A Fontes, M L Barjas Castro, M M Brandão, H P Fernandes, A A Thomaz, R R Huruta, L Y Pozzo, L C Barbosa, F F Costa, S T O Saad and C L Cesar
Optical tweezers are a very sensitive tool, based on photon momentum transfer, for individual, cell by cell, manipulation and measurements, which can be applied to obtain important properties of erythrocytes for clinical and research purposes. Mechanical and electrical properties of erythrocytes are critical parameters for stored cells in transfusion centers, immunohematological tests performed in transfusional routines and in blood diseases. In this work, we showed methods, based on optical tweezers, to study red blood cells and applied them to measure apparent overall elasticity, apparent membrane viscosity, zeta potential, thickness of the double layer of electrical charges and adhesion in red blood cells.
Tuesday, March 8, 2011
Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions
Jenny M. Tam, Carlos E. Castro, Robert J. W. Heath, Michael L. Cardenas, Ramnik J. Xavier, Matthew J. Lang, Jatin M. Vyas
The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.
DOI
The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.
DOI
Raman spectroscopy of individual monocytes reveals that single-beam optical trapping of mononuclear cells occurs by their nucleus
Samantha Fore, James Chan, Douglas Taylor and Thomas Huser
We show that laser tweezers Raman spectroscopy of eukaryotic cells with a significantly larger diameter than the tight focus of a single-beam laser trap leads to optical trapping of the cell by its optically densest part, i.e. typically the cell's nucleus. Raman spectra of individual optically trapped monocytes are compared with location-specific Raman spectra of monocytes adhered to a substrate. When the cell's nucleus is stained with a fluorescent live cell stain, the Raman spectrum of the DNA-specific stain is observed only in the nucleus of individual monocytes. Optically trapped monocytes display the same behavior. We also show that the Raman spectra of individual monocytes exhibit the characteristic Raman signature of cells that have not yet fully differentiated and that individual primary monocytes can be distinguished from transformed monocytes based on their Raman spectra. This work provides further evidence that laser tweezers Raman spectroscopy of individual cells provides meaningful biochemical information in an entirely non-destructive fashion that permits discerning differences between cell types and cellular activity.
DOI
We show that laser tweezers Raman spectroscopy of eukaryotic cells with a significantly larger diameter than the tight focus of a single-beam laser trap leads to optical trapping of the cell by its optically densest part, i.e. typically the cell's nucleus. Raman spectra of individual optically trapped monocytes are compared with location-specific Raman spectra of monocytes adhered to a substrate. When the cell's nucleus is stained with a fluorescent live cell stain, the Raman spectrum of the DNA-specific stain is observed only in the nucleus of individual monocytes. Optically trapped monocytes display the same behavior. We also show that the Raman spectra of individual monocytes exhibit the characteristic Raman signature of cells that have not yet fully differentiated and that individual primary monocytes can be distinguished from transformed monocytes based on their Raman spectra. This work provides further evidence that laser tweezers Raman spectroscopy of individual cells provides meaningful biochemical information in an entirely non-destructive fashion that permits discerning differences between cell types and cellular activity.
DOI
Monday, March 7, 2011
Optically levitating dielectrics in the quantum regime: Theory and protocols
O. Romero-Isart, A. C. Pflanzer, M. L. Juan, R. Quidant, N. Kiesel, M. Aspelmeyer, and J. I. Cirac
We provide a general quantum theory to describe the coupling of light with the motion of a dielectric object inside a high-finesse optical cavity. In particular, we derive the total Hamiltonian of the system as well as a master equation describing the state of the center-of-mass mode of the dielectric and the cavity-field mode. In addition, a quantum theory of elasticity is used to study the coupling of the center-of-mass motion with internal vibrational excitations of the dielectric. This general theory is applied to the recent proposal of using an optically levitating nanodielectric as a cavity optomechanical system [see Romero-Isart et al., New J. Phys. 12, 033015 (2010); Chang et al., Proc. Natl. Acad. Sci. USA 107, 1005 (2010)]. On this basis, we also design a light-mechanics interface to prepare non-Gaussian states of the mechanical motion, such as quantum superpositions of Fock states. Finally, we introduce a direct mechanical tomography scheme to probe these genuine quantum states by time-of- flight experiments.
DOI
We provide a general quantum theory to describe the coupling of light with the motion of a dielectric object inside a high-finesse optical cavity. In particular, we derive the total Hamiltonian of the system as well as a master equation describing the state of the center-of-mass mode of the dielectric and the cavity-field mode. In addition, a quantum theory of elasticity is used to study the coupling of the center-of-mass motion with internal vibrational excitations of the dielectric. This general theory is applied to the recent proposal of using an optically levitating nanodielectric as a cavity optomechanical system [see Romero-Isart et al., New J. Phys. 12, 033015 (2010); Chang et al., Proc. Natl. Acad. Sci. USA 107, 1005 (2010)]. On this basis, we also design a light-mechanics interface to prepare non-Gaussian states of the mechanical motion, such as quantum superpositions of Fock states. Finally, we introduce a direct mechanical tomography scheme to probe these genuine quantum states by time-of- flight experiments.
