Laser interference‐based technique for dynamic measurement of single cell deformation manipulated by optical tweezers

A laser interference‐based method was proposed to measure the deformation response of cell manipulated by optical tweezers. This method was implemented experimentally by integrating a laser illuminating system and optical tweezers with an inverted microscope. Interference fringes generated by the transmitted and reflected lights were recorded by a complementary metal oxide semiconductor camera. From the acquired images, cell height was calculated and cell morphology was constructed. To further validate this method, the morphological analyses of HeLa cells were performed in static state and during detachment process. Subsequently, the dynamic deformation responses of red blood cells were measured during manipulation with optical tweezers. Collectively, this laser interference‐based method precludes the requirement of complex optical alignment, allows easy integration with optical tweezers, and enables dynamic measurement of cell deformation response by using a conventional inverted microscope.     

Yoctoliter Thermometry for Single‐Molecule Investigations: A Generic Bead‐on‐a‐Tip Temperature‐Control Module

A new temperature‐jump (T‐jump) strategy avoids photo‐damage of individual molecules by focusing a low‐intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T‐jump of 65 °C within milliseconds. To measure the temperature in situ in single‐molecule experiments, the temperature‐dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead‐on‐a‐tip module and the robust single‐molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T‐jump and single‐molecule techniques.     

Plasmonic Trapping with a Gold Nanopillar

An improved ability to manipulate nanoscale objects could spur the field of nanotechnology. Optical tweezers offer the compelling advantage that manipulation is performed in a non‐invasive manner. However, traditional optical tweezers based on laser beams focused with microscope lenses face limitations due to the diffraction limit, which states that conventional lenses can focus light to spots no smaller than roughly half the wavelength. This has motivated recent work on optical trapping based on the sub‐wavelength field distributions of surface plasmon nanostructures. This approach offers the benefits of higher precision and resolution, and the possibility of large‐scale parallelization. Herein, we discuss the fundamentals of optical manipulation using surface plasmon resonance structures. We describe two important issues in plasmonic trapping: optical design and thermal management strategies. Finally, we describe a surface plasmon nanostructure, consisting of a gold nanopillar that takes these issues into consideration. It is shown to enable the trapping and rotation (manual and passive) of nanoparticles. Methods by which this concept can be extended are discussed.     

Optical tweezers for medical diagnostics

Laser trapping by optical tweezers makes possible the spectroscopic analysis of single cells. Use of optical tweezers in conjunction with Raman spectroscopy has allowed cells to be identified as either healthy or cancerous. This combined technique is known as laser tweezers Raman spectroscopy (LTRS), or Raman tweezers. The Raman spectra of cells are complex, since the technique probes nucleic acids, proteins, and lipids; but statistical analysis of these spectra makes possible differentiation of different classes of cells. In this article the recent development of LTRS is described along with two illustrative examples for potential application in cancer diagnostics. Techniques to expand the uses of LTRS and to improve the speed of LTRS are also suggested.     

Preparation of polymer optical fibers doped with nonlinear optical active organic chromophores

We synthesized two novel organic nonlinear optical chromophores—chiral S(+)‐N‐[p‐(4‐nitrostyryl) phenyl] prolinol and non‐chiral [p‐(4‐nitrostyryl) phenyl] piperdine—as potential laser‐active dyes for photonic applications. Both materials show good optical transmittance in the telecommunication frequency region, desirable solubility in acrylic polymer optical fiber matrices, and attractive fluorescence properties that are advantageous for laser‐gain materials and devices. Subsequently, these two chromophores were incorporated into poly(methyl methacrylate) and poly(ethyl methacrylate) and drawn into polymer optical fibers. The relevant properties of these organic dye‐doped fibers have been studied, revealing essential attributes of laser‐active characteristics. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 1794–1801, 2001     

开拓生命科学的海洋生理活性物质

本文介绍了几种典型的海洋生物生理活性物质——河豚毒素,西加毒素,石房蛤毒素,刺尾毒素的研究。这些生理活性物质不仅能特异地支配着离子通道,而且支配着受体和细胞机能的调节。今天,获得飞跃发展的生命科学领域,用分子和亚分子水平解释复杂的生命现象,海洋生理活性物质正是强有力的工具。因此,海洋生物的生理活性物质对生命科学方面的贡献是不可估量的。     

A Rotational BODIPY Nucleotide: An Environment‐Sensitive Fluorescence‐Lifetime Probe for DNA Interactions and Applications in Live‐Cell Microscopy

Fluorescent probes for detecting the physical properties of cellular structures have become valuable tools in life sciences. The fluorescence lifetime of molecular rotors can be used to report on variations in local molecular packing or viscosity. We used a nucleoside linked to a meso‐substituted BODIPY fluorescent molecular rotor ( dC^bdp ) to sense changes in DNA microenvironment both in vitro and in living cells. DNA incorporating dC^bdp can respond to interactions with DNA‐binding proteins and lipids by changes in the fluorescence lifetimes in the range 0.5–2.2 ns. We can directly visualize changes in the local environment of exogenous DNA during transfection of living cells. Relatively long fluorescence lifetimes and extensive contrast for detecting changes in the microenvironment together with good photostability and versatility for DNA synthesis make this probe suitable for analysis of DNA‐associated processes, cellular structures, and also DNA‐based nanomaterials.     

