Confocal Raman microscopy analysis of biological samples allows several advantages over standard dispersive Raman techniques.
Dr. Antoinette O’Grady, Princeton Instruments/Acton
Scientists
in the fields of biology and biochemistry are familiar with some of the more common
forms of spectroscopic analysis, such as absorption and emission spectroscopy. The
former measures the amount of light absorbed at a given wavelength to provide information
about a sample’s structure, and the latter produces
information regarding a substance by measuring the amount of light of a particular
wavelength that is emitted.
Raman spectroscopy — which measures the
light scattered by a substance over a defined range of wavelengths when excited
by a laser of a particular wavelength — offers
significant advantages over other forms of spectroscopy. It does not require any
sample preparation, samples are not destroyed, water bands are usually small and
easily subtracted, and Raman spectra usually contain sharp bands that are characteristic
of the specific molecules in the sample.
In biological samples such as cells and tissue,
infrared spectra often show broad spectral features that can offer information regarding
cellular components. However, Raman spectra also provide more detailed information
on the constituents of the specific components. This allows good specificity for
qualitative analysis and for discrimination among similar materials. Because the
intensity of the bands in a Raman spectrum are proportional to the concentration
of the molecules that give rise to them, it can be used for quantitative analysis.
Confocal Raman microscopy has the added
advantages of providing chemical information with microscopic resolution, allowing
specific spatial analysis of regions within a bulk sample, offering depth profiling
and improving rejection of fluorescence.
A brief history
Raman spectroscopy has been around since the beginning
of the 20th century. Sir C.V. Raman is credited with discovering the scattering
effect in 1928, when he observed it in a sample mixture of water and alcohol that
was excited by monochromated sunlight while using the eye as a crude detector.1,2
Soon after these initial experiments, a relationship between the excitation wavelength
and the scattered wavelengths was discovered.3
The differences between the incident
and scattered wavelengths (Raman shifts) were related to the vibrational energies
of molecular bonds. It was realized that Raman spectroscopy was a probe of the vibrational
energy levels within a molecule and, thus, complementary to infrared absorption
spectroscopy.
At that time, there was an increased
interest in Raman spectroscopy because it provided molecular information more easily
than infrared absorption spectroscopy and was the only way to measure low-frequency
vibrations. However, it was overshadowed during the 1940s, when advances in infrared
instrumentation resulted in the first commercially available instrument for infrared
absorption, making IR easier to use and more readily available than Raman spectroscopy.
By the 1950s, photomultiplier tubes
(point detectors) had replaced photographic film for the detection of Raman scattering.
The first commercially available Raman instrument was introduced in 1953.
It was not until the development of
the laser, with its superior power and monochromaticity, that a renaissance occurred
in Raman research. In the 1970s, the development of multichannel detectors, which
allow large numbers of wavelengths to be viewed simultaneously, resulted in another
large step forward in Raman instrumentation.4
Raman and biology
The 1990s saw the final stages in the modernization of instrumentation with the development of holographic
notch filters5 and of compact diode and diode-pumped lasers, and with the integration
of confocal microscopy into the technique. Holographic notch filters made the instrumentation
much more compact because a single filter replaced multiple dispersion stages as
a means of filtering the intense laser light and separating it from the weak inelastically
scattered Raman effect. The addition of confocal microscopy has brought Raman spectroscopy
firmly into the realm of biological analysis.
At its simplest, Raman spectroscopy is performed
by focusing laser light of the required excitation wavelength onto a sample. The
scattered light is collected and focused onto the entrance of a spectrograph, which
separates the light into individual wavelengths using a dispersion grating. A notch
filter usually is placed in the optical path of the spectrograph to reduce the amount
of Raleigh scattered (reflected) light from the laser entering
the spectrograph, and a CCD detector mounted to the spectrograph detects and measures
the Raman shifts (Figure 1). The distance from the laser line at which Raman lines
can be measured, the number of excitation wavelengths used, and the resolution will
increase the complexity and cost of the system.
Figure 1. A simple Raman setup consists of a notch filter, a spectrograph, a laser and
a CCD detector.
One of the principal obstacles in using Raman
spectroscopy for biological applications is the presence of a lot of background
noise caused by fluorescence from contaminants or background matrices in the sample.
There are many methods of overcoming fluorescence, but the most popular one is to
use an excitation wavelength that does not excite fluorescence. For this reason,
most biological applications use either UV or near-IR lasers.
In laser spectroscopy labs, resonance
Raman spectroscopy with UV excitation is widely used for probing the structure of
proteins and other biological molecules. It achieves Raman enhancement by exciting
a molecule near its transition state.
