Publications by authors named "Kyu Young Han"

33 Publications

2.5D microscopy: Fast, high-throughput imaging via volumetric projection for quantitative subcellular analysis.

ACS Photonics 2021 Mar 25;8(3):933-942. Epub 2021 Feb 25.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida 32816, United States.

Imaging-based single-cell analysis is essential to study the expression level and functions of biomolecules at subcellular resolution. However, its low throughput has prevented the measurement of numerous cellular features from multiples cells in a rapid and efficient manner. Here we report 2.5D microscopy that significantly improves the throughput of fluorescence imaging systems while maintaining high-resolution and single-molecule sensitivity. Instead of sequential z-scanning, volumetric information is projected onto a 2D image plane in a single shot by engineering the emitted fluorescence light. Our approach provides an improved imaging speed and uniform focal response within a specific imaging depth, which enabled us to perform quantitative single-molecule RNA measurements over a 2×2 mm region within an imaging depth of ~5 μm for mammalian cells in <10 min and immunofluorescence imaging at a >30 Hz volumetric frame rate with reduced photobleaching. Our microscope also offers the ability of multi-color imaging, depth control and super-resolution imaging.
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http://dx.doi.org/10.1021/acsphotonics.1c00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412410PMC
March 2021

Instantaneous non-diffracting light-sheet generation by controlling spatial coherence.

Opt Commun 2020 Nov 30;474. Epub 2020 May 30.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida, USA.

We demonstrate single-shot non-diffracting light-sheet generation by controlling the spatial coherence of light. A one-dimensional coherent beam, created by either increasing the spatial coherence of an LED or decreasing the spatial coherence of a laser, makes it unnecessary to scan non-diffracting beams to generate light-sheets. We theoretically and experimentally demonstrate the equivalence between our method and a scanned light-sheet, and investigate the characteristics of the light-sheet in detail. Our method is easily implementable and universally applicable for high-resolution multicolor light-sheet fluorescence imaging.
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http://dx.doi.org/10.1016/j.optcom.2020.126154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412415PMC
November 2020

Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

Opt Lett 2020 Dec;45(23):6470-6473

We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high throughput, and uniform TIRF imaging of cells over a large field of view. Our versatile method will enable quantitative shadowless TIRF imaging.
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http://dx.doi.org/10.1364/OL.411296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8323474PMC
December 2020

Optimizing the performance of multiline-scanning confocal microscopy.

J Phys D Appl Phys 2020 Mar 23;54(10). Epub 2020 Dec 23.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida, USA.

Line-scanning confocal microscopy provides high imaging speed and moderate optical sectioning strength, which makes it a useful tool for imaging various biospecimens ranging from living cells to fixed tissues. Conventional line-scanning systems have only used a single excitation line and slit, and thus have not fully exploited benefits of parallelization. Here we investigate the optical performance of multi-line scanning confocal microscopy (mLS) by employing a digital micro-mirror that provides programmable patterns of the illumination beam and the detection slit. Through experimental results and optical simulations, we assess the depth discrimination of mLS under different optical parameters and compare it with multi-point systems such as scanning disk confocal microscopy (SDCM). Under the same illumination duty cycle, we find that mLS has better optical sectioning than SDCM at a high degree of parallelization. The optimized mLS provides a low photobleaching rate and video-rate imaging while its optical sectioning is similar to single line-scanning confocal microscopy.
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http://dx.doi.org/10.1088/1361-6463/abc84bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412417PMC
March 2020

Fluorescence imaging with tailored light.

Nanophotonics 2019 Dec 28;8(12):2111-2128. Epub 2019 Sep 28.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida, USA.

Fluorescence microscopy has long been a valuable tool for biological and medical imaging. Control of optical parameters such as the amplitude, phase, polarization and propagation angle of light gives fluorescence imaging great capabilities ranging from super-resolution imaging to long-term real-time observation of living organisms. In this review, we discuss current fluorescence imaging techniques in terms of the use of tailored or structured light for the sample illumination and fluorescence detection, providing a clear overview of their working principles and capabilities.
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http://dx.doi.org/10.1515/nanoph-2019-0227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409798PMC
December 2019

Low-photobleaching line-scanning confocal microscopy using dual inclined beams.

J Biophotonics 2019 10 14;12(10):e201900075. Epub 2019 Jun 14.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida.

