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CHAPTER 3
RAMAN TWEEZERS-An Overview
Raman tweezers setup is a hybrid system which exploits the nonphysical
trapping capabilities of optical tweezers and analytical advantages of Raman
spectroscopy to study the biochemical changes in soft matter to single cell level
while keeping the cells under a physiological environment to mimics the in vivo
conditions. Raman tweezers is a technique capable of gathering Raman
fingerprints from live cells to facilitate identification, characterization and sorting
of cells/microorganisms and more importantly it can perform disease diagnosis to
single cell level. This chapter details the works done in various fields using Raman
tweezers as a tool and the advancements in the technique that project its potential.
D E S I G N A N D D E V E L O P M E N T O F O P T I C A L T W E E Z E R S I N
C O M B I N A T I O N W I T H R A M A N S P E C T R O S C O P Y
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3.1. INTRODUCTION
Even though tissue Raman spectroscopy has provided a good understanding about
the biochemistry of certain disorders [1-8], better information can be gathered from the
studies performed on cellular and sub-cellular level. It is not necessary that the tissue
under study has the cells of one type and/or in the same stage of growth. Depending on the
cell type and the growth phase they are in, the biochemistry of cells varies. When one
records the Raman spectra from a tissue, the derived signal is a combination of signals
from different cells illuminated by the laser focal spot. This result into the lowered spatial
resolution and such measurements will give limited information. This limitation has been
overcome by micro-Raman spectroscopy which uses microscope objectives to target
isolated single cells with a laser beam focused to sub-cellular dimensions. But, the
technique requires immobilization of cells which brings them in direct contact with
surface of the container. The physical or chemical methods of immobilization are
commonly followed in micro-Raman measurements. Even though, the cells are maintained
in appropriate media, micro-Raman spectroscopy measurements confirm that the cells are
settled at the bottom of the container. However, in many cases the physical stress derived
from the adhesion of cells to the surface or the chemical stress because of the change in
chemical environment around the cells may induce undesirable, non-specific perturbations
in the structure of biomolecules inside the living cells. Since, the cells are of micron size,
even slight change in the surrounding environment makes drastic changes in cell
organelles/cytoplasmic content. If one has to perform Raman spectroscopy measurements
on the cells while retaining their natural environment, then it is crucial to design the
favorable experimental procedures.
Relatively recent development of optical tweezers, an optical way of immobilizing
the cells has made a breakthrough in the single cell studies. Raman tweezers is a technique
which combines the nonphysical trapping capabilities of optical tweezers with Raman
spectroscopy to understand the biochemistry of single cell, sub-cellular organs and
microorganisms levitated in a physiological medium. In other words, Raman tweezers
spectroscopy is a special type of micro-Raman spectroscopy technique where the
biological sample under study is maintained closer to the in vivo conditions during the
course of experiment. Conventional micro-Raman spectroscopy also confines the studies
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to cellular and/or sub-cellular levels, but limited by a major drawback that the sample
under study is not necessarily in its normal/live state. In conventional technique the
cells/microorganisms are fixed either mechanically or chemically on to the glass surface
which most of the times brings the cells under mechanical/chemical stress. Hence we end
up recording the spectrum of a cell which is away from its physiological environment and
the biochemical information we gather might be far away from reality.
Even though optical tweezers, in its early days of growth, was utilized to perform
Raman spectroscopy of optically trapped single particles/droplets in 1990s [9-14],
Changan Xie and co-workers [15] in 2002, extended the application of Raman tweezers
spectroscopy in biological studies. They exploited the technique to record the Raman
spectra of blood cells and yeast cells optically levitated in saline solution and a yeast
solution respectively. The paper also reports the spectroscopic difference between live and
dead yeast cells; where the cell death was affected by heating. Near-infrared (NIR)
wavelength at 785 nm was used for both trapping and Raman excitation to overcome the
possible photodamage. The calibration and alignment of the laser tweezers Raman
spectroscopy (LTRS) system was performed by recording the Raman spectra of a
polystyrene bead of ~2 µm diameter suspended in water. One month before to Xie’s work
in 2002, K. Ajito and K. Torimitsu [16] reported LTRS studies on synaptosomes, the
nerve-ending particles (about 500–700 nm in diameter) isolated from a neuron of rat brain,
dispersed in the phosphate buffer solution (PBS). The study revealed the content of
synaptosomes through the Raman peaks at 1445 cm-1
and 1657 cm-1
corresponding to CH2
deformation mode in lipids and the amide I mode in proteins respectively.
