The best of both worlds

Resolution and analytical power are combined in confocal Raman spectroscopy - but there are some problems. However, these can be overcome says Dr Olaf Hollricher   

Confocal Raman imaging opened the door for many applications in Raman spectroscopy and imaging that were previously unavailable for measurement with conventional (non-confocal) Raman methods. However, high confocality always results in high focus sensitivity and this can make measurements difficult with rough or inclined samples. The new, patent pending, True Surface imaging extension resolves these problems and extends Raman imaging to large scale (> 1x1mm²) samples without extensive tilt alignment or sample preparation and without comprising the advantages of confocal imaging.

Raman spectroscopy is a non-destructive and non-invasive technique that delivers detailed chemical information about the components involved in the scattering process, without sample preparation or labelling.

The Raman effect occurs during the interaction of electromagnetic waves (light) with matter in which a vibrational quantum is excited (Stokes Raman scattering) or annihilated (Anti-Stokes Raman scattering). The incident light causes the molecules to vibrate and the energy shift between the exciting and scattered photons is characteristic for the type and coordination of the molecules involved in the scattering process. The energy shift is a function of the mass of the involved atoms and the strength and configuration of their bonds, so that every chemical species shows its own unique spectrum.

Although the Raman Effect had been discovered in 1928 by Chandrasekhara Raman, who was awarded the Nobel Prize in Physics two years later, routine Raman spectroscopic experiments were not possible until the laser was developed in the 1960s. Since then, Raman has developed into an extremely powerful spectroscopic technique that is used to determine the chemical and crystallographic structure of a wide variety of samples.

Principal setup of a confocal microscope. Light originating from an out-of-focus plane is not in focus in the pinhole plane and is therefore strongly rejected.

If heterogeneous samples are to be analysed, the distribution of specific materials can be very important. However, in most spectroscopy setups spatial resolution is very poor, because the exciting laser spot diameter is in the order of 100µm.

Optical microscopy on the other hand is capable of providing a spatial resolution below 200nm using visible light excitation. By combining a research-grade confocal optical microscope with a high-sensitivity Raman spectrometer, it is not only possible to obtain Raman spectra from extremely small sample volumes, but also to collect high-resolution Raman images that show the distribution of chemical species, as well as their crystallographic properties with sub-µm resolution.

A confocal microscope is of course an optical microscope with three-dimensional imaging capability. This can be achieved by illuminating the sample with a laser and subsequent detection of the scattered light through a pinhole, placed in the image plane of the microscope. The pinhole rejects out-of-focus light and ensures that only light originating from the image focal plane can reach the detector (Figure 1). In contrast, it is not possible to determine the in-focus position of a homogenous fluorescing layer with a standard optical microscope.

To obtain an image, either the laser or the sample has to be scanned and the image is acquired point-by-point and line-by-line. With this technique the majority of the out-of-focus light is rejected and the image contrast is greatly enhanced. By taking a stack of images with different focal positions, the geometry of samples can be reconstructed in 3D.

A confocal Raman microscope is a combination of a Raman spectrometer with a confocal microscope, so that the resolution of the optical microscope is combined with the analytical power of Raman spectroscopy. The sample is scanned point-by-point and line-by-line, and at every image pixel a complete Raman spectrum is taken. This process is also called hyperspectral imaging. These multi-spectrum files are then analysed to display the distribution of chemical sample properties.

Setup of a confocal Raman microscope

In the confocal Raman microscope of the WITec alpha300R/500R series, the laser light is guided to the microscope with a single-mode optical fibre (Figure 2). These single-mode fibres are designed to carry only a single transversal mode (TEM00) which can be focused to a diffraction limited spot and therefore act as perfect point light sources. This light is focused onto the sample using a dichroic beam-splitter that reflects the exciting laser beam, but is fully transparent for the frequency shifted Raman light.

In our case, the sample is scanned with a piezo-electric scan table with capacitive feedback correction for high resolution, or with a stepper motor driven xy-stage for large area measurements. The Raman scattered light is collected with the same objective and is focused into the core of another optical fibre that is connected to a spectrometer with CCD camera. Only the core of the fibre guides the light and therefore acts as a pinhole for confocal microscopy. The fibre core also doubles as an entrance slit for the spectrometer, so that no additional optical components are necessary between microscope and spectrometer. The light is dispersed inside the spectrometer and the spectra are acquired with an ultra-sensitive, back-illuminated CCD camera. At every image pixel a complete Raman spectrum is acquired and used to extract the relevant chemical information from the sample.

