Instituci Catalana de Recerca

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Raúl Alcaide Hijano UPC, Barcelona
Anna Morales Drets UAB, Barcelona
Sergi Mercadal UPC, Barcelona
Asier Elejalde UPC, Barcelona
Dmitri Petrov ICFO Institut de Ciéncies
Fotòniques and ICREA Institució Catalana de
Recerca i Estudis Avançats Caitriona Creely (PhD
Ireland) PostDoc ICFO Gajendra Singh (Master
India) PhD ICFO Giovanni Volpe (Master
Italy) PhD ICFO
Nereida Rodriguez UPC, Barcelona
Florian Buttner, Student, Ruprecht-Karls
Universität, Heidelberg, Germany
Gregory Kozyreff, Visiting Scientist
Institució Catalana de Recerca i Estudis Avançats
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Motivation Raman micro-spectrometer combined
with optical tweezers Real time monitoring of
hyperosmotic stress response in a single
cell Lag phase and G1 phase of a single yeast
cell Raman images of a single living cell in
suspension multi-beams optical trap
Institució Catalana de Recerca i Estudis Avançats
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Motivation
Current biochemical assays give statistical
information about a whole population of cells,
however, we wished to be able to select and
study a single cell at a time. Living cells grow
in different environments some cells grow
attached to a surface, while others flourish in
suspension.
Cells move due to motility and Brownian motion
Cells should be in a suitable environment before
carrying out biochemical analysis. For this
reason we applied Raman spectroscopy- a highly
sensitive analytical tool- together with Optical
Tweezers for immobilization of a single cell in
suspension for the experiments and then obtaining
time-resolved information on biochemical changes.
Institució Catalana de Recerca i Estudis Avançats
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Raman Microscope and Optical Tweezers
Physics of optical trapping
An optical beam carries both the electromagnetic
energy and MECHANICAl MOMENTUM (and TORQUE)
FtotalFgradFscat
Institució Catalana de Recerca i Estudis Avançats
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Raman Microscope and Optical Tweezers
Olympus IX 51
TRITOR 100 Piezosystem Jena
FORTE 1064 Laser Quantum UK
Acton SpectraPro 2500i and Princeton Instruments
Spec-10 CCD (-100 C)
Notch filter TYDEX, J.S.Co. St. Petersburg
CrystaLaser LG, USA
Institució Catalana de Recerca i Estudis Avançats
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Raman Microscope and Optical Tweezers
Institució Catalana de Recerca i Estudis Avançats
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Raman spectra of a polysterene bead trapped
during one hour
beam excited Raman spectrum
Institució Catalana de Recerca i Estudis Avançats
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Real-time detection of hyperosmotic stress
response in a single Saccharomyces yeast cell

• the cell stress response is the reaction of a
  living cell to ambient changes which are
  potentially harmful
• for example, an increase in temperature, pH,
  saline concentration, the presence of toxins

• the Saccharomyces yeast cells are widely used as
  a model for fundamental cell processes as
• many fundamental cellular processes are conserved
  from yeast to human cells

• the basic reaction at the fermentation process is
  the reasponse of the Saccharomyces yeast cells
• on the hyperosmotic stress

ethanol C2H5OH
glucose
glycerol C3H8O3
Institució Catalana de Recerca i Estudis Avançats
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10 pure ethanol and glycerol in water
30 min after the experiment starts
Detection area 1
femtoL Detectable concentration 100
atto_mol Detectable number of molecules 108
difference spectrum of a single yeast cell under
the hyperosmotic pressure
Institució Catalana de Recerca i Estudis Avançats
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Lag phase and G1 phase of a single yeast cell
proteins and lipids
mRNA
During the lag phase the mRNA is translated to
synthesize the required proteins
Institució Catalana de Recerca i Estudis Avançats
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Lag phase and G1 phase of a single yeast cell
activity of mitochondrion
During the G1 phase the required
proteins continue to produce
Institució Catalana de Recerca i Estudis Avançats
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Institució Catalana de Recerca i Estudis Avançats
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Institució Catalana de Recerca i Estudis Avançats
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We modified a program to control a SLM
http//www.physics.gla.ac.uk/jonathan/slmcontrol/
adding a microscope image, back-and forth
movements of the multi-beams trap and a
spectrsoscopic software WINSPEC
An object for spectrscopic study
Beam for excitation Raman spectra
Institució Catalana de Recerca i Estudis Avançats
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Multi-beams optical trap
Budding yeast cell moved by
One beam
one beam
two beams
two beams
three beam
Institució Catalana de Recerca i Estudis Avançats
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Institució Catalana de Recerca i Estudis Avançats