In 2003 Xie’s group reported the real-time Raman spectroscopic measurements
performed during a heat induced protein denaturation process in yeast cell and bacteria
when the temperature of the surrounding medium is increased [17]. In this study the
protein denaturation was spectroscopically monitored with the temperature dependent
change in intensity of the phenylalanine peak at 1004 cm-1
. The findings in this study were
cross confirmed by recording Raman spectra of native and heat-denatured solutions of
bovine serum albumin and pure phenylalanine. A similar change in the 1004 cm-1
peak is
observed in BSA spectra, whereas, no change is observed in phenylalanine spectra.
Considering the weak nature of Raman scattering, Alexander et al [18] recorded the
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surface enhanced Raman spectra of single optically trapped bacterial spores. SERS
enhancement was realized by keeping the bacterial spores in vicinity of the SERS
substrate prepared by conjugating the gold colloids (60 nm) on the glass surface.
3.2. RAMAN SPECTROSCOPY OF CELL ORGANELLES
Spectroscopic studies of living cellular organelles provide information on the
biomolecular dynamics during cellular functions. Raman tweezers is a promising tool to
study the biochemistry of functional organelles in a living cell. Moreover the low
micrometer focal spots can target cellular organelles precisely and provide high spatial
resolution. Xie et al presented the Raman spectra of nucleus in an optically trapped pine
cell. H. Tang and co-worker [19] could record the Raman signatures from an intact
mitochondria isolated from heart, kidney and liver tissues of a rat and on comparison, the
mitochondria from 3 tissue types showed variation in their lipid concentrations. They also
examined the differences between the Raman spectra of intact and Ca2+
damaged
mitochondria and found that most of the Raman peaks from phospholipids and proteins
disappeared after 60 minutes of exposure to Ca2+
solution.
In another study, Huang et al [20] recorded the space-resolved Raman spectra of
living S. Pombe (yeast) cells at different stages of cell cycle, with an intention to elucidate
the molecular compositions of organelles, including nuclei, cytoplasm, mitochondria, and
septa. The observed spatial changes in the Raman signature were correlated to the
organelle specific chemical transformation inside the cell during different stages of cell
cycle. In another study, Ojeda et al [21] recorded the Raman spectroscopy signatures of an
optically trapped chromosome and with the help of generalized discriminate analysis
(GDA) of the Raman spectra, they could distinguish between chromosome #1, #2, and #3.
The chromosome numbering was identified by G-banding. Tatischeff et al [22]
characterized the cell-derived extracellular vesicles (EVs) of Dictyostelium discoideum, an
amoeba and human urinary exosomes using Raman tweezers spectroscopy. Cells release
diverse types of membrane vesicles of endosomal and plasma membrane origin called
extracellular vesicles (EVs) [23]. EVs play an important role in intercellular
communication by serving as transport vehicles for cytosolic proteins, lipids, and RNA
between the cells.
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3.3. RAMAN SPECTROSCOPY FOR CELL GROWTH MONITORING
Raman tweezers has also been applied to study the growth related biochemical
changes inside the cells. The micro-chemical environment inside the cell experiences a
continuous change during the different phases of cell growth. Better understanding of the
biochemical processes involved in cell growth will help cell biologists to design effective
methodologies for controlling and manipulating the cell cycles. Many Raman tweezers
spectroscopy based reports have been published in this regard [24-31]. G. P. Singh et al
[24] recorded the real-time Raman spectra of optically trapped single, live yeast cell in G0
phase of its cell growth and G1 phase of its cell cycle. Raman spectroscopic monitoring
witnessed the changes in relative intensity of peaks corresponding to lipids, proteins and
RNA. This increase/decrease in peak intensities was correlated to the increase/decrease in
the concentration of respective biomolecules in the G0 phase and late G1 phase.
