Beruflich Dokumente
Kultur Dokumente
Microscopy
Essential
Knowledge
Briefings First Edition, 2017
2 Raman Microscopy
© 2017 John Wiley & Sons Ltd, The Atrium, Southern Gate,
Chichester, West Sussex PO19 8SQ, UK
Microscopy EKB Series Editor: Dr Julian Heath
Spectroscopy and Separations EKB Series Editor: Nick Taylor
Raman Microscopy 3
CONTENTS
4 INTRODUCTION
6 HISTORY AND BACKGROUND
12 IN PRACTICE
25 PROBLEMS AND SOLUTIONS
30 WHAT’S NEXT?
INTRODUCTION
The absorption and scattering of sunlight by matter is a
phenomenon known to all on an everyday basis. It is the reason
why the sky and seas are blue, why plants are green, why rain-
bows have their brilliant colors and why sunglasses protect
your eyes. Some of these everyday phenomena involve elastic
scattering – scattering with no loss of energy of the emitted
light. But sometimes the light that is emitted is of a different
wavelength because it has lost some of its energy; this is called
inelastic scattering.
The Raman effect is one form of inelastic scattering of
light and is the basis of a powerful form of non-destructive
chemical analysis called Raman spectroscopy. By measuring
the degree of energy loss of light, the analyst can identify specific
chemical bonds in a sample and obtain a chemical fingerprint
of the molecules in the sample, thus allowing its identification.
Furthermore, by performing Raman spectroscopy on a micro-
scope the analyst can also reveal the spatial distribution of mol
ecular components within the sample at micrometer or even
nanometer resolution. This is Raman microscopy or Raman
microspectroscopy and is the subject of this EKB.
This EKB starts with the discovery of elastic and inelastic
scattering and outlines the physics of the interaction of light
and molecules. It then goes on to explain how these principles
are incorporated into modern light microscopes to allow Raman
imaging or Raman microspectroscopy. This is followed by a
detailed explanation of the main ways in which researchers use
Raman microscopy, from preparing samples for analysis to
obtaining Raman spectra and maps. The EKB also discusses
Raman Microscopy 5
Virtual state
laser
Energy laser laser Anti-Stokes
Ray
Stokes
=1
Vib =0
Stokes Rayleigh Anti-Stokes
scattering scattering scattering
Stokes = laser – vib Ray = laser Anti-Stokes = laser + vib
Figure 2. Jablonski diagram showing energy levels in Stokes, Rayleigh and anti-Stokes
scattering. (© Thermo Fisher Scientific, used by permission)
⁄(cm-1) = 10 ⁄ in nanometers.
7
IN PRACTICE
Raman microscopy can be performed on any standard upright
or inverted light microscope equipped with the usual comple-
ment of optics and accessories for high-resolution widefield or con-
focal imaging.
These may include inter alia a motorized stage, low (eg 10x,
0.25NA) or high power (eg 100x 1.4NA oil immersion) objectives
and a digital camera to take images of the sample. For Raman spec-
troscopy four additional components are required: a laser, sets of
optical filters, a spectrometer or spectrograph, and a detector. A
shroud (or class 1 laser safety enclosure) to exclude extraneous light
and avoid operator exposure to laser light is also standard on most
commercial Raman microscopes.
For thick samples, confocal optics are used to allow optical
sectioning through planes in the sample, a technique called depth
Aperture Filter
Grating
Sample
Laser
Multichannel detector
Figure 4. The basic optical path for a Raman microspectrometer. The scattered light
is directed through a filter that eliminates the Rayleigh light. The Raman signal then
passes the entrance slit or aperture and strikes a dispersive element, shown here as a
simple grating. The dispersed light is then directed onto a multichannel detector
14 Raman Microscopy
Light from the laser is directed through the objective lens and
focused in a small spot on the sample. The sample is scanned under
the laser beam using the motorized stage: the operator can then
choose to do spot scans (called point and shoot), line scans or a 2D
map or image scan of the sample. At each point or pixel of the scan,
all the light reflected from the sample is collected by the lens and
passes to the spectrometer where the Rayleigh scatter is blocked by
an edge filter (preventing detector overload) allowing only the
Raman scattered light to be dispersed into its separate wavelengths
and detected by a detector (CCD camera).
