Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

Kikuchi, Kai; Adair, Liam D.; Lin, Jiarun; New, Elizabeth J.; Kaur, Amandeep

doi:10.1002/anie.202204745

Cited by 30 publications

(38 citation statements)

References 257 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This requires a low On/Off duty cycle (defined as the fraction of time a fluorophore spends in the On state) and long-lived dark states. Other requirements are high photon counts per switching event and generally a high number of switching cycles [ 32 , 111 ].…”

Section: Fluorescent Probes For Single Molecule Microscopymentioning

confidence: 99%

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Spagnolo

Luin

2022

IJMS

View full text Add to dashboard Cite

Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications.

show abstract

Section: Fluorescent Probes For Single Molecule Microscopymentioning

confidence: 99%

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Spagnolo

Luin

2022

IJMS

View full text Add to dashboard Cite

show abstract

“…In turn, the inability to visualize the sample complicates the identification of regions within the field of view that would be appropriate for activation. In spite of these complications, photocaged acridinones, azafluorenones, benzothiazoles, borondipyrromethenes (BODIPYs), coumarins, cyanines, diazaxanthilidenes, fluoresceins, pyrazolines, resorufin, rhodamines, salens, tetraethylenes, and xanthene have already been employed successfully to monitor diverse dynamic events in developing embryos and live cells on the basis of FPD. − …”

Section: Fluorescence Photoactivation To Monitor Dynamicsmentioning

confidence: 99%

“…Conventional fluorophores produce fluorescence upon irradiation at an appropriate excitation wavelength (λ Ex ) . Photoactivatable fluorophores, instead, emit significantly only if illumination at λ Ex follows irradiation at a suitable activation wavelength (λ Ac ). − Indeed, absorption of radiation at λ Ac initiates the photochemical conversion of a nonemissive reactant into an emissive product, which is then excited at λ Ex to generate fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Photoactivatable Fluorophores for Bioimaging Applications

Zhang

Zheng

Tomassini

et al. 2023

ACS Appl. Opt. Mater.

View full text Add to dashboard Cite

Photoactivatable fluorophores provide the opportunity to switch fluorescence on exclusively in a selected area within a sample of interest at a precise interval of time. Such a level of spatiotemporal fluorescence control enables the implementation of imaging schemes to monitor dynamic events in real time and visualize structural features with nanometer resolution. These transformative imaging methods are contributing fundamental insights on diverse cellular processes with profound implications in biology and medicine. Current photoactivatable fluorophores, however, become emissive only after the activation event, preventing the acquisition of fluorescence images and, hence, the visualization of the sample prior to activation. We developed a family of photoactivatable fluorophores capable of interconverting between emissive states with spectrally resolved fluorescence, instead of switching from a nonemissive state to an emissive one. We demonstrated that our compounds allow the real-time monitoring of molecules diffusing across the cellular blastoderm of developing embryos as well as of polymer beads translocating along the intestinal tract of live nematodes. Additionally, they also permit the tracking of single molecules in the lysosomal compartments of live cells and the visualization of these organelles with nanometer resolution. Indeed, our photoactivatable fluorophores may evolve into invaluable analytical tools for the investigation of the fundamental factors regulating the functions and structures of cells at the molecular level.

show abstract

“…Photoactivatable fluorophores (PAFs) switch from a nonemissive to an emissive state under illumination at an appropriate activation wavelength (λ Ac in Figure 3 ) [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. The latter species produces fluorescence upon irradiation at the corresponding excitation wavelength (λ Ex in Figure 3 ).…”

Section: Photoactivatable Fluorophoresmentioning

confidence: 99%

Photoactivatable BODIPYs for Live-Cell PALM

et al. 2023

View full text Add to dashboard Cite

Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.

show abstract

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

Cited by 30 publications

References 257 publications

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Photoactivatable Fluorophores for Bioimaging Applications

Photoactivatable BODIPYs for Live-Cell PALM

Contact Info

Product

Resources

About