Optical Switch Probes Research Articles

High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.

We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C12-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy.

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens.

Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems

Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

Synthesis and spectroscopic characterization of red-shifted spironaphthoxazine based optical switch probes

Spironaphthoxazine (NISO) is an efficient optical switch probe that has applications in high contrast detection of Förster resonance energy transfer (FRET) using optical lock-in detection (OLID). NISO exists in two distinct states, spiro (SP) and merocyanine (MC), that can be independently controlled by using alternate irradiation with near ultraviolet and visible light. Unfortunately, the SP-state of NISO has an absorption centered at 350 nm, which may lead to phototoxic effects when manipulating the probe within a living cell. To overcome this problem we introduce new, red-shifted amino-substituted NISO probes compared to NISO that undergo an efficient SP to MC transition in response to irradiation by using 405-nm light, which is less damaging to living cells. This study details the synthesis of amino-substituted NISO and their N-hydroxysuccinimide ester and maleimide derivatives and their use in generating covalent attached protein conjugates. This study also presents a characterization of the spectroscopic and optical switching properties of these red-shifted NISO probe in solution.

Rational design, synthesis, and characterization of highly fluorescent optical switches for high-contrast optical lock-in detection (OLID) imaging microscopy in living cells

A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-resolution images of specific proteins and their complexes. Auto-fluorescence however, can severely compromise image contrast and represents a fundamental limitation for imaging proteins within living cells. We have previously shown that optical switch probes and optical lock-in detection (OLID) image microscopy improve image contrast in high background environments. Here, we present the design, synthesis, and characterization of amino-reactive and cell permeable optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR-NISO undergoes rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with 365 and >530nm light. In the MC-state, the TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET) to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal. Irradiation of TMR-NISO with a defined sequence of 365 and 546nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High fidelity optical switching of TMR fluorescence is shown for TMR-NISO probes in vitro and for membrane permeable TMR-NISO within living cells.

Preparation, Characterization, and Application of Optical Switch Probes

Optical switches represent a new class of molecular probe with applications in high contrast imaging and optical manipulation of protein interactions. Small molecule, organic optical switches based on nitrospirobenzopyran (NitroBIPS) and their reactive derivatives and conjugates undergo efficient, rapid and reversible, orthogonal optically-driven transitions between a colorless spiro (SP) state and a colored merocyanine (MC) state. The excited MC-state also emits fluorescence, which serves as readout of the state of the switch. Defined optical perturbations of SP and MC generate a defined waveform of MC-fluorescence that can be isolated against unmodulated background signals by using a digital optical lock-in detection approach or to control specific dipolar interactions on proteins. The protocols describe general procedures for the synthesis and spectroscopic characterization of NitroBIPS and specifically labeled conjugates along with methods for the manipulation of dipolar interactions on proteins and imaging of the MC-state of NitroBIPS within living cells.

High-Contrast Fluorescence Imaging Using new Optical Switches and Optical Lock-in Detection Imaging Microscopy

We have developed a new microscope imaging technique to isolate specific fluorescence signals from background signals by using lock-in detection of the modulated fluorescence of a unique class of optical probe. This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of an optical switch probe through defined optical manipulation of its fluorescent and non-fluorescent states - a digital lock-in detection method is employed to isolate the modulated signal of interest from non-modulated signals in the sample, such as conventional fluorescent probes and natural fluorophores. I will discuss the spectroscopic and photochemical properties of several new synthetic and genetically-encoded optical switches and illustrate their applications in high contrast (OLID) imaging studies of specific structures and proteins in cultured cells and in living organisms.

RsCherryRev and NISO Red-shifted Optical Switch Probes for Optical Lock-in Detection (OLID) Imaging and 2-colour OLID-FRET

Optical lock-in detection (OLID) microscopy using synthetic or genetically encoded optical switches is was developed by our group to generate high-contrast images of the distributions and interactions of proteins in the presence of high and time-varying background signals such as those found in living cells in culture and in live tissue. OLID requires an optical switch probe whose fluorescence intensity can be modulated through deterministic optical control of its fluorescent and non-fluorescent states. Our initial studies focused on Dronpa, while in this study we show how the genetically-encoded optical switch rsCherryRev can be used to extend the wavelength region of OLID to the red (>550 nm) and for 2-colour OLID imaging in combination with Dronpa.Optical lock in detection (OLID) of Foerster resonance energy transfer, OLID-(FRET) using optical switches as acceptor probes can overcome several limitations of FRET imaging of protein interactions in living cells, including detecting low levels of protein complexes that result from endogenous unlabeled proteins, and non-stoichiometric formation of protein complexes between donor and acceptor probes. We have previously used NitroBIPS as an optically switchable acceptor probe for GFP, and in this study we show how the synthetic, red-shifted optical switch spironaphthoxazine (NISO) serves as a switchable acceptor for both GFP and mCherry in OLID-FRET. These new probes are used for 2-colour OLID-FRET of protein interactions in living cells.