DOI
Optical tweezers and non-ratiometric fluorescent-dye-based studies of respiration in sperm mitochondria
Timothy Chen, Linda Z Shi, Qingyuan Zhu, Charlie Chandsawangbhuwana and Michael W Berns
The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and a non-ratiometric fluorescent probe, DiOC6(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of 450 mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force from the laser tweezers were measured. Human (Homo sapiens), dog (Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated with DiOC6(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Sperm from all three species exhibited an increase in fluorescence when treated with the DiOC6(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all three species exhibited a reduction in fluorescence to pre-dye levels. With respect to VCL and escape force, the CCCP had no effect on dog or human sperm, suggesting a major reliance upon anaerobic respiration (glycolysis) for ATP in these two species. Based on the preliminary study on drill sperm, CCCP caused a drop in the VCL, suggesting potential reliance on both glycolysis and aerobic respiration for motility. The results demonstrate that optical trapping in combination with DiOC6(3) is an effective way to study sperm motility and energetics.
DOI
The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and a non-ratiometric fluorescent probe, DiOC6(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of 450 mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force from the laser tweezers were measured. Human (Homo sapiens), dog (Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated with DiOC6(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Sperm from all three species exhibited an increase in fluorescence when treated with the DiOC6(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all three species exhibited a reduction in fluorescence to pre-dye levels. With respect to VCL and escape force, the CCCP had no effect on dog or human sperm, suggesting a major reliance upon anaerobic respiration (glycolysis) for ATP in these two species. Based on the preliminary study on drill sperm, CCCP caused a drop in the VCL, suggesting potential reliance on both glycolysis and aerobic respiration for motility. The results demonstrate that optical trapping in combination with DiOC6(3) is an effective way to study sperm motility and energetics.
DOI
A Promiscuous DNA Packaging Machine from Bacteriophage T4
Zhihong Zhang, Vishal I. Kottadiel, Reza Vafabakhsh, Li Dai, Yann R. Chemla, Taekjip Ha, Venigalla B. Rao
Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%–25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.
DOI
Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%–25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.
DOI
Selective optical trapping based on strong plasmonic coupling between gold nanorods and slab
Y. J. Zheng, H. Liu, S. M. Wang, T. Li, J. X. Cao, L. Li, C. Zhu, Y. Wang, S. N. Zhu, and X. Zhang
A resonance plasmon mode is formed between a gold nanorod and an infinite slab in infrared range, with local electric field enhancement factor over 40. A strong optical attractive force is exerted on the rod by the slab at resonance frequency. Based on Maxwell stress tensor method and numerical simulations, the optical force was calculated to be over 2.0 nN/(mW/μm2). For a fixed incident wavelength, the enhanced optical force is obtained only for the rods with particular length when the diameter is fixed. This strong optical force could be used as a possible selective optical trapping technique in the future.
DOI
A resonance plasmon mode is formed between a gold nanorod and an infinite slab in infrared range, with local electric field enhancement factor over 40. A strong optical attractive force is exerted on the rod by the slab at resonance frequency. Based on Maxwell stress tensor method and numerical simulations, the optical force was calculated to be over 2.0 nN/(mW/μm2). For a fixed incident wavelength, the enhanced optical force is obtained only for the rods with particular length when the diameter is fixed. This strong optical force could be used as a possible selective optical trapping technique in the future.
DOI
Optical tweezers for studying taxis in parasites
A A de Thomaz, A Fontes, C V Stahl, L Y Pozzo, D C Ayres, D B Almeida, P M A Farias, B S Santos, J Santos-Mallet, S A O Gomes, S Giorgio, D Feder and C L Cesar
In this work we present a methodology to measure force strengths and directions of living parasites with an optical tweezers setup. These measurements were used to study the parasites chemotaxis in real time. We observed behavior and measured the force of: (i) Leishmania amazonensis in the presence of two glucose gradients; (ii) Trypanosoma cruzi in the vicinity of the digestive system walls, and (iii) Trypanosoma rangeli in the vicinity of salivary glands as a function of distance. Our results clearly show a chemotactic behavior in every case. This methodology can be used to study any type of taxis, such as chemotaxis, osmotaxis, thermotaxis, phototaxis, of any kind of living microorganisms. These studies can help us to understand the microorganism sensory systems and their response function to these gradients.
