Single-fluorophore Imaging Research Articles

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

ABSTRACTFluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 μm2/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 μm2/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms.

Tethered Fluorophore Motion: Studying Large DNA Conformational Changes by Single-fluorophore Imaging

We have previously introduced tethered fluorophore motion (TFM), a single-molecule fluorescence technique that monitors the effective length of a biopolymer such as DNA. TFM uses the same principles as tethered particle motion (TPM) but employs a single fluorophore in place of the bead, allowing TFM to be combined with existing fluorescence techniques on a standard fluorescence microscope. TFM has been previously been used to reveal the mechanism of two site-specific recombinase systems, Cre-loxP and XerCD-dif. In this work, we characterize TFM, focusing on the theoretical basis and potential applications of the technique. Since TFM is limited in observation time and photon count by photobleaching, we present a description of the sources of noise in TFM. Comparing this with Monte Carlo simulations and experimental data, we show that length changes of 100 bp of double-stranded DNA are readily distinguishable using TFM, making it comparable with TPM. We also show that the commonly recommended pixel size for single-molecule fluorescence approximately optimizes signal to noise for TFM experiments, thus enabling facile combination of TFM with other fluorescence techniques, such as Förster resonance energy transfer (FRET). Finally, we apply TFM to determine the polymerization rate of the Klenow fragment of DNA polymerase I, and we demonstrate its combination with FRET to observe synapsis formation by Cre using excitation by a single laser. We hope that TFM will be a useful addition to the single-molecule toolkit, providing excellent insight into protein-nucleic acid interactions.

Live cell single‐molecule detection in systems biology

In this review, we describe technology and use of single-fluorophore imaging and detection in living cells with regard to application in systems biology and medicine. Because all biological reactions occur under aqueous conditions, the realization of single-fluorophore imaging using an optical microscope has led to the direct observation of biological molecules at work. Today, we can observe single molecules in individual living cells and even higher multicellular organisms. Using single-molecule imaging, we can determine the absolute values of kinetic and dynamic parameters of molecular reactions as a whole and during fluctuations and distribution. In addition, identification of the coordinate of single molecules has enabled super-localization techniques to virtually improve spatial resolution of optical microscopy. Single-molecule detection that depends on point detection instead of imaging is also useful in detecting concentrations, diffusive movements, and molecular interactions in living cells, especially in the cytoplasm. The precise and absolute values of positional, kinetic, and dynamic parameters that are determined by single-molecule imaging and detection in living cells constitute valuable data on unitary biological reactions, because they are obtained without destroying the integrity of complex cellular systems. Moreover, most parameters that are determined by single-molecule measurements can be substituted directly into equations that describe kinetic and dynamic models in systems biology and medicine.

Intracellular Myosin Motor Protein Motion Using Laser Scanning Confocal Microscopy

We focus on live cell single molecule imaging and tracking with particular focus on measuring single myosin V steps along actin filaments within live cells. The goal of the work is to acquire information that will offer new insights into the mechanism of these motors' processivity. Conventionally, single molecule Myosin studies have been accomplished through total internal reflection fluorescence microscopy (TIRF).1,2 However, TIRF methods have very limited applicability to live cell in vivo studies. As such, we apply confocal laser scanning microcopy (CLSM) to imaging and tracking of Myosin V. CLSM also offers the capability to provide full 3-dimensional tracking of biomolecular motors. Our experiments have confirmed step size results from previous TIRF experiments. In terms of instrumentation, we overcome the nominally slow scanning speed of CLSM by the use of a fiber scanning technique that allows us to scan with image acquisition times of less than 100 ms. We make use of home-made streptavidin-functionalized quantum dots in conjunction with this tracking technique to increase the quantum efficiency and stability of the fluorophores. The QD fabrication and sample preparation techniques will also be presented.1. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization,” Science 300 (5628), 2061-2065 (2003).2. T. Sakamoto, A. Yildez, P. R. Selvin, and J. R. Sellers, “Step-size is determined by neck length in myosin V,” Biochemistry 44 (49), 16203-16210 (2005).

Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging

Bipolarity of the meiotic spindle, required for proper chromosome segregation, is maintained throughout cell division despite rapid microtubule turnover. How this is achieved has remained mysterious, as determining the organization of individual spindle microtubules has been difficult. Here, we develop single-fluorophore speckle imaging to examine microtubule organization in the vertebrate meiotic spindle. We find that the mean length of microtubules is approximately 40% of spindle length. Long and short filaments distribute randomly throughout the spindle and those in close proximity can move in the same direction with highly heterogeneous velocities. The ratio between microtubule and spindle lengths remains unchanged as spindles elongate upon dynein-dynactin inhibition. However, maintaining this ratio depends on proper kinesin-5 function. Our data suggest that force transmission within the spindle must be understood in terms of the crosslinking dynamics of a tiled array of individual filaments, most of which do not span the distance from the pole to the metaphase plate.

Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system

Direct imaging or counting of RNA molecules has been difficult owing to its relatively low electron density for EM and insufficient resolution in AFM. Bacteriophage phi29 DNA-packaging motor is geared by a packaging RNA (pRNA) ring. Currently, whether the ring is a pentagon or hexagon is under fervent debate. We report here the assembly of a highly sensitive imaging system for direct counting of the copy number of pRNA within this 20-nm motor. Single fluorophore imaging clearly identified the quantized photobleaching steps from pRNA labeled with a single fluorophore and concluded its stoichiometry within the motor. Almost all of the motors contained six copies of pRNA before and during DNA translocation, identified by dual-color detection of the stalled intermediates of motors containing Cy3-pRNA and Cy5-DNA. The stalled motors were restarted to observe the motion of DNA packaging in real time. Heat-denaturation analysis confirmed that the stoichiometry of pRNA is the common multiple of 2 and 3. EM imaging of procapsid/pRNA complexes clearly revealed six ferritin particles that were conjugated to each pRNA ring.

膜分子のホップ拡散の普遍性 : 1蛍光分子観察と高分解能1粒子追跡による研究

膜分子のホップ拡散の普遍性 : 1蛍光分子観察と高分解能1粒子追跡による研究

27] Single-particle tracking image microscopy

The techniques of single particle tracking (SPT) and optical force microscopy (OFM) as described above allow direct imaging of the motion of molecules in the membrane of live cells, and provide a means of controlling the movement by an almost noninvasive method. Combination of these techniques with other single-molecule methods, such as single-fluorophore imaging, allows direct comparison of motion at video rate (because faster than video rate imaging of fluorophore is still not generally feasible) to determine any effect due to the attached colloidal gold particle. Also, simultaneous use of the two techniques allows for monitoring two molecules, one at high time resolution. As such, the system can then be used in conjunction with green fluorescent protein (GFP) transfection to watch simultaneously the motion of an internal component of, say, a signaling pathway while seeing the motion of the transmembrane signaling receptor.

2P109クラスリン被覆ピットへ集合するアダプターと受容体の1分子運動観察

2P109クラスリン被覆ピットへ集合するアダプターと受容体の1分子運動観察

Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging.

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F(1)-ATPase revealed that the subunit rotates in the molecule in discrete 120 degrees steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120 degrees stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

Single-fluorophore imaging with an unmodified epifluorescence microscope and conventional video camera

Single fluorophores in aqueous solution were imaged in real time with a conventional silicon-intensified target video camera connected to an unmodified commercial microscope (IX70, Olympus) with epifluorescence excitation with a high-pressure mercury lamp. Neither a powerful laser nor an extremely sensitive video camera was required. Three experimental systems were used to demonstrate quantitatively that individual, moving or stationary Cy3 fluorophores could be imaged with the microscope: Cy3-gelsolin attached to an actin filament sliding over heavy meromyosin, sliding actin filaments sparsely labelled with Cy3, and heavy meromyosin labelled with one or two Cy3 fluorophores. The results should encourage many laboratories to attempt 'single-molecule physiology' in which the functions and mechanisms of molecular machines are studied at the single-molecule level in an environment where the biological machines are fully active.

Axial rotation of sliding actin filaments revealed by single-fluorophore imaging.

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of approximately 72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of approximately 1 microm, which is much greater than the 72 nm pitch. Thus, myosin does not "walk" on the helical array of actin protomers; rather it "runs," skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each "kick" of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.

Focusing on an individual.

Every element of life, at all levels of hierarchy down to a single molecule, is a functional entity that by itself performs a delicate task (s). Understanding of the mechanism of biological machinery therefore requires a close observation of the performance of individual cells, organelles, or molecules. Three examples are presented, in which single-cell or single-molecule observation under an optical microscope was crucial in the analysis: ionic regulation of the acrosome reaction in starfish sperm revealed by multi-view microscopy, elucidation of elementary steps of actomyosin motors using optical tweezers, and detection of molecular rotation via single fluorophore imaging.