Imaging Fluorescence Blinking of a Mitochondrial Localization Probe: Cellular Localization Probes Turned into Multifunctional Sensors

Du, Zhuofei; Piguet, Joachim; Baryshnikov, Glib; Tornmalm, Johan; Demirbay, Barış; Ågren, Hans; Widengren, Jerker

doi:10.1021/acs.jpcb.2c01271

Cited by 8 publications

(15 citation statements)

References 37 publications

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“…With the excitation modulation systematically varied on the time scales of the fluorophore dark-state kinetics, rapid blinking kinetics can be quantified without the need for time-resolved detection. 37 − 42 …”

Section: Methodsmentioning

confidence: 99%

“…In TRAST measurements, fluorophore blinking kinetics are determined by recording the average fluorescence intensity from an ensemble of fluorophores subject to modulated excitation. With the excitation modulation systematically varied on the time scales of the fluorophore dark-state kinetics, rapid blinking kinetics can be quantified without the need for time-resolved detection. − …”

Section: Methodsmentioning

confidence: 99%

“…37−42 TRAST measurements were carried out on a home-built TRAST setup based on an inverted epi-fluorescence microscope (Olympus, IX70) and modified from a previously described arrangement. 37 In short, fluorescence is excited by a beam of a diode laser (638 nm Cobolt, 06-MLD, 200 mW, Cobolt, 06-MLD, 730 nm, 100 mW, or 785 nm 06-MLD, 250 mW) passing appropriate excitation filters (Semrock Bright-Line 637/7, Chroma ET740/40x). The 638 nm and 785 nm laser beams were modulated by acousto-optic modulators (AOM; AA Opto Electronics, MQ180-A0,25-VIS and MT110-A1-IR), while the 730 nm laser was directly modulated by external triggering.…”

Section: ■ Introductionmentioning

confidence: 99%

“…37−42 TRAST measurements were carried out on a home-built TRAST setup based on an inverted epi-fluorescence microscope (Olympus, IX70) and modified from a previously described arrangement. 37 TRAST Analysis. To calculate the recorded fluorescence intensity in the TRAST experiments, we used the reduced photophysical model for SCy7 in Figure 3A, with two emissive states, the all-trans state, N, and the double-cis state, P 2 , and with two (mono-)cis states of SCy7 nonluminescent, for simplicity represented as one state, P 1 .…”

Section: ■ Introductionmentioning

confidence: 99%

“…In this work, we studied the photodynamics of the heptamethine NIR cyanine dye, Sulfo-Cy7 (SCy7), by transient state (TRAST) spectroscopy, − together with fluorescence correlation spectroscopy (FCS). In TRAST, reversible transitions of long-lived dark states of fluorescent molecules can be characterized from how the time-averaged fluorescence intensity from the fluorophores varies with the modulation of the laser excitation intensity.…”

Section: Introductionmentioning

confidence: 99%

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Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging

et al. 2023

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Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

“…37−42 TRAST measurements were carried out on a home-built TRAST setup based on an inverted epi-fluorescence microscope (Olympus, IX70) and modified from a previously described arrangement. 37 TRAST Analysis. To calculate the recorded fluorescence intensity in the TRAST experiments, we used the reduced photophysical model for SCy7 in Figure 3A, with two emissive states, the all-trans state, N, and the double-cis state, P 2 , and with two (mono-)cis states of SCy7 nonluminescent, for simplicity represented as one state, P 1 .…”

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging

et al. 2023

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Interplay between a Heptamethine Cyanine Dye Sensitizer (IR806) and Lanthanide Upconversion Nanoparticles

Liu,

Kulkarni,

Kostiv

et al. 2024

Advanced Optical Materials

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Lanthanide‐doped upconversion nanoparticles (UCNPs) have attractive emission properties but suffer from weak light‐absorbing capacities and thereby relatively low brightnesses. This motivates using strongly absorbing dye molecules as antennas and sensitizers. However, despite much effort, understanding of this dye‐UCNP interplay is still limited. Major sensitization mechanisms are still under discussion, largely because there is a lack of effective means to observe key factors such as dark state transitions within the dyes. Here, a combined spectroscopic procedure is established to systematically investigate the photophysics behind the dye‐UCNP interaction, embracing fluorescence‐based transient‐state excitation‐modulation, lifetime and correlation spectroscopy, and spectrofluorometry/spectrophotometry. With this procedure the heptamethine cyanine dye IR806, a typical UCNP sensitizer is studied, its photophysical model is established, its photophysics in UCL‐sensitization‐related environments is deciphered, and the energy transfer from the IR806 singlet excited state to Yb3+ (UCNP sensitizer ion) can be identified as the dominant sensitization mechanism. These studies suggest that IR806 can form non‐emissive H‐aggregates at the nanoparticle surfaces, which can be dissociated after certain light excitation duration (typically>100 µs). Moreover, buildup of a non‐fluorescent, photo‐redox state of IR806 after longer irradiation times (10–100 ms) can deleteriously affect its UCL sensitization effect, inferring an optimal excitation duration for dye‐sensitized UCNPs, relevant for, e.g., optical imaging applications.

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Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters

Sandberg,

Demirbay,

Kulkarni

et al. 2023

J. Phys. Chem. B

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Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based superresolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a barcoding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans−cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

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Imaging Fluorescence Blinking of a Mitochondrial Localization Probe: Cellular Localization Probes Turned into Multifunctional Sensors

Cited by 8 publications

References 37 publications

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging

Interplay between a Heptamethine Cyanine Dye Sensitizer (IR806) and Lanthanide Upconversion Nanoparticles

Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters

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