DOI
In this work we present a methodology to measure force strengths and directions of living parasites with an optical tweezers setup. These measurements were used to study the parasites chemotaxis in real time. We observed behavior and measured the force of: (i) Leishmania amazonensis in the presence of two glucose gradients; (ii) Trypanosoma cruzi in the vicinity of the digestive system walls, and (iii) Trypanosoma rangeli in the vicinity of salivary glands as a function of distance. Our results clearly show a chemotactic behavior in every case. This methodology can be used to study any type of taxis, such as chemotaxis, osmotaxis, thermotaxis, phototaxis, of any kind of living microorganisms. These studies can help us to understand the microorganism sensory systems and their response function to these gradients.
DOI
Microparticle movements in optical funnels and pods
José A. Rodrigo, Antonio M. Caravaca-Aguirre, Tatiana Alieva, Gabriel Cristóbal, and María L. Calvo
Three-dimensional microparticle movements induced by laser beams with a funnel- and tubular pod-like structure, in the neighbourhood of the focal plane of an optical trapping setup, are experimentally studied. The funnel and pod beams constructed as coherent superpositions of helical Laguerre-Gaussian modes are synthesized by a computer generated hologram using a phase-only spatial light modulator. Particle tracking is achieved by in-line holography method which allows an accurate position measurement. It is experimentally demonstrated that the trapped particle follows different trajectories depending on the orbital angular momentum density of the beam. In particular applying the proposed pod beam the particle rotates in opposite directions during its movement in the optical trap. Possible applications of these single-beam traps for volumetric optical particle manipulation are discussed.
DOI
Three-dimensional microparticle movements induced by laser beams with a funnel- and tubular pod-like structure, in the neighbourhood of the focal plane of an optical trapping setup, are experimentally studied. The funnel and pod beams constructed as coherent superpositions of helical Laguerre-Gaussian modes are synthesized by a computer generated hologram using a phase-only spatial light modulator. Particle tracking is achieved by in-line holography method which allows an accurate position measurement. It is experimentally demonstrated that the trapped particle follows different trajectories depending on the orbital angular momentum density of the beam. In particular applying the proposed pod beam the particle rotates in opposite directions during its movement in the optical trap. Possible applications of these single-beam traps for volumetric optical particle manipulation are discussed.
DOI
Friday, March 4, 2011
Laser tweezers Raman spectrum of normal breast cell line and carcinoma breast cell line
Xie, Y.-A., Leng, Z.-H., Meng, L.-J. , Luo, X.-L. , Zhang, W.-M. , Kuang, Z.-P.
OBJECTIVE: To detect the interaction between Raman spectrum of normal breast cell line (HBL-100) and carcinoma breast cell line (MCF-7), as well as the change of biological tissue composition from normal cell to carcinogenesis. In the end, Characteristic Raman spectra of cancer cells were presented. METHODS: Raman spectrum of normal breast cell line and carcinoma breast cell line were recorded, The result of which was performed by principal component analysis(PCA). RESULTS: Significant differences of average Raman spectrums were founded between the normal cell and the carcinoma one; The spectra line of carcinoma cell became strong at the whole; The strength of 936, 1002, 1298 and 1445 cm-1 increased; the peak of 1102 cm-1 shifted to 1094 cm-1; the peak of 484 cm-1 disappeared; Additionally, the structure and the amount of protein, nucleic acid, lipids and other such molecules had also changed. Average Raman spectrum of monocell was analyzed with the principal component of PCA, the result of which suggested normal cell and carcinoma cell could be differentiated with PCA, The discrimination rate was 87% (13/15). CONCLUSIONS: Judging normal breast cell and carcinoma cell with laser tweezers Raman spectrums is an efficient method. Single cells laser tweezers raman spectra technique can become a new way to diagnose cancer, which has very wide prospect.
DOI
OBJECTIVE: To detect the interaction between Raman spectrum of normal breast cell line (HBL-100) and carcinoma breast cell line (MCF-7), as well as the change of biological tissue composition from normal cell to carcinogenesis. In the end, Characteristic Raman spectra of cancer cells were presented. METHODS: Raman spectrum of normal breast cell line and carcinoma breast cell line were recorded, The result of which was performed by principal component analysis(PCA). RESULTS: Significant differences of average Raman spectrums were founded between the normal cell and the carcinoma one; The spectra line of carcinoma cell became strong at the whole; The strength of 936, 1002, 1298 and 1445 cm-1 increased; the peak of 1102 cm-1 shifted to 1094 cm-1; the peak of 484 cm-1 disappeared; Additionally, the structure and the amount of protein, nucleic acid, lipids and other such molecules had also changed. Average Raman spectrum of monocell was analyzed with the principal component of PCA, the result of which suggested normal cell and carcinoma cell could be differentiated with PCA, The discrimination rate was 87% (13/15). CONCLUSIONS: Judging normal breast cell and carcinoma cell with laser tweezers Raman spectrums is an efficient method. Single cells laser tweezers raman spectra technique can become a new way to diagnose cancer, which has very wide prospect.
DOI
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