Instrumentation for conventional Raman microscopy and total internal reflection Raman
microscopy
Application Note
Raman microscopy is used in the fields of life sciences, pharmaceuticals, geology and
mineralogy, and the semiconductor industry, to name a few. A conventional dispersive
Raman microscope consists of a laser, microscope-based sample cell, spectrometer,
detector, and optics. Figure 1 shows an instrument layout where the laser is focused onto
the sample with a microscope objective and the Raman scattering is collected in the epidirection through the same objective. (Caution: The laser should be blocked from entering
the microscope eyepiece for user safety.)
The Raman signal is passed via a side port on the microscope to a collection lens with an fnumber matching the Andor HoloSpec ƒ/1.8i spectrograph. The main benefit of this
spectrograph is the modular nature of its components, namely the entrance slit and grating
are easily swapped for use with different excitation wavelengths or application
requirements. Raman Scattering is then passed through a slit and reflected off a
holographic grating. Upon reflection from the grating the diffracted light is projected onto an
Andor Newton 940 CCD with 2048 by 512 pixels. Calibration of the spectral coordinate is
performed using a standard (e.g.50/50 v/v acetonitrile toluene) with known spectral peak
locations. Figure 2 shows the spectrum of a background subtracted 1500-nm polystyrene
film obtained using 30-mW 532-nm excitation. A Raman image can be generated by raster
scanning the stage over the laser.1 Other Raman imaging techniques have also been
developed in which a tunable filter is used to image the presence of Raman scattering at a
specific wavenumber region.2,3 The entire image is taken in one exposure with an imaging
Figure 1: Instrument schematic for conventional Raman utilizing a
microscope in the epi-direction.
CCD, but the tunable filter must be adjusted for each wavenumber region collected.
Total internal reflection (TIR) Raman microscopy can be used to measure thin films and
interfacial phenomena with reduced background from the bulk medium. Examination of thin
films is an important endeavor in many fields, for example the analysis of thin film-based
electronic devices. The amount of Raman signal is decreased due to the small probe
volume in the case of thin films. A further complication to obtaining meaningful thin film
measurements is a signal from the bulk, including the background from optics and the bulk
medium, that may dominate the signal. TIR Raman microscopy has the benefit of probing
the sample with an evanescent wave as opposed to direct illumination, thus limiting the
distance over which the Raman signal is obtained. TIR Raman microscopy can be used to
enhance the signal from the thin film and reject background signals.
Figure 3: Schematic of a prism sample interface showing conditions for
refracted and reflected light.
TIR requires the laser be
directed onto an interface
between medium 1 and 2
(Figure 3) with indices of
refraction 1 > 2 at an
incident angle (i) greater
than or equal to the
critical angle (c) as
determined by Snell’s
law: _c=sin^(-1)(_2/_1 ).
Two routes are available
to achieve TIR: sending
Figure 2: Raman spectrum of a 1500 nm polystyrene film displaying its
characteristic peaks.
the laser through the objective or using an external prism. Through the objective requires a collimated laser that is directed off axis through a high numerical
aperture (generally 1.45 or higher) objective. TIR through the objective has the benefit of easy setup, but is limited by the effective angle range, which may be
problematic if the critical angle cannot be reached for a given interface. The index of refraction of most objectives typically limits the usefulness to aqueous
systems. Alternatively the sample may be optically coupled to an external prism. Prism-based TIR allows for a wide range of usable angles and the choice of
prism materials. One downside is the complexity of the instrumentation involved to deliver the laser to the prism. This can be mitigated by using a fiber
introduction as described below. Figure 4 shows the schematic for a prism-based TIR Raman microscope. The laser is focused onto a fiber optic that has the
other end terminated with a variable collimator to focus the laser on a high index of refraction hemispherical prism. An arm mounted on a rotary stage is set
so the center of rotation is at the sample plane and does not move. The laser is then introduced at the same position on the prism regardless of angle. The
collection of the Raman scattering is the same as described above for conventional Raman microscopy.
Figure 4: Schematic for prism based scanning angle Raman/reflectivity instrument utilizing laser introduction via a fiber optic
The TIR Raman spectra for a sapphire/polystyrene/air interface are shown in Figure 5 (15 mW 532-nm excitation). An enhancement in the measured Raman
signal occurs as the laser’s incident angle approaches 64°, which is close to the critical angle for a sapphire/polystyrene interface. TIR Raman microscopy is
quite sensitive and can be used to measure a single monolayer.4
Figure 5: SA measurements of an 800-nm film of polystyrene film ranging just below to above the critical angle.
Acknowledgments
Written by Michael D. Lesoine and Emily A. Smith. Construction of the Raman instrument was carried out by and for Professor Emily Smith’s research group
at The Ames Laboratory and was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences,
Geosciences, and Biosciences. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. DE-AC0207CH11358.
References
1. Ploetz, E.; Laimgruber, S.; Berner, S.; Zinth, W.; Gilch, P. Applied Physics B: Lasers & Optics 2007, 87, 389.
2. Bowden, M.; Bowden, D. J.; Gardiner, G.; Rice, D.; Gerrard, M. Journal of Raman spectroscopy 1990, 21, 37.
3. Veirs, D. K.; Chia, V. K. F.; Rosenblatt, G. M. Appl. Opt. 1987, 26, 3530.
4. McKee, K. J.; Meyer, M. W.; Smith, E. A. Analytical Chemistry 2012, 84, 4300.