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line-scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2-fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single-molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three-dimensional single-molecule RNA imaging in mammalian cells.
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http://dx.doi.org/10.1002/jbio.201900075DOI Listing
October 2019

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging.

J Vis Exp 2019 04 8(146). Epub 2019 Apr 8.

CREOL, The College of Optics and Photonics, University of Central Florida;

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.
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http://dx.doi.org/10.3791/59360DOI Listing
April 2019

Nuclear speckle fusion via long-range directional motion regulates speckle morphology after transcriptional inhibition.

J Cell Sci 2019 04 17;132(8). Epub 2019 Apr 17.

Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2 µm/min and 1.5 µm/min. Frequently, three or even four speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer, but larger, speckles after transcriptional inhibition. These speckle movements are not actin dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle marker protein SON. Similar long-range speckle movements and fusion events were observed after heat shock or heavy metal stress, and during late G2 and early prophase. Our observations suggest a mechanism for long-range, directional nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.226563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503955PMC
April 2019

Facile single-molecule pull-down assay for analysis of endogenous proteins.

Phys Biol 2019 03 22;16(3):035002. Epub 2019 Mar 22.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, FL, United States of America.

The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane-Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab') or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.
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http://dx.doi.org/10.1088/1478-3975/ab0792DOI Listing
March 2019

Recent advancement of light-based single-molecule approaches for studying biomolecules.

Wiley Interdiscip Rev Syst Biol Med 2019 07 6;11(4):e1445. Epub 2019 Feb 6.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida.

Recent advances in single-molecule techniques have led to new discoveries in analytical chemistry, biophysics, and medicine. Understanding the structure and behavior of single biomolecules provides a wealth of information compared to studying large ensembles. However, developing single-molecule techniques is challenging and requires advances in optics, engineering, biology, and chemistry. In this paper, we will review the state of the art in single-molecule applications with a focus over the last few years of development. The advancements covered will mainly include light-based in vitro methods, and we will discuss the fundamentals of each with a focus on the platforms themselves. We will also summarize their limitations and current and future applications to the wider biological and chemical fields. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods.
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http://dx.doi.org/10.1002/wsbm.1445DOI Listing
July 2019

Flat-field illumination for quantitative fluorescence imaging.

Opt Express 2018 Jun;26(12):15276-15288

The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.
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http://dx.doi.org/10.1364/OE.26.015276DOI Listing
June 2018

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

Biochemistry 2018 03 28;57(10):1572-1576. Epub 2018 Feb 28.

Department of Biophysics and Biophysical Chemistry , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States.

Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.
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http://dx.doi.org/10.1021/acs.biochem.7b01293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149537PMC
March 2018

Endogenous Alpha-Synuclein Protein Analysis from Human Brain Tissues Using Single-Molecule Pull-Down Assay.

Anal Chem 2017 12 29;89(24):13044-13048. Epub 2017 Nov 29.

Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida , Orlando, Florida 32827, United States.

Alpha-synuclein (α-SYN) is a central molecule in Parkinson's disease pathogenesis. Despite several studies, the molecular nature of endogenous α-SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of biospecimens. Here, we introduce α-SYN single-molecule pull-down (α-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual α-SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. This powerful single-molecule assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.
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http://dx.doi.org/10.1021/acs.analchem.7b04335DOI Listing
December 2017

Spatially encoded fast single-molecule fluorescence spectroscopy with full field-of-view.

Sci Rep 2017 09 8;7(1):10945. Epub 2017 Sep 8.

CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida, USA.

We report a simple single-molecule fluorescence imaging method that increases the temporal resolution of any type of array detector by >5-fold with full field-of-view. We spread single-molecule spots to adjacent pixels by rotating a mirror in the detection path during the exposure time of a single frame, which encodes temporal information into the spatial domain. Our approach allowed us to monitor fast blinking of an organic dye, the dissociation kinetics of very short DNA and conformational changes of biomolecules with much improved temporal resolution than the conventional method. Our technique is useful when a large field-of-view is required, for example, in the case of weakly interacting biomolecules or cellular imaging.
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http://dx.doi.org/10.1038/s41598-017-10837-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591195PMC
September 2017

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.

J Am Chem Soc 2016 Apr 25;138(13):4512-21. Epub 2016 Mar 25.

Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.
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http://dx.doi.org/10.1021/jacs.5b13449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852477PMC
April 2016

RESOLFT nanoscopy with photoswitchable organic fluorophores.

