Dual Laser Excitation Research Articles

Inflow nano-optics from the near-to the far-field detection based on Metal-Enhanced Fluorescence signaling

In this communication we evaluated the detection and tracking of Ultraluminescent gold core–shell nanoplatforms based on Metal-Enhanced Fluorescence (MEF) within colloidal dispersion by In-Flow Cytometry and Imaging Flow Cytometry (IFC). The enhanced properties were developed by the incorporation of Rhodamine B (RhB) as a laser dye reporter at variable controlled silica spacer lengths (Au@SiO2-RhB) from a gold core template. Thus, SiO2 was varied from 6 to 7 nm to 25 nm in order to place the laser dye sensitizer at different lengths of the higher electromagnetic fields surrounding the 45-nm gold core diameter. By Laser Fluorescence Microscopy, high intense emissions were shown at shorter SiO2 lengths, while lower emissions were found towards longer ones. These characteristics related to MEF were evaluated by in-flow cytometry with dual laser excitation and fluorescence detection, and by an IFC set-up. By In-flow cytometry, different scattered-light distributions were shown, depending on the nanoparticle concentrations in the colloidal dispersion and size of the Au@SiO2-RhB. Moreover, varied fluorescent event detections and counting were recorded according to the SiO2 lengths applied. A ratio of fluorescent event detection counting of core–shell/core-less of 40–42 was established for optimal MEF Enhancement Factors (MEFEF) of 41–43 determined by Laser Fluorescence Microscopy. Core-less nanoparticles showed a marked decrease of fluorescent event detection counting caused by higher photobleaching and absence of the MEF effect. Hence, an improved detection effect was shown within in-flow based on MEF from single nanoplatforms. Therefore, the Ultraluminescence emission of core–shell nanoarchitectures within in-flow techniques was shown and discussed. A non-classical light in real time within colloidal gold core–shell nanoparticles was generated by accurate control of their architectures.

In Memoriam: Marvin A. Van Dilla: 1919-2019.

© 2020 International Society for Advancement of Cytometry.

Fatigue crack detection using dual laser induced nonlinear ultrasonic modulation

In this study, a nonlinear ultrasonic modulation technique based on dual laser excitation is proposed for fatigue crack detection. Two pulse lasers are shot on the target specimen for ultrasonic generation. The corresponding ultrasonic responses are measured by a laser Doppler vibrometer (LDV) and analyzed to extract the crack induced nonlinear ultrasonic modulation. First, the effect of the pulse laser beam size on the frequency content of the generated ultrasonic waves is numerically and experimentally investigated. Then, this finding of the laser beam size effect is utilized to generate wideband (WB) and narrowband (NB) ultrasonic waves by adjusting the laser beam sizes of the two pulsed excitation lasers. Nonlinear ultrasonic modulation results from the interaction of WB and NB ultrasonic waves when a fatigue crack exists in the target specimen. The fatigue crack is then detected by comparing the spectral responses obtained under a single WB input and both WB and NB inputs. In the end, a fully noncontact dual laser ultrasonic system is developed and used to detect micro fatigue cracks in aluminum and steel plate specimens.

Conventional fluorescence microscopy below the diffraction limit with simultaneous capture of two fluorophores in DNA origami.

A conventional fluorescence microscope was previously constructed for simultaneous imaging of two colors to gain subdiffraction localization. The system is predicated on color separation of overlapping Airy discs, construction of matrices of Cartesian coordinates to determine locations as well as centers of the point spread functions of fluorophores. Quantum dots that are separated by as little as 10 nm were resolved in the x-y coordinates. Inter-fluorophore distances that vary by 10 nm could also be distinguished. Quantum dots are bright point light source emitters that excite with a single laser and can serve as a label for many biomolecules. Here, alterations in the method are described to test the ability to resolve Atto 488 and Atto 647 dyes attached to DNA origami at ~40 nm spacing intervals. Dual laser excitation is used in tandem with multi-wavelength bandpass filters. Notwithstanding challenges from reduced intensity in Atto labeled DNA origami helical bundles compared to quantum dots, preliminary data show a mean inter-fluorophore distance of 56 nm with a range (14-148 nm). The range closely matches published results with DNA origami with other methods of subdiffraction microscopy. Sub-diffraction simultaneous two-color imaging fluorescence microscopy acronymically christened (SSTIFM) is a simple, readily accessible, technique for measurement of inter-fluorophore distances in compartments less than 40 nm. Preliminary results with so called nanorulers are encouraging for use with other biomolecules.

Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC-1 and Multilaser Excitation.

The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨ(m)). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, ΔΨ(m) along with other biological parameters, such as apoptosis and the production of reactive oxygen species.

Dual-laser homo-FRET on the cell surface

Inhomogeneous broadening and red-edge effects have been detected on a highly mobile system of fluorescently conjugated mAbs targeted to cell surface receptors. By exploiting site-selective spectroscopy and the characteristic loss of homo-FRET on increasing excitation and decreasing emission wavelengths, contributions of physical rotation and homo-FRET to the depolarization of fluorescence anisotropy have been separated. Absolute homo-FRET efficiency has been determined by ratioing two anisotropies: a homo-FRET-sensitive one, which is excited at the absorption main band and detected at the long wavelength region of emission, and a homo-FRET-insensitive one, which is excited at the long wavelength region of absorption and detected at the short wavelength region of emission. Because the anisotropies are simultaneously detected in a unified detection scheme of a dual T-format arrangement, the method is applicable for the real-time tracking of dynamical changes of physical rotations and proximities. The utility of the method is demonstrated in the context of the MHCII molecule and the heavy and light chains of the MHCI molecule, a system of three receptors with well-characterized close mutual proximities. Although the method is presented for a flow cytometer, it can also be realized in a fluorescence microscope capable for dual-laser excitation and dual-anisotropy detection.

Spatially compressed dual-wavelength excitation Raman spectrometer.

The design and operation of a novel dual-laser excitation Raman instrument is described. The use of two lasers of differing wavelengths allows for a Raman spectrum covering all fundamental modes of vibration to be collected while minimizing fluorescence and allowing for spatial compression of the spectrum on an imaging detector. The use of diode lasers with integrated distributed Bragg reflector gratings facilitates the use of an integrated thermoelectric cooler to allow collection of shifted excitation spectra for both of the lasers, further enhancing the rejection of fluorescence. An example is given, which uses seven excitation wavelengths for each laser to reconstruct the Raman spectrum of a solvent in the presence of a highly fluorescent dye by using a sequentially shifted excitation Raman reconstruction algorithm.

Optically Modulatable Blue Fluorescent Proteins

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Modulated Fluorophore Signal Recovery Buried within Tissue Mimicking Phantoms

Optically modulated fluorescence from ∼140 nM Cy5 is visualized when embedded up to 6 mm within skin tissue mimicking phantoms, even in the presence of overwhelming background fluorescence and scatter. Experimental and finite element analysis (FEA)-based computational models yield excellent agreement in signal levels and predict biocompatible temperature changes. Using synchronously amplified fluorescence image recovery (SAFIRe), dual-laser excitation (primary laser: λ = 594 nm, 0.29 kW/cm(2); secondary laser: λ = 710 nm, 5.9 kW/cm(2), intensity-modulated at 100 Hz) simultaneously excites fluorescence and dynamically optically reverses the dark state buildup of primary laser-excited Cy5 molecules. As the modulated secondary laser both directly modulates Cy5 emission and is of lower energy than the collected Cy5 fluorescence, modulated Cy5 fluorescence in phantoms is free of obscuring background emission. The modulated fluorescence emission due to the secondary laser was recovered by Fourier transformation, yielding a specific and unique signature of the introduced fluorophores, with largely background-free detection, at excitation intensities close to the maximum permissible exposure (MPE) for skin. Experimental and computational models agree to within 8%, validating the computational model. As modulated fluorescence depends on the presence of both lasers, depth information as a function of focal position is also readily obtained from recovered modulated signal strength.

Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

Circularly polarized emission from ensembles of InAs/GaAs quantum dots

We present a low-temperature micro-photoluminescence (μ-PL) study of ensembles of InAs/GaAs quantum dots (QDs) with respect to its circular polarization (ρc) for a manifold of experimental conditions such as single or dual laser excitation, different excitation energies (hυex), varying excitation powers (Pex) of both lasers, and with or without an external magnetic field (Bext). It is demonstrated that an essential ρc (≤ 40%) could be recorded depending on Pex, even at Bext = 0 for hυex exceeding the PL energy of the wetting layer (EWL), while ρc remains negligible for hυex < EWL. To explain the data obtained, a model is developed according to which a nuclear magnetic field (BN) is created in the QDs by spin-polarized electrons. The BN plays a crucial role in the preservation of the electron spin, which otherwise effectively relaxes due to the presence of the anisotropic electron-hole exchange interaction (ωex). The application of an additional infra-red laser gives rise to a population of excess holes in the QDs, thus producing positively charged excitons. In this case, ωex = 0 and accordingly, ρc ≈ 40% at Bext = 0 is recorded, even for excitation with hυex < EWL.

Formation of NR1/NR2 and NR1/NR3 Heterodimers Constitutes the Initial Step in N-Methyl-D-aspartate Receptor Assembly

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.

Effects of Separate Carrier Generation on the Emission Properties of InAs/GaAs Quantum Dots

Individual quantum dots have been studied by means of microphotoluminescence with dual-laser excitation. The additional infrared laser influences the dot charge configuration and increases the dot luminescence intensity. This is explained in terms of separate generation of excess electrons and holes into the dot from the two lasers. With increasing dot density and/or sample temperature, the increase of the luminescence intensity vanishes progressively, while the possibility to control the dot charge remains.

Three-dimensional confocal thermal imaging using anti-Stokes luminescence

High-resolution thermal images of dye-stained live cells are obtained using confocal anti-Stokes luminescence on a confocal laser scanning microscope. Stokes and anti-Stokes fluorescence signal ratio imaging, using dual laser excitation with a fast alternate line scan, is used to monitor dynamical temperature changes at submicrometer resolution. Temperature mapping of a dye-doped polymer film with local heating is shown as an example. Three-dimensional mapping of local temperature changes in living cells induced by an external laser as the heating source, is obtained with temperature resolution better than 0.5 K.

Effective optical manipulation of the charge state and emission intensity of the InAs∕GaAs quantum dots by means of additional infrared illumination

InAs quantum dots (QDs) at different levels of density have been studied by means of photoluminescence, when in addition to the main laser, a second infrared (IR) laser is employed to excite the QD. It is demonstrated that the IR laser considerably affects the QD charge state as well as the emission intensity level (an increase greater than fivefold was observed). These effects are explained in terms of separate generation of excess electrons and holes provided under dual-laser excitation. However, these effects progressively vanish with increasing QD density. The results obtained unambiguously imply that the emission intensity from the QD can be effectively enhanced by purely optical means.

Quantitative histology by multicolor slide-based cytometry.

In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings. We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single- (FITC) up to four-color (FITC/PE/PECy5/APC) stained specimen. Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

Frequency and Voltage Dependence of the Dielectrophoretic Trapping of Short Lengths of DNA and dCTP in a Nanopipette

The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.

Multiparameter flow cytometry of fluorescent protein reporters.

Reporters based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells. Stable cell lines were generated from the Sp2/0-Ag14 hybridoma that express up to three spectral enhanced versions of GFP, the enhanced cyan fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), and/or a variant of the Discosoma coral red fluorescent protein (DsRed). The panel of lines was used to demonstrate a flow cytometric procedure for simultaneous analysis of all four fluorescent proteins that utilizes dual-laser excitation at 488 nm and 407 nm. Additional schemes for simultaneous detection of two, three or four of these fluorescent proteins are also presented.

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins.

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.