Yeast glycolysis is one of the best studied metabolic pathways and is a particularly good model system to study oscillatory behaviour, due to the tendency of yeast populations to synchronise their oscillations1. To resolve the question whether isolated yeast cells can oscillate, we studied yeast in micro-fluidic cells, under conditions that prevent cell-cell communication (low cell density, high flow rate). Thus, we could separate oscillations from synchronisation, which is not possible in typical population studies where a population average is monitored (i.e. where only synchronised cultures can be studied). After characterising the yeast oscillations in isolated cells, it is now important to allow cell-cell communication in the system to study the synchronisation characteristics.
A setup, consisting of an optical tweezers system and microfluidic devices coupled with fluorescence imaging was designed to perform a time dependent observation during artificially induced glycolytic oscillations. Multi-channel flow devices and diffusion chambers were fabricated using soft lithography. Automatized pumps controlled specific flow rates of infused glucose and cyanide solutions, used to induce the oscillations. Flow and diffusion in the microfluidic devices were simulated to assure experimentally the desired coverage of the solutions across the yeast cells, a requirement for time dependent measurements.
Using near infrared optical tweezers, yeast cells were trapped and positioned in array configurations, ranging from a single cell to clusters of various symmetries, in order to obtain information about cell-cell communications during the metabolic cycles.
Single cell analysis techniques provide a unique opportunity of determining the intercellular heterogeneity in a cell population, which due to genotype variations and different physiological states of the cells i.e. size, shape and age, cannot be retrieved from averaged cell population values. In order to obtain high-value quantitative data from single-cell experiments it is important to have experimental platforms enabling high-throughput studies. Here, we present a microfluidic chip, which is capable of capturing individual cells in suspension inside separate traps. The device consists of three adjacent microchannels with separate inlets and outlets, laterally connected through the V-shaped traps. Vshaped traps, with openings smaller than the size of a single cell, are fabricated in the middle (main) channel perpendicular to the flow direction. Cells are guided into the wells by streamlines of the flows and are kept still at the bottom of the traps. Cells can then be exposed to extracellular stimuli either in the main or the side channels. Microchannels and traps of different sizes can be fabricated in polydimethylsiloxane (PDMS), offering the possibility of independent studies on cellular responses with different cell types and different extracellular environmental changes. We believe that this versatile high-throughput cell trapping approach will contribute to further development of the current knowledge and information acquired from single-cell studies and provide valuable statistical experimental data required for systems biology.
In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress.
Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast “High Osmolarity Glycerol” (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level.
The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.
Yeast glycolytic oscillations have been studied since the 1950s in cell free extracts and in intact cells. Until recently,
sustained oscillations have only been observed in intact cells at the population level. The aim of this study was to
investigate sustained glycolytic oscillations in single cells.
Optical tweezers were used to position yeast cells in arrays with variable cell density in the junction of a microfluidic
flow chamber. The microfluidic flow chambers were fabricated using soft lithography and the flow rates in the different
inlet channels were individually controlled by syringe pumps. Due to the low Reynolds number, the solutions mixed by
diffusion only. The environment in the junction of the chamber could thus be controlled by changing the flow rates in the
inlet channels, with a complete change of environment within 2 s. The optimum position of the cell array was determined
by simulations, to ensure complete coverage of the intended solution without any concentration gradients over the cell
array. Using a DAPI filter set, the NADH auto fluorescence could be monitored in up to 100 cells simultaneously.
Sustained oscillations were successfully induced in individual, isolated cells within specific flow rates and
concentrations of glucose and cyanide. By changing the flow rates without changing the surrounding solution, it was
found that the cell behavior was dependent on the concentration of chemicals in the medium rather than the flow rates in
the range tested. Furthermore, by packing cells tightly, cell-to-cell interaction and synchronization could be studied.
The use of phase only spatial light modulators for holographic optical trapping results in the appearance of ghost orders,
creating unwanted traps with uncontrolled intensity and causing variations in the intensity of the desired traps. By
introducing dummy areas in the diffraction plane during the hologram optimization, the intensity in the ghost orders can
be significantly reduced. By directing a variable fraction of the light to the dummy area, the optical power in the traps
can be controlled independently and kept constant also while moving traps to different arrangements. We present and
evaluate an algorithm for hologram generation which utilizes dummy areas and allows arbitrary spot positioning in three
dimensions. The method enables the use of holographic optical trapping for applications requiring precise control of the
intensity in traps, such as optical force measurement.