It is very important to optimise the throughput and sensitivity of the entire instrument. If only a single spectrum is to be acquired, it is usually not important whether the integration time is 10 seconds or 1 minute. Even much longer integration times are in many cases acceptable. This changes completely if a Raman image is to be taken. Even a short integration time of only 1s per spectrum leads to a total acquisition time of nearly 3 hours in a Raman image consisting of 100 x 100 pixels = 10,000 spectra.

Typical integration times in a confocal Raman microscope are between 700µs and 100ms per spectrum, so that a complete Raman image of 10,000 spectra takes between a few seconds and 20 minutes. The shortest time is limited by the readout speed of the CCD camera (and the available Raman signal), while the longest integration time is determined only by the required signal and the patience of the user.

Working principle of a confocal chromatic sensor

Confocal microscopy is superior to standard microscopy because of its ability to strongly suppress out-of-focus light. In many cases, even on flat samples, this is important to improve the signal-to-noise (or signal-to-background) ratio.

Nevertheless, confocal microscopy can be challenging if large samples or rough surfaces have to be analysed, because only those parts of the sample that are in focus contribute to the image. In these cases, WITec´s new patent pending True Surface Microscopy option is a solution as it allows confocal Raman imaging guided by the surface topography. With this the focus follows the surface topography with high precision, so that even rough or inclined samples always stay in focus.

Geological sample (Josefsdal Chert 99SA07, sample courtesy Frances Westall, CNRS Orleans, France) with a strongly tilted and very rough surface

To achieve this unique capability, the WITec alpha500 series can be equipped with a highly precise sensor for optical profilometry. The topographic coordinates from the profilometer measurement are used to perfectly follow the sample surface in confocal Raman imaging mode. The result is an image revealing chemical properties at the surface of the sample, even if this surface is rough or inclined. The topography sensor of the WITec alpha500 works using the confocal chromatic sensor principal (Figure 3). A white-light point source is focused onto the sample with a hyperchromatic lens assembly, a lens system that has a good point mapping capability, but a strong linear chromatic error. Every colour has therefore a different focal distance. The light reflected from the sample is collected with the same lens and focused through a pinhole onto a spectrometer. As only one specific colour is in focus at the sample surface, only this light can pass through the confocal pinhole. The detected wavelength is therefore related to the surface topography.

Scanning the sample in the XY plane (up to 50x100mm) reveals a topographic map of the sample. This topography can then be followed in a subsequent Raman imaging measurement so that the laser is always kept in focus with the sample surface (or at any distance below the surface). Depending on the type of sensor used, a lateral resolution of 10-25µm and a vertical resolution of 40-120nm can be achieved at a measurement range of 1-3mm and a working distance of 10-16mm (other sensors with different ranges and resolutions are available on request).

Topography as measured with the confocal chromatic sensor

Figure 4 shows a geological sample with an arrow pointing to the area of interest, as well as the topography as measured with the optical profilometer.  The two images below show the same area measured in confocal Raman imaging mode (left) and using True Surface imaging option (right) as well as the spectra of the three different components that were found on this surface. The scan range was 2x2mm2 and the topography range was 1.7mm, a nearly 45 degree incline. As can be seen, True Surface imaging keeps the focus always on the surface and maintains the advantage of using a confocal microscope even on very rough or inclined surfaces.

Figure 5a shows a height profile of a pharmaceutical tablet (aspirin) and Figure 5b the same profile with the confocal Raman measurement overlaid. The drugs are labelled red and blue respectively, while the excipient is shown in green

The confocal Raman images below are taken without True Surface microscopy correction

(Figure 5c). Topographic variation was more than 300µm.

True Surface Raman imaging allows the measurement of rough or inclined samples while maintaining the advantages of confocal imaging. Large area scans, which were very demanding before are now possible without sample preparation and alignment by following the topography previously acquired with a confocal chromatic sensor.

The topographic sensor used for True Surface Microscopy is also an ideal supplement to atomic force microscopy (AFM), which provides topographic information on small sample areas (< 100 µm) with ultra-high precision (< 1 nm).

Dr. Olaf Hollricher
Contact:
t: +49 (0) 731 140 700
f: +49 (0) 731 140 70200
e: olaf.hollricher@witec.de