Z. Tao et al [25] made a remarkable study on Rhodotorula glutinis (R. glutinis), a
pigmented yeast species, using Raman tweezers by monitoring the change in intensity of
Raman peaks of carotenoids along with those of nucleic acids and lipids. Rhodotorula
glutinis is a pigmented yeast species which produces large amount of carotenoids and has
industrial applications. The carotenoid accumulation in R. glutinis is known to depend on
the culture conditions and stage of the cell growth. Tao’s data showed that the carotenoid
production is more in the late exponential and stationary phases of cell growth represented
by the increase in lipid concentration with concomitant decrease in DNA/RNA
concentration. Biochemical synthesis in glucose stimulated rat β-cells was monitored
using Raman tweezers spectroscopy by X. Rong and coworkers [26]. The data in this
study suggested a time dependent increase in intensity of Raman peaks corresponding to
proteins and lipids as a representative of the synthesis of those biomolecules in glucose
stimulated cells.
H. Wu et al [27] demonstrated the possibility of using Raman tweezers for real-
time, in vivo lipidomics in oil-producing microalgae, the future source of bio-fuels. He
addressed the problem of extracting the lipids from microalgae to check the production
efficiency for various culture conditions and at different phases of cell growth. Raman
tweezers can replace the time consuming chemical techniques to provide relatively fast
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monitoring of lipid concentration in microalgae. In an another report, Yan Li and group
[28] studied the growth dynamics for over 40 min and heterogeneity in two interacting
microbial cells using dual trap Raman tweezers setup which can simultaneously record the
spectra from both the cells. Biochemical changes related to the growth dynamics of a
daughter and parent cells in a budding yeast cell have been monitored with time and
heterogeneity in the cell content was presented.
Heterogeneity in the release of CaDPA during the germination of two Bacillus
spores derived from identical microenvironment was also monitored. P. Zhang et al [29]
also studied the dynamics and heterogeneity during the germination of individual bacterial
spores using Raman tweezers and quantitative differential interference contrast
microscopy (DICM). Another study lead by L. Kong et al [30] combined phase contrast
microscopy with Raman tweezers to monitor a variety of changes that occurred during the
germination of single Bacillus spores in both nutrient (l-alanine) and non-nutrient (Ca-
dipicolinic acid) germinants with a temporal resolution of 2s. A study by Zhou et al [31],
combining Raman tweezers and DICM showed that the kinetics of germination in
individual G. stearothermophilus spores are generally similar to that of Bacillus species.
The study could find Raman markers for germination of G. Stearothermophilus spores at
different activation temperatures. A recent study by same group extended the use of
Raman tweezers and DICM to compare the kinetics of germination in Clostridium difficile
spores with that in Bacillus subtilis [32]. Clostridium difficile is a leading cause of
nosocomial diarrhea and germination of Clostridium difficile spores by the bile salts of
gastrointestinal tract initiate the infection.
3.4. SPECTROSCOPIC MONITORING OF THE CELL UNDER STRESS
Biological cells experience many types of stress induced by the extracellular and
intracellular environment [33]. Some aspects which can induce stress in cells include the
hyperosmotic pressure [34, 35], chemical stress because of reactive oxygen species [33],
mechanical stress because of cell packing and cell movement in constricted blood veins
[36]; stress induced by temperature and pH changes [37, 38] etc. Under these stress
conditions, cells will experience drastic changes in structures and conformations of
various intracellular biomolecules. These biochemical changes can represent either the
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surrender of cells to the stress or cells’ protective response to the stress for their survival.
Careful monitoring of the biochemical changes can be helpful to address stress related
disorders. Application of Raman tweezers left no biological field untouched because of its
high specificity and versatility [39]. Again Raman tweezers wins the race over other
techniques to monitor cells’ stress response because it can extract in situ biochemical
fingerprint from a single cell.