Optical tweezers applied to a microfluidic system

We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.     

Interactions,Structure, and Microscopic Response: Complex Fluid Rheology Using Laser Tweezers

Optical trapping techniques are emerging as significant research tools in complex fluids, offering the ability to probe nano‐ and microscopic interactions, structures, and responses that govern the rheology of complex fluids. In combination with real‐space imaging, microstructural response of these fluids can be directly and quantitatively correlated to imposed microscopic stresses and strains. Thus, laser tweezers are enabling us to bridge multiple length scales in colloid and polymer rheology and should be highly useful for investigating the mechanisms of linear and nonlinear rheology. In this article, we briefly review the theory and practice of using optical traps in complex fluids. We discuss the characteristics of the gradient force trap, practical concerns in trapping experiments, and applications, including measurements of micromechanics and microrheology in colloid and polymer gels.     

Non‐covalent Versus Covalent Control of Self‐Assembly and Chirality of Nile Red‐modified Nucleoside and DNA

A DNA‐based covalent versus a non‐covalent approach is demonstrated to control the optical, chirooptical and higher order structures of Nile red ( Nr ) aggregation. Dynamic light scattering and TEM studies revealed that in aqueous media Nr ‐modified 2′‐deoxyuridine aggregates through the co‐operative effect of various non‐covalent interactions including the hydrogen bonding ability of the nucleoside and sugar moieties and the π‐stacking tendency of the highly hydrophobic dye. This results in the formation of optically active nanovesicles. A left‐handed helically twisted H‐type packing of the dye is observed in the bilayer of the vesicle as evidenced from the optical and chirooptical studies. On the other hand, a left‐handed helically twisted J‐type packing in vesicles was obtained from a non‐polar solvent (toluene). Even though the primary stacking interaction of the dye aggregates transformed from H→J while going from aqueous to non‐polar media, the induced supramolecular chirality of the aggregates remained the same (left‐handed). Circular dichroism studies of DNA that contained several synthetically incorporated and covalently attached Nr ‐modified nucleosides revealed the formation of helically stacked H‐aggregates of Nr but—in comparison to the noncovalent aggregates—an inversed chirality (right‐handed). This self‐assembly propensity difference can, in principle, be applied to other hydrophobic dyes and chromophores and thus open a DNA‐based approach to modulate the primary stacking interactions and supramolecular chirality of dye aggregates.     

Optically-actuated translational and rotational motion at the microscale for microfluidic manipulation and characterization

The single beam optical trap (optical tweezers), a highly focused beam, is on its way to revolutionizing not only the fields of colloidal physics and biology, but also materials science and engineering. Recently, spatially-extended three-dimensional light patterns have gained considerable usage for exerting force to alter, manipulate, organize and characterize materials. To advance the degree of manipulation, such as rotation of materials in microfluidic environments along with spatial structuring, other beam parameters such as phase and polarization have to be configured. These advances in optical tweezers' technology have enabled complex microfluidic actuation and sorting. In addition to remotely (in a non-contact way) applying force and torques in three-dimensions, which can be continuously varied unlike mechanical manipulators, optical tweezers-based methods can be used for sensing the force of interaction between microscopic objects in a microfluidic environment and for the characterization of micro-rheological properties. In this review, we place emphasis on applications of optical actuation based on novel beams in performing special functions such as rotation, transportation, sorting and characterization of the microscopic objects. Further, we have an extended discussion on optical actuation (transport and rotation) with fiber optic microbeams and spectroscopic characterization in the microfluidic environment. All these advancements in optical manipulation would further facilitate the growing use of optical tools for complex microfluidic manipulations.     

Nucleic Acid‐Barcoding Technologies: Converting DNA Sequencing into a Broad‐Spectrum Molecular Counter

The emergence of high‐throughput DNA sequencing technologies sparked a revolution in the field of genomics that has rippled into many branches of the life and physical sciences. The remarkable sensitivity, specificity, throughput, and multiplexing capacity that are inherent to parallel DNA sequencing have since motivated its use as a broad‐spectrum molecular counter. A key aspect of extrapolating DNA sequencing to non‐traditional applications is the need to append nucleic‐acid barcodes to entities of interest. In this review, we describe the chemical and biochemical approaches that have enabled nucleic‐acid barcoding of proteinaceous and non‐proteinaceous materials and provide examples of downstream technologies that have been made possible by DNA‐encoded molecules. As commercially available high‐throughput sequencers were first released less than 15 years ago, we believe related applications will continue to mature and close by proposing new frontiers to support this assertion.     

