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

Abstract

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 nm and >530 nm 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 nm and 546 nm 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.

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

“…Optical switching of the high quantum yield fluorescence emission of Cy in the hybrid probe is achieved through a photochromic FRET mechanism [16,19,21]. In particular, excitation of the highly fluorescent SP state (no-FRET) with a short (100 ms), low-power pulse between 365 and 405 nm generates the non--fluorescent MC state (FRET) of Cy-NISO ( figure 1a,b).…”

“…(b) Synthesis of optical switch probes Details will be presented elsewhere, although the synthetic methods used for the preparation of Cy-NISO, Cy-NISO-Nhydroxysuccinimide ester (NHS), Cy3-NISO-benzylguanosine (BG) and NISO-NHS are similar to those used in our earlier papers [18,19].…”

Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy

“…The rational design of highly fluorescent fluorophore compounds with optical switches for high-contrast optical lock-in detection used in imaging microscopy in living cells was reported by Marriott et al [142,143]. They designed a highly fluorescent probe consisted of tetramethylrhodamine (TMR) and spironaphthoxazine (NISO) or (TMR-NISO) which was an optical switch to improve the image contrast.…”

“…The TMR-NISO molecule integrated a reversible excited-state and a transition is carried out between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with near ultraviolet (λ=365 nm) or with visible (λ>530 nm) light respectively ( Figure 12). In the MC-state, the TMR (donor) emission is almost completely extinguished by FRET to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal [142]. Irradiation of TMR-NISO with a defined sequence of λ=365 nm and λ=546 nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal.…”

Flipping the Photoswitch: Ion Channels Under Light Control

“…Relaxation to the thermodynamically favoured SP can be accelerated by green light or occurs thermally. Large changes in geometry and polarity, compatibility with two-photon excitation and fluorescence of the MC isomer make SPs attractive photochromes for biological applications Marriott et al 2008;Petchprayoon et al 2011). HTIs were synthesized and studied in detail recently (Figure 1c) (Cordes et al 2007;Eggers et al 2001;Herre 2005;Mostoslavskii 1970;Regner et al 2012).…”