Fig 1a: Three dimensional reconstruction image of the distribution of the oil, alkane and water (GreOil, Red: Alkane, Blue: Water, 30 x 30 x 11.5 µm, 150 x 150 x 23 pixel, 517 500 Raman spectra, total acquisition time of the stack: 23 min)
In the Life Sciences and Bio-medical Research, the characterization of chemical compounds within cells or tissues is one of the most important and difficult tasks for researchers to perform. The development of pharmaceuticals such as drug delivery coatings or capsules also requires as much information as possible about their chemical and structural compositions. For the fields of nanotechnology and materials science, a thorough knowledge of the samples' surface morphology on a sub-micron scale is essential. A variety of techniques are available for such high resolution nano-scale imaging and component analysis, including fluorescence microscopy, electron microscopy coupled with energy dispersive X-ray analysis or electron microprobe and other related techniques. Quite often these techniques are somewhat destructive and abrasive, or require labor intensive and complex sample preparation. Most importantly though, these analytical methods are vacuum or high vacuum techniques, therefore imposing limitations on the investigation of e.g. wet, out-gassing samples, or they induce structural changes. Confocal Raman Imaging and/or Atomic Force Microscopy applied in ambient conditions can help overcome these drawbacks and allow for detailed structural and compositional investigation prior to the possible application of invasive techniques.
Fig 1b: Corresponding spectra (de-mixed, GreOil, Red: Alkane, Blue: Water).
The Raman Effect and its application in microscopy – 3D Chemical Imaging
In Raman spectroscopy, a vibrational quantum state is excited or annihilated within a molecule, leading to an energy shift between the incident light and the scattered light. This energy shift is unique to each molecule and allows the chemical identification of compounds within a sample. By integrating a Raman spectrometer within a state-of-the-art confocal microscope setup, Raman imaging with a spatial resolution down to 200 nm laterally and 500 nm vertically can be achieved using visible light excitation. Only light from the image focal plane can reach the detector, which strongly increases image contrast and slightly increases resolution. Special filters are used to suppress the reflected laser light while enabling the Raman scattered light to be detected with a spectrometer/CCD camera combination. With this setup, a complete Raman spectrum is acquired at each image pixel, typically taking between 0.7 and 100 ms. The individual spectra are combined to form Raman images consisting of tens of thousands of spectra. From this multi-spectrum file, an image is generated by integrating over a certain Raman line in all spectra or by evaluating the various peak properties such as peak-width, min/max analysis or peak position. Due to the confocal arrangement, even depth profiling and 3-D imaging are possible if the sample is transparent.
Fig 2a: WITec Project Plus software screenshot showing the cluster tree of a cluster analysis of a toothpaste sample
Sensitive Setup and Ultrafast 3D Image Acquisition
The latest spectroscopic detector technology combined with a high-throughput confocal microscope are the crucial points in the recent improvements in sensitivity allowing acquisition times for a single Raman spectrum to be reduced to 0.7 milliseconds. Using such a sensitive setup can also be an advantage when performing measurements on delicate and precious samples requiring the lowest possible levels of excitation power. Time resolved investigations of fast dynamic processes can also benefit from the ultrafast spectral acquisition times. In the following study, the ultrafast Confocal Raman Imaging capabilities of the alph300 R were used to analyze an oil-alkane-water emulsion three dimensionally. In a volume of 30 x 30 x 11.5 µm, 23 confocal Raman scans were acquired in z-direction leading to 23 Raman images each consisting of 150 x 150 pixel (22 500 spectra). The total acquisition time for one image was 60 s resulting in 23 min for the acquisition of the complete stack (517 000 Raman spectra). Using a 3D reconstruction software, a three dimensional image of the distribution of the three compounds can be created as shown in Fig 1 a (green: oil, red: alkane, blue: water).Fig. 1 b show the corresponding Raman spectra (de-mixed).
Fig 2b: Corresponding Raman spectra as a result of the cluster analysis procedure
As a Raman image typically consists of tens of thousands of spectra, a powerful data analysis software is essential in order to extract the information of interest. Hidden structures in the images should ideally be visualized automatically ensuring an objective and consistent interpretation of the imaging data. WITec Project Plus is a unique software tool for advanced microscopic data processing. It features a Cluster Analysis algorithm which automatically identifies similar spectra and classifies them into a user defined number of clusters. Each cluster is represented by an average spectrum and a map showing the spatial distribution of these similar spectra. This technique is a very powerful tool to determine the number of components in a certain sample volume. It does not require any a-priori knowledge about the sample, the user only has to guess the number of components he expects in the sample. In the following example, cluster analysis was applied to a confocal Raman imaging data set acquired at a toothpaste sample. The scan range of the image was 20 x 20 µm with 200 x 200 pixel (40 000 spectra). The acquisition time for one spectrum was 0,76 ms resulting in a total acquisition time of 42 s for the image. Fig. 2 a shows the cluster tree of the WITec Project Plus software with the distribution of 6 selected components. The average spectra for each cluster are shown in Fig.2 b. By combining the image clusters of the 6 components, the complete distribution of the phases in the toothpaste can be visualized as shown in Fig. 2 c.
Fig 2c: Color coded Raman Image of the lateral distribution of the toothpaste compounds
Combining Raman Imaging and Atomic Force Microscopy
For high resolution surface topography imaging along with the chemical information derived from Raman imaging, the Confocal Raman microscope can be transformed into an Atomic Force Microscope (AFM) by simply rotating the microscope turret when using a modular instrument configuration. Atomic Force Microscopy is employed to trace the topography of samples with extremely high resolution by recording the interaction forces between the surface and a sharp tip mounted on a cantilever. The sample is scanned under the tip using a piezo-driven scan-stage and the topography is reproduced with specialized software tools which translate this information into images. Structures below the diffraction limit can be visualized using this imaging technique. As an example, phase separation of a spin-coated film of a polymer blend PMMA-SBR (poly-methyl-methacrylate – styrol-butadien rubber) on glass is studied with a combination of AFM for ultimate resolution and Raman Imaging for chemical information. The topographic image obtained with the AFM (Fig. 3) reveals round features 20-30nm in height with diameters varying from 150nm to 4µm, surrounded by a network-like structure. From the AFM measurements, the phase separation can be clearly seen but it cannot be determined whether or not the phases are completely separated and which material forms the round features and which the network.
Fig 3: AFM image of a PMMA-SBR polymer blend, spin coated on glass. 20x20µm scan, 30nm topographic scale.
A Raman image of the structure (Fig. 4) clearly reveals the chemical structure of the phases. The Raman image was obtained using a 100x, NA=1,25 oil immersion objective with 532nm excitation. The image consists of 200x200 spectra taken with 70ms integration time per spectrum. One can clearly see the complete phase separation of the polymers. It is also obvious that the PMMA forms the round, flat structures while the rubber is present in the network structures.
Fig 4: Raman image of the PMMA-SBR polymer blend. The PMMA is color-coded blue, whereas the SBR is shown in red. Imaging parameters: 200x200 spectra, 70ms integration time per spectrum.
Knowledge of chemical components and their distribution in a sample is of immense importance for R&D as well as quality control in various research and industry fields. It was demonstrated that confocal Raman imaging provides the ability to non-invasively image the chemical properties of emulsions three dimensionally with the highest resolution. The advantage of computerized data processing with a chemometric software tool for Raman imaging data evaluation could be additionally shown.