G.P Singh et al [40] have performed Raman tweezers study on hyperosmotic stress
condition by taking yeast cell as a model. In this report they induced hyperosmotic stress
on yeast cells by diluting them in 10% glucose solution made up in synthetic defined
complete (SDC) media. They observed an enhanced production of glycerol and ethanol in
the yeast cells that are subjected to hyperosmotic stress conditions. The effect of
hyperosmotic alcohol solution on single human red blood cells (RBCs) was studied by J.
L. Deng et al [41]. Real-time monitoring of Raman fingerprints from a trapped RBC in
20% alcohol solution showed a time dependent decrease in intensity of 752 cm-1
peak
arriving from porphyrin breathing mode. The rate of decrease further increased with
increase in concentration of alcohol solution and RBCs got ruptured (lysed) after being 3
minutes in 30% alcohol solution.
Raman tweezers, mostly in its dual or multiple trap mode has been successively
employed to study the in situ biochemical response of a cell to the mechanically induced
stress [42-46]. Most of these studies used erythrocytes/red blood cells (RBCs) as model
cell, because the mechanical rigidity of RBCs leads to the oxygen transport related
complications in many diseases including malaria, spherocytosis, elliptocytosis, sickle cell
anemia etc. Erythrocytes adopt different squeezing/stretching states during their passage
through tiny blood capillaries to deliver oxygen and hence the study of mechanochemical
processes associated with deformation of RBCs is necessary. The results of two
independent studies conducted by Satish Rao et al [43] and G. Rusciano [44] showed that
the mechanical stretching of RBCs induces hemoglobin transition from oxygenated state
to the deoxygenated state. Whereas, the Raman tweezers experiments performed by S. Raj
et al [45, 46] on the RBCs under varying percentage of mechanical stretching showed a
significant, load dependent, increase in the intensity of 1035 cm-1
peak corresponding to
in-plane CH2 asymmetric deformation and/or phenylalanine along with changes in some
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other peaks. These studies postulated the possible role of membrane proteins in controlling
the RBC shape change by releasing the spectrin protein [45, 46].
Many biological systems experience oxidative stress and have their own
antioxidative system to suppress its adverse effects. Oxidative stress pathway is through
the production of intermediate reaction species like peroxides, free radicals etc collectively
called reactive oxygen species (ROS) which damage the structure and properties of cell
components [47, 48]. Either the elevated level of these species inside the cell or failure of
its antioxidant system leads to the permanent cell damage. Apart from their negative role,
reactive oxygen species play a pivotal role in immune system to attack and kill the
pathogens [49] . There are techniques to probe the effect of oxidative stress on cells and/or
the protective response of cells to it, but most of these techniques will be employed on a
large population of cells and lack in situ measurements. Raman tweezers can focus its
attention on single, live cell and has capabilities to depict the stress dependent biochemical
reactions [48, 50].
3.5. IDENTIFICATION, CHARACTERISATION AND SORTING OF
MICROBES
The advancements in socially vital fields like clinical diagnosis, pharmaceutical
industry, food and water processing industries etc demand a rapid tool for detection,
identification and characterization of both good and bad microorganisms. In this aspect
Raman tweezers will play a crucial role as it can give very specific and in situ information
about different species of microorganisms in comparatively less time. Rapid microbial
identification is more necessary in medicine for fast diagnosis and early medical
intervention to various infections. Researchers have already initialized Raman tweezers
based fundamental studies in this direction [51-56]. The studies conducted by C. Xie and
coworkers [52] demonstrated the identification of optically trapped bacterial spores based
on their Raman signatures and also achieved the optical sorting of the spores by optically
manipulating them through the micro-channel into a clean collection chamber. In another
study, W. E. Huang et al [55] identified and sorted two strains of bacteria (P. Fluorescens
and E.coli ) on the basis of 13
C uptake affected red shift of phenylalanine peak from 1001
cm-1
to 965 cm-1
in P. Fluorescens Raman spectra. To demonstrate the specificity of
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Raman tweezers, optically separated bacteria were later utilized for single strain
cultivation and single cell genome amplification. The study implies that the Raman
tweezers approach has potential application in picking and studying the nonculturable
microorganisms that make 99% of all microbes present in the environment. Recently, Pilát
et al [57] added their contribution to this field by developing and demonstrating an
analytical sorting system combining optical trapping and Raman spectroscopy which
works in micro-fluidic environment to identify and sort living cells of various prokaryotic
and eukaryotic origins. A report by Huang et al [58] have projected the potential
applications of Raman-activated cell sorting (RACS) in single cell biotechnology with
emphases on development of bench top, integrated Raman tweezers systems capable of
extracting DNA/RNA from single cell for sequencing.