Most commercial Raman microscopes can be configured
with one or more lasers, which may be gas-based (eg He–Ne) or
solid-state (diode lasers, such as Ti:sapphire, Nd:YVO4). While each
laser emits essentially a single frequency, the varied lasers enable
users to select from a wide range of excitation wavelengths from
ultraviolet (UV), through visible (Vis) to the near infrared (NIR).
The Raman system is equipped with Rayleigh filters and spectro-
graphs specific to the laser choice, to allow fast switching of wave-
lengths during an experiment. The multiple laser offerings provide
the needed trade-off between Raman efficiency and fluorescence, as
discussed in the Problems and Solutions section of this EKB.
Optical filters are placed between the sample and the spec-
trometer to reject the excitation wavelengths from the laser and
allow through only the Raman scattered emission wavelengths.
These filters are of the edge or notch types that allow transmis-
sion of only light of longer wavelengths or of a specific band-
width, respectively.
Raman spectrometers operate in one of two modes: dispersive
or interferometric (also called Fourier transform [FT]). The choice
Raman Microscopy 15
Input Cylindrical
aperture lens
Grating
Secondary
#2
2048
pixel
detector
Primary Secondary #1
mirror
Figure 5. The actual light path is more complex than shown in Figure 4. Multiple
mirrors control the spatial and spectral integrity of the signal. The spectrograph
module of the Thermo Scientific DXR2 Raman microscope is shown on the right
1.00
0.70
0.40
0.30
0.20
0.10
Figure 6. Raman spectrum of benzene (C6H6) showing C–H and C–C bond stretching
Asprin
Acetaminophen
Caffeine
Titanium dioxide
TNT
PETN
1500
1000
500
-0
Explosive slide 6 - C-4 organic
6000 C-4
Raman intensity
4000
2000
-0
3000 2000 1500 1000 500
Raman shift (cm-1)
The figure shows how successful this can be. The image shows 20μm
TNT particles from which Raman spectra were collected. The spectra reveal
TNT (2,4,6 trinitrotoluene), PETN (pentaerythritol tetranitrate), and C-4
(RDX: 1,3,5-trinitroperhydro-1,3,5-triazine).
Raman Microscopy 23
shift as the basis for generating Raman images it is possible to get a 3D spatial
distribution of the stress around the inclusion. In addition, photoluminescence
in diamonds is commonly observed, due to crystal defects such as substitution
of carbon for another element, like nitrogen. Many different photoluminescence
peaks have been observed for diamond and the specific 613nm peak observed
in the spectra around the inclusion (shown in violet) is consistent with a previ-
ously reported photoluminescent peak in diamond.
Inclusion
Stress
Photoluminescence
Diamond
Raman imaging goes beyond just identification. It can be used for visualizing
physical properties revealed in the Raman spectra. The colors show different
types of information and images from a single data set. Figure from Thermo
Fisher Scientific, used by permission
Raman Microscopy 25
Sample degradation
Lasers produce very intense beams of light that are capable of
causing damage to the sample. It is important to select a laser power
(wattage) that does not cause any changes in the sample during the
investigation. At worst the laser could cause burning of the sample
but the damage is usually more subtle. Samples can show a change
in phase due to recrystallization, and living cells are very sensitive
to phototoxicity and their macromolecules to radiolysis.
Fortunately in most cases this is not a problem: although
Raman scattering intensity is inversely proportional to wavelength,
the spectra (peak locations) are not wavelength dependent and so the
investigator can often simply reduce laser power or change to a
longer wavelength to avoid this type of sample damage.
Anecdotal reports of samples being destroyed or even catching
fire are common, usually resulting from high laser power hitting a
deeply colored material, such as ink on a tablet.
Brightness of lasers
There are two ways of looking at the suitability of lasers for
Raman microscopy (other than the obvious wavelength dependen-
cies already noted): power (wattage) and brightness. Power is sim-
ply the output of the laser. Brightness describes the power density
at the sample. Lower power lasers can often deliver more brightness
at the sample by using a smaller beam divergence resulting in
greater Raman emission. The tight focus of these lasers and small
spot sizes also contribute to high spatial resolution (Table 1).