The precise cutting of axons in C. elegans using short laser pulses permits the investigation of parameters that may influence axonal regeneration. This study began by building and optimizing a femtosecond laser axotomy setup
that we first used to monitor the effect of cutting axons near or far from the cell body of the PLM mechanosensory
neurons in C. elegans. To assess regeneration, we developed a scoring system where the angle between the
regenerating trajectory and its direct line to the target is measured; we called this measurement the "angle of
regeneration". The results indicate that axons cut near the cell body regenerate better than those cut far from the
cell body but nearer their target. The role of teneurins, which are transmembrane proteins with a large extracellular
domain that are thought to regulate the remodelling of the extracellular matrix, has not yet been explored as a
potential contributor to axon regeneration. We cut PLM axons in wild-type or ten-1 mutant worms, and measured
the angle of regeneration 48 hours later, and the frequency of reconnection to the target. Our results show that
functional ten-1 contributes to successful axon regeneration.
We have implemented several algorithms for hologram generation, aimed for holographic optical tweezers
applications, using the parallel computing architecture CUDA. We compare required computation time for different
implementations of the Gerchberg-Saxton algorithm and provide guidelines for choosing the best suited version with
respect to the application. We also show that additional calculations, compensating for limitations in the used spatial
light modulator and optical system, can be included in the hologram generating software with little or no loss in computational speed.
Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements
of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have
been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to
depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular
location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the
depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in
human skin.
This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a
GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during
changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in
single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's
or Huntington's disease.
Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology
and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells,
large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge
efforts and could also include a bias, which complicates the use and comparison of data for further simulations or
modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of
CellStress.
CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell
boundaries using bright field images, and thus reduces the fluorescent labeling needed.
The positioning accuracy when a phase-only one dimensional spatial light modulator (SLM) is used for beam
steering is limited by the number of pixels and their quantized phase modulation. Optimizing the setting of the
SLM pixels individually can lead to the inaccuracy being a significant fraction of the diffraction limited spot size.
This anomalous behaviour was simulated numerically, and experiments showed the same phenomena with very
good agreement. However, by including an extra degree of freedom in the optimization of the SLM setting, we
show that the accuracy can be improved by a factor proportional to the number of pixels in the SLM.
Optical manipulation techniques have become an important research tool for single cell experiments in microbiology.
Using optical tweezers, single cells can be trapped and held during long experiments without risk of cross contamination
or compromising viability. However, it is often desirable to not only control the position of a cell, but also to control its
environment. We have developed a method that combines optical tweezers with a microfluidic device. The microfluidic
system is fabricated by soft lithography in which a constant flow is established by a syringe pump. In the microfluidic
system multiple laminar flows of different media are combined into a single channel, where the fluid streams couple
viscously. Adjacent media will mix only by diffusion, and consequently two different environments will be separated by
a mixing region a few tens of micrometers wide. Thus, by moving optically trapped cells from one medium to another
we are able to change the local environment of the cells in a fraction of a second. The time needed to establish a change
in environment depends on several factors such as the strength of the optical traps and the steepness of the concentration
gradient in the mixing region. By introducing dynamic holographic optical tweezers several cells can be trapped and
analyzed simultaneously, thus shortening data acquisition time. The power of this system is demonstrated on yeast
(Saccharomyces cerevisiae) subjected to osmotic stress, where the volume of the yeast cell and the spatial localization of
green fluorescent proteins (GFP) are monitored using fluorescence microscopy.
In recent years there has been a growing interest in the use of optical manipulation techniques, such as optical
tweezers, in biological research as the full potential of such applications are being realized. Biological research is
developing towards the study of single entities to reveal new behaviors that cannot be discovered with more
traditional ensemble techniques. To be able to study single cells we have developed a new method where a
combination of micro-fluidics and optical tweezers was used. Micro-fluidic channels were fabricated using soft
lithography. The channels consisted of a Y-shaped junction were two channels merged into one. By flowing
different media in the two channels in laminar flow we were able to create a sharp concentration gradient at the
junction. Single cells were trapped by the tweezers and the micro-fluidic system allowed fast environmental
changes to be made for the cell in a reversible manner. The time required to change the surroundings of the cell
was limited to how sharp mixing region the system could create, thus how far the cells had to be moved using
the optical tweezers. With this new technique cellular response in single cells upon fast environmental changes
could be investigated in real time. The cellular response was detected by monitoring variations in the cell by
following the localization of fluorescently tagged proteins within the cell.