The gene expression in living organisms is accompanied by a lot of biochemical
changes in intracellular environment. Raman tweezers spectroscopy was applied to
monitor the dynamics of gene expression and protein synthesis in live E. Coli bacterial
cells by triggering the over expression of Myelin oligodendrocyte glycoprotein [MOG (1-
120)] on exposure to isopropyl thiogalactoside (IPTG) [59]. The study observed a time-
dependent increase in the intensity of characteristic Raman peaks of vibrations in proteins.
The in vivo, real-time uptake and metabolism of trehalose, a disaccharide having two α-
glucose units by Sinorhizobium meliloti bacteria is quantified by holding the live
bacterium in place with an optical trap and recording the Raman spectra at various time
intervals after supplementing the medium with trehalose [60]. In general, this approach
gives real-time chemical information and nullifies the toxic effects of isotopic probes
involved in cellular uptake studies.
On the other hand, Raman tweezers have been used to determine the spectral
changes associated with biochemical response of bacteria to the antibiotics [61, 62]. In a
recent study by Bernatová et al [63, 64], the influence of selected bacteriostatic and
bactericidal antibiotic agents on Staphylococcus epidermidis bacteria is monitored using
Raman tweezers. They observed little change in the Raman signals of DNA in the spectra
of Staphylococcus epidermidis treated with a bacteriostatic agent whereas the action of
bactericidal agent decreased the signal strength from DNA drastically; suggesting DNA
fragmentation as one of the pathways of bactericidal action. The study also extended to
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demonstrate the ability of Raman tweezers to distinguish between biofilm-positive and
biofilm-negative strains of Staphylococcus epidermidis with the help of principal
component analysis (PCA) [65, 66].
3.6. IDENTIFICATION AND CHARACTERISATION OF DISEASES
Single cell Raman spectroscopy studies will provide better insight into biochemical
modifications due to various diseases and thereby provide adequate, useful resources for
drug design. Even the action of drugs to repair the disease induced biochemical damage
can be well understood from the Raman spectroscopy studies on drug treated cells
maintained under in vivo conditions. Lots of attempts have been made to identify and
characterize the Raman shift markers for various diseases including cancer and mode of
action of drugs on these diseases. In some early attempts, K. E. Hamden et al applied
Raman tweezers to characterize Kaposi sarcoma (KS), a tumor caused by human
herpesvirus-8 (HHV8) [67] and the study revealed a significant variations in intensity of
some peaks between the Raman spectra of normal and infected cells. In another interesting
study on Kaposi’s sarcoma, the Raman fingerprint of cells supporting Kaposi’s sarcoma-
associated herpesvirus (KSHV) reactivation has been reported by recording the Raman
spectra from trapped normal and KSHV infected cells [68].
Studies carried out by K. Chen et al [69] and F. Zheng et al [70] applied the
technique for diagnosis of colorectal cancer. The former study used PCA as a
discrimination tool, whereas the later study used artificial neural networking for the
classification of spectral features from cancerous and noncancerous cells. J. W. Chan and
co-workers have reported the studies on Raman tweezers spectroscopy of normal and
leukemic cells [71-73] . In their first study [71] they compared the Raman spectral features
of leukemic cells derived from transformed Raji (B) and Jurkat (T) cell lines with those of
normal cells derived from blood of healthy volunteers. The second report from Chan et al
[72] reported the Raman spectroscopic analysis of the hematopoietic cells obtained from
four healthy individuals and three leukemia patients. The third study focuses on the
advantages of Raman spectroscopy of optically trapped cells over that of chemically fixed
cells and the possible anomalies in the spectroscopic discrimination of para-formaldehyde
and methanol fixed normal and leukemia cells. Raman spectroscopic identification of
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different healthy and unhealthy single cells in a suspension prepared using peripheral
blood of leukemia patients have been achieved [74]. A. Y. Lau et al [75] integrated
multichannel micro-fluidics with Raman tweezers for identification, and simultaneous
sorting of individual cells from two leukemia cell lines based on their Raman shift
markers. Raman Tweezers literature also quotes the identification and characterization of
carcinoma of prostate [76, 77], bladder [76], brain [78], pharynx [79], and liver [80].
