Fluorescence Lifetime Probes Research Articles

A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria

A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria

Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

Monitoring cancer treatments in melanoma cells using a fluorescence lifetime nanoprobe based on a CdSe/ZnS quantum dot functionalized with a peptide containing D-penicillamine and histidine

Anticancer therapies with cisplatin and volasertib (BI-6727) were monitored by fluorescence lifetime imaging microscopy (FLIM) in live SK-Mel-2 melanoma cells. A CdSe/ZnS quantum dot functionalized with a peptide containing D-penicillamine and histidine (CdSe/ZnS-PH) was used as intracellular pH fluorescent probe. A faster cytosol acidification was observed for cells treated with cisplatin when compared to volasertib. The first changes in the intracellular pH were found after 2 hours of treatment with cisplatin and 8 hours with volasertib. Additionally, the relationship between cytosol acidification and apoptosis was investigated using an innovative methodology based on time-resolved fluorescence measurements. Similar low percentages of apoptotic cells were observed after short incubation periods (2 – 8 hours) with both drugs. In contrast, late apoptosis and death were found for a large fraction of cells during 24-hour incubation with cisplatin but not volasertib. Thus, the early acidification observed in cisplatin treatment could accelerate apoptosis and cell death. Despite volasertib treatment shows slower mechanism of action than cisplatin, similar inhibitory effects were found for both drugs at longer incubation periods (72 hours). This study proves the potential of CdSe/ZnS-PH nanoparticle as a fluorescence lifetime probe in the study of the mechanism of action of antitumor drugs.

Unexpected omega-3 activities in intracellular lipolysis and macrophage foaming revealed by fluorescence lifetime imaging

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo.

A targetable fluorescent probe for detecting mitochondrial viscosity in live cells by using fluorescence lifetime imaging

A D–π–A typed fluorescence lifetime probe for sensitively detecting viscosity has been designed and synthesized. BSOH has been successfully applied to real-time monitoring mitochondrial viscosity in live cells by fluorescence lifetime imaging.

Visualizing pH and Mass Transport at Hydrogen Evolving Electrodes with Fluorescence Lifetime Microscopy

The chemical industry needs electrochemical solutions to avoid large CO₂ emissions. However, many of these electrochemical processes suffer from poor mass transport around the catalyst. To optimize these technologies, we need to gain a better understanding of the local processes at the electrode. This could give us ideas of new catalyst and reactor designs to enhance mass transport.The pH is a parameter that helps us to understand these processes. This is because the catalyst stability and selectivity of many reactions is strongly dependent on the local pH. You need a non-invasive technique to measure in operando spatial maps of pH. Optical techniques are a logical route for this.Fluorescence Lifetime Imaging Microscopy (FLIM) is a technique that measures the fluorescence lifetime of a probe molecule. It is friendly to use, as it is self-referencing (i.e. you do not need to calibrate your system for every new measurement). Many electrochemical applications, like seawater electrolysis, CO2 reduction and alkaline water electrolysis, tend to work in alkaline conditions (pH >8). Unfortunately commercial FLIM probes are only available in neutral and mildly acidic pH (4 to 8), owing to their use in biological systems.We have developed new fluorescence lifetime probes based on the 1-methyl-7-amino-quinolinium fluorophore molecules, that allow pH measurements in the range of pH 5.5-13. We have used the probes to visualise the local pH around a hydrogen evolving electrode and can show the boundary layer that forms close to the electrode and investigate how it is disturbed by gas bubbles at a rate of 3 fps and resolution of 4 μm. Figure 1

A Comparative Study of High-Contrast Fluorescence Lifetime Probes for Imaging Amyloid in Tissue.

Optical imaging of protein aggregates in living and post-mortem tissue can often be impeded by unwanted fluorescence, prompting the need for novel methods to extract meaningful signal in complex biological environments. Historically, benzothiazolium derivatives, prominently Thioflavin T, have been the state-of-the-art fluorescent probes for amyloid aggregates, but their optical, structural, and binding properties typically limit them to in vitro applications. This study compares the use of novel uncharged derivative, PAP_1, with parent Thioflavin T as a fluorescence lifetime imaging probe. This is applied specifically to imaging recombinant α-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to that of Thioflavin T, PAP_1 binds to α-synuclein aggregates with an affinity several orders of magnitude greater than Thioflavin T; thus, we observe a specific decrease in the fluorescence lifetime of PAP_1 bound to α-synuclein aggregates, resulting in a separation of >1.4 standard deviations between PAP_1-stained brain tissue background and α-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between highly fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for α-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.

A coumarin-based TICT fluorescent probe for real-time fluorescence lifetime imaging of mitochondrial viscosity and systemic inflammation in vivo.

