device is the unique combination of piezo-controlled linear translation stages used for both xy  and z  directions.

•The step interval of the piezo drive can be set to be as small as 50 nm, to allow the sample (which was mounted on the metal post) to gently approach the flat sensing bottom of the ATR crystal.

•The soft contact-piezo controlled system is ideal for very soft, delicate or more fragile samples

soft-contact piezo controlled macro-ATR
Figure 6. Translational stage, crystals and magnetic mount used for soft-contact piezo controlled macro-ATR

Sample mounting for the soft-contact piezo controlled macro ATR is largely sample specific, so please discuss this with a member of the IRM beamline staff.

Examples of samples for soft-contact piezo controlled macro-ATR:

  • Insect wings and gecko skin (superhydrophobic wax coatings)
  • biopolymer gels
Recent Macro-ATR FTIR Publications from Australian Synchrotron IRM beamline

1.    N. L. Benbow, J. L. Webber, P. Pawliszak, D. A. Sebben, T. T. M. Ho, J. Vongsvivut, M. J. Tobin, M. Krasowska, and D. A. Beattie, “A Novel Soft Contact Piezo-Controlled Liquid Cell for Probing Polymer Films under Confinement using Synchrotron FTIR Microspectroscopy,” Scientific Reports, (2019), in-press.

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2.    Y. P. Timilsena, J. Vongsvivut, M. J. Tobin, R. Adhikari, C. Barrow, and B. Adhikari, “Investigation of Lipid and Protein Distribution in Spray-Dried Chia Seed Oil Microcapsules Using Synchrotron-FTIR Microspectroscopy,” Food Chemistry, 275, 457-466 (2019).

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3.    B. Dorakumbura, R. Boseley, T. Becker, D. Martin, A. Richter, M. J. Tobin, W. van Bronswijk, J. Vongsvivut, M. Hackett, and S. Lewis, “Revealing the Spatial Distribution of Chemical Species within Latent Fingermarks Using Vibrational Spectroscopy,” Analyst, 143, 3961-4208 (2018), doi: 10.1039/C7AN01615H.

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4.    P. Seredin, D. Goloshchapov, Y. Ippolitov, and J. Vongsvivut, “Pathology-Specific Molecular Profiles of Saliva in Patients with Multiple Dental Caries—Potential Application for Predictive, Preventive and Personalised Medical Services,” EPMA Journal, 9(2), 195-203 (2018), doi: 10.10. 1007%2Fs13167-018-0135-9.

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5.    J. Vongsvivut, V. K. Truong, M. A. Kobaisi, S. Maclaughlin, M. J. Tobin, R. J. Crawford, and E. P. Ivanova, “Synchrotron Macro ATR-FTIR Microspectroscopic Analysis of Silica Nanoparticle-Embedded Polyester Coated Steel Surfaces Subjected to Prolonged UV and Humidity Exposure,” PLoS ONE, 12 (12), e0188345 (2017), doi: 10.1371/journal.pone.0188345.

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6.    M. Ryu, H. Kobayashi, A. Balčytis, X. Wang, J. Vongsvivut, J. Li, N. Urayama, M. Norio, V. Mizeikis, M. J. Tobin, S. Juodkazis, and J. Morikawa, “Nanoscale Chemical Mapping of Laser-Solubilized Silk,” Material Research Express, 4 (11), 115028 (2017), doi: 10.1088/2053-1591/aa98a9.

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7.    M. Ryu, A. Balčytis, X. Wang, J. Vongsvivut, Y. Hikima, J. Li, M. J. Tobin, S. Juodkazis, and J. Morikawa, “Orientational Mapping Augmented Sub-Wavelength Hyper-Spectral Imaging of Silk,” Scientific Reports, 7, 7419 (2017), doi: 10.1038/s41598-017-07502-3.

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8.    S. Nunna, C. Creighton, B. L. Fox, M. Naebe, M. Maghe, K. Bambery, M. J. Tobin, J. Vongsvivut, and N. Hameed, “The Effect of Thermally Induced Chemical Transformations on the Structure and Properties of Carbon Fiber Precursors,” Journal of Materials Chemistry A, 5, 7372-7382 (2017), doi: 10.1039/C7TA01022B.

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9.    M. J. Tobin, K. R. Bambery, D. E. Martin, L. Puskar, D. A. Beattie, E. P. Ivanova, S. H. Nguyen, H. K. Webb, and J. Vongsvivut, “Attenuated Total Reflection FTIR Microspectroscopy at the Australian Synchrotron,” In Light, Energy and the Environment, OSA Technical Digest (online); Optical Society of America: Leipzig, paper FTu2E.5 (2016), doi: 10.1364/FTS.2016.FTu2E.5.