Probing the response of cancer cells to various treatments including chemotherapy,
radiotherapy and drugs is also necessary. These studies provide the information which
plays a vital role in deciding the dose, efficiency of the treatment, post treatment effects
and counter actions. The induced apoptosis in gastric carcinoma cells by 5-FU (5-
fluorouracil), a drug that is known to induce the apoptosis of cancer cells has been
analyzed using Raman tweezers [81]. The observations of the study suggested a significant
decrease in the Raman peak intensity of cellular lipids, proteins and nucleic acids owing to
the cell death. Tobias J. Moritz et al [82] performed the Raman spectroscopic analyses of
leukemic T cells exposed to the chemotherapy drug doxorubicin at different time points
over 72 hours. They reported the increase in intensities of lipid and DNA Raman peaks
with drug exposure time and concentration. A recent study in this category is reported by
Liu et al [83], which focuses on Raman spectroscopic probing of oxygenation response of
optically trapped single, RBCs derived from normal adult, sickle cell anaemia, and cord
blood to an applied mechanical force. A force dependent increase in the deoxygenation is
observed in all the three cell types; with sickle RBCs showing more deoxygenation,
normal RBCs showing less deoxygenation and cord blood RBCs showing least
deoxygenation for the same optical forces.
3.7. ADVANCES IN RAMAN TWEEZERS
Researchers are in verge of developing many complimentary techniques to
increase the capabilities of Raman tweezers. One of such approaches is the development
of computer algorithms to control the positioning of stage and acquisition of Raman
spectra in holographic Raman tweezers by hand tracking, gestures recognition, eye
tracking and speech recognition [84, 85]. The other report is on developing a lab-on-chip
optical trapping and Raman spectroscopy setup using micro-fabricated dual waveguides
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[86]. In this chip, a loop shaped waveguide in which the trapping and Raman excitation
laser is coupled bisects a microfluidic channel where both the waveguide and channel are
fabricated monolithically on a SiO2 substrate. The light coming from two arms of the
waveguide traps and Raman excites the micro-particles flowing through the channel. The
scattered Raman signals in the transverse direction were collected using a microscope
objective and fed into a spectrograph. Another study reports the Raman spectroscopic
characterization and identification of micro-particles that are trapped and propelled along
the optical waveguides due to radiation pressure generated by evanescent field that is built
above the waveguide surface [87]. Lihui Ren and co-workers [88] designed an automatic
system for Raman-based single-cell phenotyping and demonstrated its use in
discriminating between Saccharomyces cerevisiae and BY4743 Streptococcus sanguinis
both in tube and slide environment. The automation software framework includes
algorithms for instrument control, image analysis, Raman profiling, database update and
database search. In an interesting study, Wheaton et al [89] have used double-nanohole
optical tweezers to record the extraordinary acoustic Raman (EAR) spectra of trapped
polystyrene nanosphere, titania nanosphere and globular proteins (carbonic anhydrase and
conalbumin). Double-nanohole optical tweezers has also been used to record the Raman
vibrational spectra of a single MS2 bacteriophage [90]. In another report, Yuan and co-
workers have claimed the designing of an integrated microscopy system that has
capabilities to perform Raman tweezers spectroscopy measurements on a single cell along
with the ability to capture the real-time images from trapped cell in both reflectance
confocal microscopy (RCM) mode and two photon fluorescence (TPF) mode [91].
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