Raman Spectroscopy and some
experimental results
By
Ansam Jameel Talib
Molecular Physics
Course
Professor
Dr. HANS SCHUESSLER
History of Raman Scattering
• 1923 – Inelastic light scattering predicted by A.
Smekel
C. V. Raman
• 1928 – Landsberg and Mandelstam saw unexpected
frequency shifts in scattering from quartz
• 1928 – C.V. Raman and K.S. Krishnan saw “feeble
fluorescence” from neat solvents
• 1930 – C.V. Raman wins Nobel Prize
• http://www.aps.org/publications/apsnews/200902/physicshistory.cfm
http://bwtek.com/raman-theory-of-raman-scattering/
Raman Spectroscopy
a spectroscopic technique used to observe vibrational, rotational, and other low-frequency
modes in a system.
Raman spectra are similar to infrared spectra .
Useful for functional group detection and fingerprint regions that permit the identification
of specific compounds. The advantages: small sample requirement, minimal sensitivity
toward interference by water, and high conformational and environmental sensitivity.
•
http://www.inphotonics.com/raman.htm
Can Raman spectra be obtained from
solids, liquids and gases
Solids
Liquids
Gels
Slurries
powders
films, etc.
Raman spectra can even be obtained from some metals.
It is possible to obtain Raman spectra of gases.
However, since the concentration of molecules in gases is generally very low, this typically
requires special equipment, such as long pathlength cells.
https://depts.washington.edu/ntuf/facility/docs/NTUF-Raman-Tutorial.pdf
Types of Raman Spectroscopy
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1- Surface-enhanced Raman spectroscopy (SERS)
2- Resonance Raman spectroscopy
3- Angle-resolved Raman spectroscopy
4- Hyper Raman
5- Spontaneous Raman spectroscopy (SRS)
6- Optical tweezers Raman spectroscopy (OTRS)
7- Stimulated Raman spectroscopy
8- Spatially offset Raman spectroscopy (SORS)
9- Coherent anti-Stokes Raman spectroscopy (CARS)
10- Raman optical activity (ROA)
11. Transmission Raman
12. Inverse Raman spectroscopy
13- Tip-enhanced Raman spectroscopy (TERS)
14- Surface plasmon polariton enhanced Raman scattering (SPPERS)
15- Stand-off Remote Raman
16- Confocal Raman
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http://en.wikipedia.org/wiki/Raman_spectroscopy
Tip-enhanced Raman spectroscopy (TERS)
• Tip-Enhanced Raman (TERS or nanoRaman): chemical imaging at the nanoscale
• TERS (or nano-Raman) brings you the best of both worlds: the chemical specificity
of Raman spectroscopy with imaging at spatial resolution typically down to 10nm.
This technique can be demonstrated on various samples ranging from nanotubes to
DNA.
• TERS has been shown to have sensitivity down to the single molecule level and
holds some promise for bioanalysis applications
•
http://www.intechopen.com/books/electronic-properties-of-carbon-nanotubes/detection-of-carbon-nanotubes-using-tip-enhanced-raman-spectroscopy
http://www.intechopen.com/books/elec
tronic-properties-of-carbonnanotubes/detection-of-carbonnanotubes-using-tip-enhanced-ramanspectroscopy
http://www.asdn.net/asdn/nanotools/spm.shtml
Confocal Raman
• Couples a Raman spectrometer to a standard optical microscope, allowing high
magnification visualization of a sample and Raman analysis with a microscopic laser
spot.
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Raman microscopy is easy: simply place the sample under the microscope, focus,
and make a measurement.
• Just adding a microscope to a Raman spectrometer does not give a controlled
sampling volume - for this a spatial filter is required. Confocal Raman microscopy
refers to the ability to spatially filter the analysis volume of the sample, in the XY
(lateral) and Z (depth) axes.
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http://www.horiba.com/us/en/scientific/products/raman-spectroscopy/raman-academy/raman-faqs/what-is-confocal-raman-microscopy/
http://www.horiba.com
Confocal Principle
The LabRAM HR Evaluation is an
integrated Raman system.
The
microscope is coupled to a 800 mm
focal length spectrograph equipped
with two switchable gratings.