We explore the Generalized Phase Contrast (GPC) approach for optical sorting in microfluidic systems. A microsystem is used in which two streams meet, interact and separate in an X-shaped channel. When the flow in the two arms of the X is balanced, the laminar flow that exists at very low Reynolds numbers ensures minimal stream blending and the fluid separates without mixing (i.e. diffusion is negligible). Optical forces due to an intensity pattern can be fashioned to induce a selective deflection of particles between the two streams. This method is known as optical fractionation (OF). In brief, OF uses the same mechanisms as optical tweezers to exert forces upon microscopic particles. OF has been shown to have an exponential size selectivity. This means that the interaction between the streams can be made to discriminate by particle size at a critical flow velocity. With correctly adjusted flow velocity, particles with a certain size will more often shift to the other stream than another particle size. One method for creating the light pattern is by interference of several beams that are variably attenuated using mechanical means. However, this approach offers low
optical efficiency and is not easily reconfigured. The GPC method offers a solution that gives the possibility to instantaneously reconfigure the intensity pattern by a method that is inherently computer-controllable. This enables one to rapidly test various intensity patterns to optimize sorting of particles.
The average environmental response of red blood cells (RBCs) is routinely measured in ensemble studies, but in such investigations valuable information on the single cell level is obscured. In order to elucidate this hidden information is is important to enable the selection of single cells with certain properties while subsequent dynamics triggered by environmental stimulation are recorded in real time. It is also desirable to manipulate and control the cells under phsyiological conditions. As shown here, this can be achieved by combining optical tweezers with a confocal Raman set-up equipped with a microfluidic system. A micro-Raman set-up is combined with an optical trap with separate optical paths, lasers and objectives, which enables the acquisition of resonance Raman profils of single RBCs. The microfluidic system, giving full control over the media surrounding the cell, consists of a pattern of channels and reservoirs produced by electron beam lithography and moulded in PDMS. Fresh Hepes buffer or buffer containing sodium dithionite are transported through the channels using electro-osmotic flow, while the direct Raman response of the single optically trapped RBC is registered in another reservoir in the middle of the channel. Thus, it is possible to monitor the oxygenation cycle in a single cell and to study photo-induced chemistry. This experimental set-up has high potential for monitoring the drug response or conformational changes caused by other environmental stimuli for many types of single functional cells since "in vivo" conditions can be created.
We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.
In this paper we will describe a system designed to combine optical tweezers and laser scalpels with confocal as well as epi-fluorescence microscopy. A continuos wave Nd:YVO4 laser is used to produce a dual optical tweezers, where each trap can be individually controlled. A second optical tweezers setup is based on a tunable titanium sapphire laser, which allows us to adjust the wavelength to minimize the damage to the cell under investigation. A pulsed nitrogen laser working at 337 nm forms a laser scalpel. The tweezers and scalpels are both incorporated in an inverted microscope equipped with epi-fluorescence and confocal imaging capabilities. In order to further control the sample we have developed a technique to tailor make the environment closest to the studied objects. Micrometer-sized structures such as channels and reservoirs have been produced in rubber silicon using lithographic methods. In combination with a micro-manipulator, our system can be used to extract single cells from a population of billions for further studies or growth.
It has recently been shown that the combination of Raman spectroscopy and optical tweezers constitute a powerful tool for biological studies. Raman spectra of single cells immobilized in a sterile surrounding can then be recorded without the risk of surface-induced morphological cell changes. Further, the complete cellular environment can be changed while measuring dynamics in real time. We here introduce a novel Raman tweezers set-up ideal for resonance Raman studies of single cells. The system differs from earlier set-ups in that two separate laser beams, used for trapping and Raman excitation, are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually, for example in order to optimize the functionality of the set-up or to record resonance Raman profiles from the same trapped cell. Further, the tweezers can be removed from the system without affecting the spectrometer configuration. Trapping is achieved by tightly focusing IR diode laser radiation (830 nm) through an inverted oil immersion objective with high numerical aperture (NA = 1.25), while Raman scattering is excited by the lines of an ArKr ion-laser. The backscattered Raman signal is collected by a single-grating spectrometer equipped with a microscope and a 60x water-immersion objective (NA = 0.9). The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using differen excitation lines (488, 514.5 568.2 nm) in resonance with the heme moiety and by studying the spectral evolution during illumination.
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