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10.  V. K. Truong, M. Stefanovic, S. Maclaughlin, M. Tobin, J. Vongsvivut, M. Al Kobaisi, R. J. Crawford, and E. P. Ivanova, “The Evolution of Silica Nanoparticle-Polyester Coatings on Surfaces Exposed to Sunlight,” Journal of Visualized Experiments, 116, e54309, 1-11 (2016), doi: 10.3791/54309.

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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 require use of our Grazing Angle objective (GAO) (see below). 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 
Grazing Incidence

The IRM beamline has a Bruker Vis/IR Grazing Angle Objective (GAO, X15) 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 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 tranflectance 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.


Figure 7. Grazing angle objective
Figure 7. The grazing angle objective (15 X magnification) is ideal for thin films down to monolayers on a reflective substrate

​​Examples of samples grazing angle experiments:

  • Thin (sub micron) coatings on metallic or other highly IR-reflective surfaces
  • Corrosion products on metallic surfaces
FT-IR Rapid Scan; Time-Resolved Spectroscopy

Time resolved spectroscopy (TRS) using a Fourier Transform Infrared 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 changes induced by an electrochemical potential event, to provide an example.  More information on rapid scan FTIR spectroscopy can be found on the Bruker website

Accessories available

A number of different accessories can be coupled to our FTIR microscopes to assist with experiments. These include:

  • 15x and 36x objectives/condensers (numerical aperture, NA=0.60) for reflection and transmission studies
  • 15x Grazing Angle objective (GAO) for thin layer analysis (polarisers are available to enhance absorptions)
  • a 20x objective for macro-Attenuated Total Reflection (ATR) studies (i.e. for hard/solid samples)
  • Polarisers for analysis of structural information and molecular orientation within samples
  • a micro-compression cell for the analysis of small solids and geological samples; can be fitted with diamond windows
  • a Linkam sample stage (FTIR600) that can heat samples up to 600°C and cool them to -196°C/liquid nitrogen temperatures; can be used with samples in either transmission or reflection mode
  • Hyperion 3000 Focal Plane Array (FPA) FTIR microscope is also installed offline at the beamline and is available by request during allocated beamtime

For the analysis of living biological cells, we offer:

Liquid flow-through cell to monitor living systems in close to real-time

A Bioptechs flow cell designed for light microscopy has been adapted for FTIR microspectroscopy; see figure 8 below. The design uses a peristaltic pump to flow a film of media around live biological cells. Their response to changes in this media can then be measured in almost real time. Please contact a Beamline Scientist if you wish to use this sample holder. For more details on the cell itself, please see the Bioptechs website.

The modified Bioptechs liquid flow-through cell
Figure 8. The modified Bioptechs liquid flow-through cell

Liquid compression cells for the study of live microbiological systems.

A major restriction to studying living systems by IR is the strong absorbance of water in the mid-IR region which overlays the amide I band (1650 cm-1) of proteins and complex biological materials. This limits measurements to samples with a path length of <10 microns, however a liquid cell designed at the Australian Synchrotron (Figure 9) enables the collection of high quality, mid-IR spectra of live mammalian cells at these small path lengths. The design utilises a micro-compression cell to sandwich biological cells between two calcium fluoride windows (13 mm in diameter), one of which is printed with a micro-fabricated spacer 6 microns in thickness (Figure 10). The s-channels in the spacer allow excess water to squeeze out from between the plates when a cell suspension is compressed. The design has approximately a 40-60 minute hold time before the water evaporates completely. NOTE This set up is not always available for use. 

Another sample holder also designed and available at the beamline sandwiches biological samples between two calcium fluoride plates (40 mm in diameter) but uses spacers made out of Mylar (6 µm thickness) or Kapton (7 µm thickness), which you lay onto the bottom window before adding your sample. This setup can be used in both static and flow-through modes.

Please contact a Beamline Scientist if you wish to use either of these sample holders and for more detailed information on these designs.

NOTE PC2 cell culture facilities are available for use onsite to help set up these types of experiments.

The liquid compression cell
Figure 9. The liquid compression cell

Calcium fluoride window with a 6 um micro-fabricated spacer
Figure 10. Calcium fluoride window with a 6 µm micro-fabricated spacer


Beamtime Guide and Technical Information

Samples, Data Analysis, Publications