Optical Lock-in Detection (OLID) and OLID-FRET Imaging Microscopy

The success of imaging fluorescent proteins within living cells is limited by the presence of background signals - while some improvements to image contrast can be achieved through background subtraction, these benefits diminish when the background signal varies in time and space. Further this approach does not allow imaging below the background signal, a regime of interest for single molecule biology. We have developed an optical lock-in detection (OLID) approach and associated signal analysis that involve modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and non-fluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from non-modulated background signals. The lock-in detection method involves pixel-by-pixel based cross-correlation analysis that maps the correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching, and a reference waveform that represents the defined response of the switch probe to the switching cycle. The fundamentally new approach to imaging, which is similar in principle to radar, allows for the selective detection of emission from optical switch probes even in the presence of a larger population of conventional fluorescent probes. We have developed new synthetic and genetically-encoded optical switches for OLID and OLID-FRET imaging in living cells and tissues including nitrospirobenzopyran and naphthozaxine based probes as well as fusion proteins of Dronpa and a new optically switchable mcherry. OLID imaging using these probes is remarkably efficient at imaging specific structures and proteins in high and time varying background signal environments such as in living cells, in culture, in brain slices and in live Xenopus embryos and zebrafish larvae.

Optical Switchable Spironaphthoxazine (NISO)-derived Probes for Optical Lock-in Detection (OLID) Imaging Microscopy and OLID-FRET

An optical switch probe based on spironaphthoxazine (NISO) harboring a O6-benzylguanine (BG) functionality was synthesized and characterized for applications in optical lock-in detection (OLID) Image microscopy. NISO undergoes rapid and reversible, high-fidelity transitions between a colorless spiro (SP) state and a blue colored MC-state that serves as an acceptor probe in Forster resonance energy transfer (FRET) with GFP, YFP and other green or red emitting donor probes. The transition from SP to MC is brought about with high quantum yield by exciting SP with 365 nm or 720 nm (2-photon) while the MC to SP transition is rapidly affected upon excitation of MC with 543∼632 nm light. Thus a defined waveform of optical or opto-thermal manipulations of the NISO switch provides a simple means to modulate the intensity of a donor probe via FRET. NISO was linked to BG via a polyethylene glycol (PEG) linker and this substrate was shown to efficiently label O6-benzylguanine-DNA alkyltransferase (AGT) and AGT-fusion proteins. The optical switching properties BG-PEG-NISO coupled to AGT in fusion proteins with GFP (GFP-AGT/PEG-NISO) and mCherry-(mCherry-AGT/PEG-NISO) were studied. In vitro OLID-FRET imaging studies showed the suitability of NISO as an acceptor probe for GFP and mcherry. The R0 for FRET between GFP and MC-NISO is 4.7 nm and GFP-AGT fusion protein covalently labeled with BG-PEG-NISO exhibited a 55% decrease in GFP fluorescence intensity upon conversion of SP to MC. Other examples of in vitro and in vivo OLID-FRET using GFP-AGT and mcherry-AGT labeled with NISO-BG will be presented.

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.

Probing Conformational Changes of Prestin with Thiol-Reactive Optical Switches

Thiol-reactive optical switch probes were used to examine conformational changes of prestin-based membrane motor. Because this motor is based on mechanoelectric coupling similar to piezoelectricity, the motile activity can be monitored by charge movements across the plasma membrane, which appears as nonlinear capacitance. When the plasma membrane is conjugated with the probes, optically induced spiro-merocyanine transition positively shifted nonlinear capacitance of outer hair cells and prestin-transfected cells by ∼10mV. These shifts were reversible and were eliminated by pretreatment with iodoacetamide. However, they were little affected by pretreatment with biotin maleimide, which cannot reach the cytoplasmic surface. Our results showed that merocyanine states, with a larger dipole moment, interact with the motor's extended conformation stronger than with the compact conformation by 1.6×10−21J/molecule. The interaction sites are near the cytoplasmic side of the motor protein.

Optical Lock-In Detection of FRET Using Synthetic and Genetically Encoded Optical Switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.

Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation.

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical cis/trans-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.