Sci Rep 2015 Dec 7;5:17804. Epub 2015 Dec 7.

Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Korea.

Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.
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http://dx.doi.org/10.1038/srep17804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671063PMC
December 2015

Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

Sci Rep 2015 Nov 27;5:17295. Epub 2015 Nov 27.

Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.
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http://dx.doi.org/10.1038/srep17295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661537PMC
November 2015

Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser.

Opt Lett 2015 Jun;40(11):2653-6

We describe a dual-color three-dimensional stimulated emission depletion (3D-STED) microscopy employing a single laser source with a repetition rate of 80 MHz. Multiple excitation pulses synchronized with a STED pulse were generated by a photonic crystal fiber, and the desired wavelengths were selected by an acousto-optic tunable filter with high spectral purity. Selective excitation at different wavelengths permits simultaneous imaging of two fluorescent markers at a nanoscale resolution in three dimensions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849877PMC
http://dx.doi.org/10.1364/OL.40.002653DOI Listing
June 2015

An improved surface passivation method for single-molecule studies.

Nat Methods 2014 Dec 12;11(12):1233-6. Epub 2014 Oct 12.

1] Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2] Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [3] Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [4] Howard Hughes Medical Institute, Urbana, Illinois, USA. [5] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

We report a surface passivation method based on dichlorodimethylsilane (DDS)-Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS-Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.
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http://dx.doi.org/10.1038/nmeth.3143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245390PMC
December 2014

Understanding the photophysics of the spinach-DFHBI RNA aptamer-fluorogen complex to improve live-cell RNA imaging.

J Am Chem Soc 2013 Dec 10;135(50):19033-8. Epub 2013 Dec 10.

Howard Hughes Medical Institute , Urbana, Illinois 61801, United States.

The use of aptamer-fluorogen complexes is an emerging strategy for RNA imaging. Despite its promise for cellular imaging and sensing, the low fluorescence intensity of the Spinach-DFHBI RNA aptamer-fluorogen complex hampers its utility in quantitative live-cell and high-resolution imaging applications. Here we report that illumination of the Spinach-fluorogen complex induces photoconversion and subsequently fluorogen dissociation, leading to fast fluorescence decay and fluorogen-concentration-dependent recovery. The fluorescence lifetime of Spinach-DFHBI is 4.0 ± 0.1 ns irrespective of the extent of photoconversion. We detail a low-repetition-rate illumination scheme that enables us to maximize the potential of the Spinach-DFHBI RNA imaging tag in living cells.
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http://dx.doi.org/10.1021/ja411060pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908778PMC
December 2013

Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake.

Mol Biosyst 2013 May;9(5):1004-11

WCU Department of Biophysics and Chemical Biology, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 151-747, Republic of Korea.

Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.
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http://dx.doi.org/10.1039/c2mb25431jDOI Listing
May 2013

STED nanoscopy with time-gated detection: theoretical and experimental aspects.

PLoS One 2013 18;8(1):e54421. Epub 2013 Jan 18.

Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy.

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054421PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548795PMC
July 2013

Effect of single-base mutation on activity and folding of 10-23 deoxyribozyme studied by three-color single-molecule ALEX FRET.

J Phys Chem B 2012 Mar 27;116(9):3007-12. Epub 2012 Feb 27.

Department of Chemistry, Seoul National University, Seoul 151-747, Korea.

We investigated the effect of single-base mutation on the RNA-cleaving activity and ion-induced folding of 10-23 deoxyribozyme at the single-molecule level by 3-color ALEX FRET (alternating laser excitation fluorescence resonance energy transfer). We found that substitution or deletion of a single base in the active region of the enzyme leads to a different folding pathway and enzymatic activity for all three mutants studied, but the severity of the effect was dependent on the type of mutation and the mutation site. We suggest that mutation of even a single base may result in a considerably different ionic and hydrogen-bonding interactions. Structural changes of 10-23 deoxyribozyme as it successively binds with Mg(2+) and the substrate were also unambiguously identified by the current single-molecule-detection method.
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http://dx.doi.org/10.1021/jp2117196DOI Listing
March 2012

Quantitative genotyping of single nucleotide polymorphism by single-molecule multi-color fluorescence resonance energy transfer.

Chem Commun (Camb) 2011 Oct 17;47(37):10362-4. Epub 2011 Aug 17.

Department of Chemistry, Seoul National University, Seoul 151-747, Korea.