Single molecule studies of DNA binding proteins using optical tweezers

Optical tweezers have become a versatile tool in the biological sciences. Combined with various types of optical microscopy, they are being successfully used to discover the fundamental mechanism of biological processes. Recently, the study of proteins acting on DNA was aggressively undertaken at the single-molecule level. Here, we review the most recent studies which have revealed the dynamic behavior of individual protein molecules at work on DNA, providing detailed mechanistic insight that could not be revealed, at least not easily, using bulk-phase or ensemble approaches.     

拉曼镊子在生物单细胞中的应用与进展

拉曼镊子(Raman tweezers)是将激光光镊(Optical tweezers)与显微拉曼光谱(Raman spectroscopy)结合的光学技术,可以在接近自然状态下研究单个生物细胞或细胞器.因其有无直接接触、无损伤、快速识别、实时追踪等特点,广泛用于生物细胞的识别、探测、筛选等.研究显示,拉曼镊子在微观生物研究的应用中,可提高拉曼光谱的信噪比,也能实现生化动力学过程的实时跟踪,从而能深刻了解细胞内生物大分子的活动规律.本文着重介绍了拉曼镊子的起源、原理及其在单细胞中的应用以及展望.     

Optische Einzelmolekülspektroskopie. Einblicke in den Nanokosmos

Single‐molecule fluorescence spectroscopy evolved to a variety of tools to investigate molecular dynamics in thermodynamic equilibrium and to reveal subpopulations in heterogeneous molecular distributions which usually remain hidden in bulk experiments. Applications of single‐molecule experiments range from life sciences and material sciences to photo‐physics and photo‐chemistry. Some of these research fields, like chemical catalysis, have just recently been entered. This article summarizes major principles of single‐molecule fluorescence spectroscopy and gives an overview on some important applications up to the development of novel microscopic techniques with nanometer resolution.     

Biohybrid fluorescent nanodiamonds as dual‐contrast markers for light and electron microscopies

Cathodoluminescence (CL) and correlative light‐electron microscopy (CLEM) are two useful analytical tools in diverse research areas. Recently, fluorescent nanodiamonds (FNDs) have emerged as promising imaging agents for both CL and CLEM owing to their exceptional photophysical and chemical properties. However, to realize their practical applications in the life sciences, surface modification and functionalization of the nanomaterials with bioactive molecules are critical and essential. Here we provide a comprehensive review on the methods of synthesizing biohybrid FNDs as well as recent advances of CL and CLEM imaging of cells with these carbon nanoparticles as dual‐contrast markers.     

A Simple,High‐Yield Synthesis of DNA Duplexes Containing a Covalent,Thermally Cleavable Interstrand Cross‐Link at a Defined Location

Interstrand DNA–DNA cross‐links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross‐links are important, but not well understood. The preparation of chemically well‐defined cross‐linked DNA substrates represents a significant challenge in the study of cross‐link repair. Here a simple method is reported that employs “post‐synthetic” modifications of commercially available 2′‐deoxyoligonucleotides to install a single cross‐link in high yield at a specified location within a DNA duplex. The cross‐linking process exploits the formation of a hydrazone between a non‐natural N⁴‐amino‐2′‐deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross‐link is stable under physiological conditions, but can be readily dissociated and re‐formed through heating–cooling cycles.     

A Simple,High‐Yield Synthesis of DNA Duplexes Containing a Covalent,Thermally Cleavable Interstrand Cross‐Link at a Defined Location

Interstrand DNA–DNA cross‐links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross‐links are important, but not well understood. The preparation of chemically well‐defined cross‐linked DNA substrates represents a significant challenge in the study of cross‐link repair. Here a simple method is reported that employs “post‐synthetic” modifications of commercially available 2′‐deoxyoligonucleotides to install a single cross‐link in high yield at a specified location within a DNA duplex. The cross‐linking process exploits the formation of a hydrazone between a non‐natural N⁴‐amino‐2′‐deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross‐link is stable under physiological conditions, but can be readily dissociated and re‐formed through heating–cooling cycles.     

Mechanical property analysis of stored red blood cell using optical tweezers

The deformation of human red blood cells subjected to direct stretching by optical tweezers was analyzed. The maximum force exerted by optical tweezers on the cell via a polystyrene microbead 5 μm in diameter was 315 pN. Digital image correlation (DIC) method was introduced to calculate the force and the deformation of the cell for the first time. Force–extension relation curves of the biconcave cell were quantitatively assessed when erythrocytes were stored in Alsever's Solution for 2 days, 5 days, 7 days and 14 days respectively. Experiment results demonstrated that the deformability of red blood cells was impaired with the stored time.