FLIM Strategies for Intracellular Sensing

Herein, we describe the synthesis and application of cross-linked polystyrene-based dual-function nano- and microparticles containing both fluorescent tags and metals. Despite containing a single dye, these particles exhibit a characteristic dual-band fluorescence emission. Moreover, these particles can be combined with different metal ions to obtain hybrid metallofluorescent particles. We demonstrate that these particles are easily nanofected into living cells, allowing them to be used for effective fingerprinting in multimodal fluorescence-based and mass spectrometry-based flow cytometry experiments. Likewise, the in situ reductions of the metal ions enable other potential uses of the particles as heterogeneous catalysts.

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“…This showed the valuable information that FLIM provides to probe the environment of the dyes used in fluorescence microscopy. 18 …”

Section: Resultsmentioning

confidence: 99%

Metallofluorescent Nanoparticles for Multimodal Applications

et al. 2018

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“…Important uncertainties may arise when one of the bands is affected by other sources within the cellular environment (quenching, photobleaching, and so forth) or by the presence of emissive interferences, such as the species causing cellular autofluorescence (i.e., structural fluorescent groups such as tryptophan and tyrosine in proteins, lipofuscin in cell membranes, NADH in the cytosol, porphyrins in the blood, albumin in serum, B vitamins, etc. ), which may alter the sensing ratio [11,12].…”

Section: Introductionmentioning

confidence: 99%

“…FLIM is inherently multidimensional since not only the PL emission intensity but also the PL lifetime values in each pixel of the image is collected, usually represented in pseudocolor image scales, serving as an extra contrast tool. Importantly, FLIM is especially suited for the quantitative real-time detection of biologically important molecules or ions within living cells [12]. The PL lifetime is a parameter that has numerous advantages, such as being independent of the fluorophore concentration and the excitation power, in addition to possible heterogeneities in the optical properties of the medium in which it is located.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines

Ripoll

Roldán

Contreras‐Montoya

et al. 2020

IJMS

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The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes.

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“…However, intracellular multiplexing using ratiometric methods would require at least four different excitation/emission channels that make it a challenging problem that suffers from numerous complications and the lack of robustness, which limits their use. A powerful alternative to ratiometric methods for intracellular sensing is fluorescence lifetime imaging microscopy (FLIM) [ 6 ]. A group of fluorescent molecules that have been promoted to the excited state by a pulse of light will emit fluorescence, usually following exponential decay kinetics, whose decay rate ( k ) defines the so-called fluorescence lifetime ( τ ) as τ = 1/ k , normally on the order of a few nanoseconds.…”

Section: Introductionmentioning

confidence: 99%

“…One of the advantages of FLIM microscopy for intracellular imaging is the possibility of discarding all interferences of cellular autofluorescence by applying a time-gated filtering method, by which the short-lived photons coming from cellular species are removed, leaving in the image only those photons arising from the species of interest [ 6 ]. However, this approach is more efficient when fluorophores exhibiting a long lifetime are employed.…”

Section: Introductionmentioning

confidence: 99%

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions

Herrero-Foncubierta

Paredes

Girón

et al. 2018

Sensors

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The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants.

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FLIM Strategies for Intracellular Sensing

Cited by 15 publications

References 161 publications

Metallofluorescent Nanoparticles for Multimodal Applications

Metallofluorescent Nanoparticles for Multimodal Applications

Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions

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