Raman Microscopy 27
Table 1. Less is more! Comparing two 780nm laser options for Raman. Table from
Thermo Fisher Scientific
WHAT’S NEXT?
At a recent analytical conference, it seemed every aisle had at
least one vendor showing new aspects of Raman systems. This
surge in use has been driven largely by technological advances,
such as new lasers, spectrometers and detectors. A major trend in
the last 10 years has been the rapid evolution of Raman spectros
copy from a technique used only by spectroscopy experts into a
tool used by scientists and engineers to solve problems. Compact,
enclosed systems, such as the Thermo Scientific DXR2 family of
spectrometers, provide the flexibility to analyze particles or image
entire tablets, with huge applications in the field of carbon-
materials, such as nanotubes and graphene. Substantial growth in
the handheld or portable markets has been driven by this same
innovation in components.
The ability of the Raman laser and Raman scatter to be directed
by glass optics (rather than the mirrors and salt plates needed for
infrared) allows novel access in multimodal analyses, where Raman
is combined with other techniques. Three examples are Rheometry-
Raman, X-ray photoelectron spectroscopy (XPS)-Raman and atomic
force microscopy (AFM)-Raman.
Rheometry involves the measurement of the shear storage
modulus G’ and shear loss modulus G” of a material. Essentially,
G’ relates to the elastic response, while G” relates to the viscous
response. As an epoxy cures, it passes from being a viscous fluid
(higher G”) to an elastic solid (high G’). By coupling a Raman spec-
trometer into the rheometer, a relationship between the physical
parameters and the underlying chemistry can be constructed. Fig-
ure 8 shows G’, G” and the Raman measurements for the freezing
of a polymer (the experiment started hot and cooled, right to left
Raman Microscopy 31
108 1400
Raman
2500 808 cm-1 peak height
1200
2000 Solid 107
1500
Solid 1000
1000
G’, G” (Pa)
1600 600
1400
1200 Transition 105 400
3000 Melt 200
10 4
2500 Melt G’ 0
G”
900 850 800 103 -200
Raman shift (cm-1) 50 100 150 200
Temperature (°C)
Figure 8. Correlation of shear storage modulus G’ and shear loss modulus G” and
corresponding Raman shifts during the melting of a polymer. Figure from Thermo
Fisher Scientific, used by permission
1.20E+06 C 1s
Name Atomic
Ag 3d %
1.00E+06 O 1s
8.00E+05 C 76.0
Counts/s
Zn2p Ni 2p
6.00E+05
O 16.8
4.00E+05
2.00E+05 Si 2s Ag 4.9
0.00E+00
1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0
Si 1.3
Binding energy (eV)
Zn 0.5
Ni 0.4
G Band 160 Radial
800 120 Breathing
Raman intensity (c/s)
Modes
80
600
100 150 200 250 300
400
1D Band
200 2D Band
Figure 9. Correlation of XPS and Raman data from SWCNTs. Figure from Thermo
Fisher Scientific, used by permission
FURTHER INFORMATION
De Gelder J, De Gussem K, Vandenabeele P, Moens L. Refer-
ence database of Raman spectra of biological molecules. J Raman
Spectrosc 2007;38:1133–47. (http://dx.doi.org/10.1002/jrs.1734)
Delhaye M, Dhamelincourt P. Raman microprobe and micro-
scope with laser excitation. J Raman Spectrosc 1975;3:33–43. (http://
dx.doi.org/10.1002/jrs.1250030105)
Fleischmann M, Hendra PJ, McQuillan AJ. Raman spectra of
pyridine adsorbed at a silver electrode. Chem Phys Lett 1974;26:163–6.
(http://dx.doi.org/10.1016/0009-2614(74)85388-1)
Landsberg G, Mandelstam L. A novel effect of light scattering
in crystals, Naturwissenschaften 1928;16:557.
Raman CV, Krishnan KS. A new type of secondary radiation.
Nature 1928;121(3048):501–2. (http://dx.doi.org/10.1038/121501c0)
Raman CV, Krishnan KS. The molecular scattering of light.
Nobel prize lecture, December 11, 1930. (http://www.nobelprize.
org/nobel_prizes/physics/laureates/1930/raman-lecture.pdf)
Essential
Knowledge
Briefings