We developed a new single nucleotide polymorphism (SNP) genotyping method based on single-molecule multi-color fluorescence resonance energy transfer (FRET). We demonstrated that this new method uses less than 1 fmol of sample and is also highly quantitative with a detection level of 1% or lower in the minor allele fraction.
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http://dx.doi.org/10.1039/c1cc12737cDOI Listing
October 2011

Metastable dark States enable ground state depletion microscopy of nitrogen vacancy centers in diamond with diffraction-unlimited resolution.

Nano Lett 2010 Aug;10(8):3199-203

Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.

Current far-field optical nanoscopy schemes overcome the diffraction barrier by ensuring that adjacent features assume different states upon detection. Ideally, the transition between these states can be repeated endlessly and, if performed optically, with low levels of light. Here we report such optical switching, realized by pairing the luminescent triplet and a long-lived dark state of diamond color centers, enabling their imaging with a resolution >10 times beyond the diffraction barrier (<20 nm).
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http://dx.doi.org/10.1021/nl102156mDOI Listing
August 2010

Single-molecule, real-time measurement of enzyme kinetics by alternating-laser excitation fluorescence resonance energy transfer.

Chem Commun (Camb) 2010 Jul 1;46(26):4683-5. Epub 2010 Jun 1.

Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Korea.

Using a single-molecule fluorescence method uniquely suitable for binding assay, alternating-laser excitation fluorescence resonance energy transfer (ALEX-FRET), we accurately measured the cleavage rate of 8-17 deoxyribozyme, an RNA-cleaving enzyme, at the single-molecule level in real time with a minimum consumption of samples, i.e., at least three orders of magnitude smaller than used in the conventional ensemble FRET method.
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http://dx.doi.org/10.1039/c002666bDOI Listing
July 2010

Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light.

Nano Lett 2009 Sep;9(9):3323-9

Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Charged nitrogen-vacancy (NV) color centers in diamond are excellent luminescence sources for far-field fluorescence nanoscopy by stimulated emission depletion (STED). Here we show that these photostable color centers can be visualized by STED using simple continuous-wave or high repetition pulsed lasers (76 MHz) at wavelengths >700 nm for STED. Furthermore, we show that NV centers can be imaged in three dimensions (3D) inside the diamond crystal and present single-photon signatures of single color centers recorded in high density samples, demonstrating a new recording scheme for STED and related far-field nanoscopy approaches. Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences.
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http://dx.doi.org/10.1021/nl901597vDOI Listing
September 2009

Conformational study of jet-cooled L-phenylglycine.

J Chem Phys 2008 May;128(18):184313

Department of Chemistry, Seoul National University, Seoul, South Korea.

We investigated the conformational structures of L-phenylglycine in the gas phase by photoionization and double resonance spectroscopy techniques as well as high-level ab initio calculations. The UV-UV and IR-UV double resonance spectroscopy suggested that there exists only one conformer that has a free OH band for the carboxyl group. Rotational contour analysis combined with ab initio calculation indicated that the conformer we detected by resonant two-photon ionization was not one of those found by Sanz et al. in their microwave spectroscopic study [Chem. Eur. J. 12, 2564 (2006)]. Different methods of vaporization along with different expansion and cooling conditions and different detection methods are believed to be the culprit for such intriguing discrepancy. The identical hydrogen bonding structure of our phenylglycine conformer with the most abundant conformer of glycine found in helium droplets and their nearly identical OH frequencies suggest that the skeletal structure of glycine is not significantly altered by phenyl substitution.
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http://dx.doi.org/10.1063/1.2918344DOI Listing
May 2008

Solvent migration from the C- to the N-terminus of amino acid in photoionization of phenylglycine-water complex.

J Chem Phys 2008 Jan;128(4):041104

Department of Chemistry, Seoul National University, Seoul 151-747, Korea.

Photo-oxidation of amino acids is known to generate reactive protein radicals that lead to lethal disorders. We investigated photoionization of hydrated phenylglycine complexes in the gas phase and found that the excess internal energy from photoionization drives decarboxylation in competition with dehydration. We also found that, in decarboxylation, the solvent migrates a large distance from the C terminus of the neutral amino acid to the N terminus of the newly formed radical cation upon ionization, prior to the departure of the carboxyl group. It is noted that a solvent does not just act as a passive medium bound to the solute molecule but actively pursues its own course of action upon external perturbation that changes its chemical environment.
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http://dx.doi.org/10.1063/1.2835351DOI Listing
January 2008
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