Laser (λ nm): power (mW)
405 nm : 0.65 mW
532 nm : 10.5 mW
660 nm : 12.3 mW
785 nm: 35.5 mW
Raman Spectroscopy and Imaging
of Red Blood Cells
• Ansam J. Talib1, Sandra C. Bustamante1 , Zachary N. Liege 1,2 ,
Sarah Ritter1, Alexander Sinyukov1, Dmitri V. Voronine1,2,
Alexei V. Sokolov1,2, Kenith Meissner1 and Marlan O. Scully1,2,3
•
1Texas A&M
University, 2 Baylor University, 3Princeton University
Motivations
• Today, more than 340 million people suffer from diabetes and
this number doubles every 15 years.*
• In the USA, 30 million people have diabetes and 87 million
more have pre-diabetes.
• Common diabetic monitoring procedures include checking
blood sugar levels multiple times a day by finger pricks using
a glucose meter or an implantable glucose biosensor.**
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*Danani G, Finucane MM, et al. National, regional,and global trends in fasting plasma gluocose and diabetes prevalence since 1980:
systematic analysis of health examination surveys and epidemiological studies with 370 country- years and 2.7 million participants.
The Lancet 2011; 378:31-40.
**Vashist SK. Non-invasive glucose monitoring technologyin diabetes management: A review. Anal Chim Acta 2012;750:16-27.
http://www.medicalmalpracticeinquirer.com/assets_c/2011/
01/iStock_000004641088Large-thumb-300x200-6230.jpg
Goals of the project
Our long-term goal is to develop red blood cells (erythrocytes)
loaded with a fluorescent dye (erythrosensors), which can be
detected with a light source through the skin and can be used
as biocompatible glucose bio-sensors (because such cells will
stay in circulation instead of triggering an immune response,
and getting out of circulation).
The specific aim is to measure the difference between FITCghost glygly and normal red blood cells.
Red blood cells (RBCs)
RBCs (also called erythrocytes) are the
most common type of blood cells and
are the vertebrate organism's principal
means of delivering oxygen to body
tissues.
RBCs are the most abundant cells in
blood, with a shape of a biconcave disk
with a flattened center (look like
donuts).
RBCs contain a special protein called
hemoglobin, which helps carrying
oxygen.
http://bio662.dyndns.info/s3b/b3n/b3n02Blood
Circulation/b3n02eBldC112BloodCells.htm
Cell Membranes
 RBC membrane plays many roles that aid in regulating
their surface deformability, flexibility, adhesion to other cells
and immune recognition.
 They can be squeezed to 3 microns.
Membranes of RBC
100x
Fluorescein isothiocyanate (FITC)
Fluorescein (mistakenly abbreviated by its
commonly-used reactive isothiocyanate form,
FITC) is a small organic molecule, and is
typically conjugated to proteins via primary
amines. Usually, between 3 and 6 FITC
molecules are conjugated to each antibody
In cellular biology, FITC is often used to label and
track cells
Excitation: max = 495 nm
Emission: max = 525 nm
http://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Product_Information_Sheet/f7250pis.pdf
RBC
Membranes
of RBC
FITC ghost
Raman Spectrum of Hemoglobin
Laser 532 nm
Slit 200
Grating 600 gr/mm
Objective 50x
Optical image and Raman spectrum
of RBC
Objective 50x
Laser 532 nm
Slit 200
Grating 600 gr/mm
Objective 50x
Raman Images of RBC
Trp: tryptophan
Protein
C-N stretch
Bimolecular class: Unknown
Cytochrome- like moiety
(resonance enhanced)
Raman Spectrum of FITC Ghost Cells
Laser 532 nm
Slit 200
Grating 600 gr/mm
Objective 50x
Optical and Raman Images of FITC Ghost Cells
Laser 532 nm
Slit 200
Grating 600 gr/mm
Objective 50x
Intensity (counts)
120
100
500
140
1 000
Raman shift (cm-¹)
1578.63
1439.66
1345.98
1251.53
1131.94
920.98
953.83
996.10
665.65
736.37
RBC
Cursor spectrum
80
1 500
Conclusions
Confocal microscopy will allow us to investigate the vesicle size and shape of
RBCs and FITC ghost.
We can get a clear understanding for the distribution of the hemoglobin inside
the RBCs and the FITC-ghost.
TERS of RBCs