Figure 2. Investigators placed a 5-μl droplet containing 20 μM of lysozyme in a
phosphate buffer onto a slide. After the protein segregated from the buffer, confocal
Raman spectroscopy helped elucidate where pure lysozyme (A) and phosphate salt (B)
remained.
Yet it does not
lend itself to use outside a highly specialized environment because of the high
cost of suitable UV lasers and because it is difficult to use notch filters in this
wavelength region.
Near-IR wavelengths have become preferred for
biological applications because of the low cost of diode lasers and the fact that
the longer wavelengths also can avoid fluorescence. It is relatively easy to integrate
these compact lasers with notch filters, a spectrograph and a CCD to provide a dedicated
Raman instrument. Most modern commercial instruments used for biological applications
also integrate a confocal microscope because it offers the added advantages of being
able to use both visible and near-IR excitation while avoiding fluorescence, increasing
spatial resolution and allowing comprehensive mapping of a sample. It also provides
a familiar interface for biologists.
Figure 3. Raman spectra taken from points A (left) and B (right) of Figure 2 contain either pure
lysozyme or only phosphate salt.
In confocal Raman microscopy, also
called Raman microspectroscopy, the microscope objective focuses the incident laser
radiation onto a point in the sample. The Raman scatter is collected by the objective
and coupled into the spectrograph through a pinhole aperture. The aperture enables
confocal detection: Raman scatter obtained from outside the focal point is out of
focus at the aperture and, thus, not detected.
Because the laser is focused to a very small point, the laser flux in the sample is high enough to
quench fluorescence in a much shorter time than in conventional Raman spectroscopy.
Also, excited electrons that might cause fluorescence can migrate to molecules with
lower energies so that a molecule outside the sampling area excited by the laser
may emit the fluorescence photon, which would miss the pinhole aperture. This occurs
because electrons within the sampling area will naturally migrate to lower-energy
molecules, which are found outside the area.
Applications
Several types of confocal Raman microscopes are
commercially available, ranging from the open modular type systems that can be reconfigured
to accommodate a wide variety of experiments and applications to dedicated turnkey
systems with sophisticated software for biological applications.
Raman spectroscopy has become a popular
analytical method for many biological applications, and as the instrumentation evolves
toward becoming a dedicated biologist’s tool, the variety and scope of applications
will only widen. Raman microspectrometry involves almost no sample preparation,
and it has better axial and spatial resolution than conventional microscopy. This
makes it possible to perform extremely detailed >analysis
of cells in their natural state.
Microscopic resolution allows the chemistry of
individual cells to be investigated and mapped images to be generated. These images
can contain full spectral information at each pixel so that the distribution of
components within the cell can be visualized based upon their Raman signature. This
is extremely valuable to researchers because biochemical changes can be observed
during a cell’s life cycle or when a cell becomes damaged or cancerous. Using
confocal Raman, the changes in a variety of cell types, including bacteria and eukaryotes,
can be monitored over time, and comparison between healthy and diseased tissue states
can be easily analyzed.
One challenge faced in analyzing cells
in their natural state is that they often will move around within a given matrix.
Researchers at the University of East Carolina in Greenville, N.C., have solved
this problem by combining a confocal Raman system with optical tweezers that can
immobilize the cells.6
Researchers at Purdue University in
West Lafayette, Ind., are using Raman microscopy for proteomic analysis of femtomole
quantities derived from microliter volumes of micromolar protein solutions.7 They
have used a dropped coating deposition method, in which a small volume of a dilute
solution of protein (and buffer) is deposited on a slide, consisting of a highly
polished stainless steel coated with nanometer Teflon coating (Figures 2 and 3).8
Meet the author
Antoinette O’Grady is spectroscopy business
manager at Princeton Instruments/Acton in Trenton, N.J.; e-mail: [email protected].
References
1. C.V. Raman and R.S. Krishnan (1928). NATURE, Vol. 121, p. 501.
2. C.V. Raman (1928). INDIAN J PHYS, Vol. 2, p. 387.
3. R.W. Wood (1928). NATURE, Vol. 122, p. 349.
4. R. Al-Wazzan (1996). PhD thesis, Queen’s University of Belfast.
5. B. Yang et al (1991). Side-on photomultiplier gating system for Thomson scattering and laser-excited atomic fluorescence spectroscopy. APPL?SPECTROS, Vol. 45, pp. 1533-1560.
6. C. Xie et al (2005). Raman sorting and identification of single living microorganisms with optical tweezers. OPT
LETT, Vol. 30, pp. 1800-1802.
7. D. Zhang et al (2004). Chemical segregation and reduction of Raman background interference using drop coating
deposition. APPL SPECTROS, Vol. 58, pp. 929-933.
8. Y. Xie et al (2004). ANALYT BIOCHEM, Vol. 332, pp. 116.