Systemic inflammation, linked with abnormal mitochondrial viscosity, is reported to be associated with cerebro-cardiovascular disease and Alzheimer's disease. Therefore, it is of great significance to detect the mitochondrial viscosity to indicate the inflammatory signal in vivo. Considering the strategies of fluorescent molecular rotors (FMRs) and fluorescence lifetime imaging microscopy (FLIM), we have rationally designed a novel mitochondrial viscosity-specific fluorescent probe Mito-VCI, based on coumarin fluorophores with benzo[e]indolium as the rotor group. In a high viscosity solution system, the fluorescence lifetime of the probe Mito-VCI was prolonged due to the planarization and rigidity enhancement of the molecular rotor. Satisfactorily, the probe was only sensitive to viscosity, instead of non-viscosity factors such as pH and polarity. Furthermore, the probe sensitively targeted mitochondria in HeLa cells with a Pearson's correlation of 0.93, and specifically detected dynamics variation of mitochondrial viscosity with FLIM imaging in HeLa cells induced by LPS. Notably, significant fluorescence lifetime changes of Mito-VCI between normal and inflammatory tissues also occurred (for example, the fluorescence lifetime in the spleen changed from 1.128 to 1.432 ns). It can be inferred from the above observations that Mito-VCI could work as an effective and sensitive fluorescent molecular rotor for mitochondrial viscosity monitoring through FLIM imaging with a systemic inflammatory response, and provide potential applications for the diagnosis of systemic inflammation in pharmacology and toxicology studies.

Fluorescence lifetime probes for detection of RNA degradation

To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.

Our group has previously established a strategy utilizing fluorescence lifetime probesto image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individualmitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performingSC-FLIM, including theevaluation of fluorescence lifetimes from the FLIM images. Tovalidatethe feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.

Targeted fluorescence lifetime probes reveal responsive organelle viscosity and membrane fluidity.

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging.

Diazaoxatriangulenium: synthesis of reactive derivatives and conjugation to bovine serum albumin.

The azaoxa-triangulenium dyes are characterised by emission in the red and a long fluorescence lifetime (up to 25 ns). These properties have been widely explored for the azadioxatrianguelnium (ADOTA) dye. Here, the syntheses of reactive maleimide and NHS-ester forms of the diazaoxatriangulenium (DAOTA) system are reported. The DAOTA fluorophore was conjugated to bovine serum albumin (BSA) and investigated in comparison to the corresponding ADOTA-BSA conjugate. It was found that the fluorescence of DAOTA experienced a significantly higher degree of solvent quenching if compared to ADOTA as non-conjugated dyes in aqueous solution, while the fluorescence quenching observed upon conjugation to BSA was significantly reduced for DAOTA when compared to ADOTA. The differences in observed quenching for the conjugates can be explained by the different electronic structures of the dyes, which renders DAOTA significantly less prone to reductive photoinduced electron transfer (PET) quenching from e.g. tryptophan. We conclude that DAOTA, with emission in the red and inherent resistance to PET quenching, is an ideal platform for the development of long fluorescence lifetime probes for time-resolved imaging and fluorescence polarisation assay.

DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins

Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

Photoactivated Green Fluorescence Emission by Femtosecond Oscillator from Indole Solutions

We reported a novel femtosecond-laser-activated fluorescence emission from indole solutions upon excitation by the second harmonic wavelength of a femtosecond oscillator. A new absorption band around 400 nm and corresponding fluorescent band in the green domain were produced after the irradiation of femtosecond laser. This femtosecond-laser-activated luminescence process that allows the use of visible wavelength as a substitute for UV light to excite fluorescence from indole would extend applications based on indole chromophore. Furthermore, the photoactived emission can act as a fluorescence lifetime probe to measure the polarity in complex biological systems since it is polarity-sensitive. High performance liquid chromatography with fluorescence detector (HPLC-FLU) and high performance liquid chromatography with mass spectrometer (HPLC-MS) analysis demonstrate that the origin of the photoactivated fluorescence is new molecular species that generated in indole solution upon femtosecond laser irradiation.

High‐aperture cryogenic light microscopy

We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.

Predicting in vivo fluorescence lifetime behavior of near-infrared fluorescent contrast agents using in vitro measurements

Fluorescence lifetime (FLT) information is complementary to intensity measurement and can be used to improve signal-to-background contrast and provide environment sensing capability. In this study, we evaluate the FLTs of eight near-infrared fluorescent molecular probes in vitro in various solvent mediums and in vivo to establish the correlation between the in vitro and in vivo results. Compared with other mediums, two exponential fittings of the fluorescence decays of dyes dissolved in aqueous albumin solutions accurately predict the range of FLTs observed in vivo. We further demonstrate that the diffusion of a near-infrared (NIR) reporter from a dye-loaded gel can be detected by FLT change in mice as a model of controlled drug release. The mean FLT of the NIR probe increases as the dye diffuses from the highly polar gel interior to the more lipophilic tissue environment. The two-point analysis demonstrates an efficient in vitro method for screening new NIR fluorescent reporters for use as FLT probes in vivo, thereby minimizing the use of animals for FLT screening studies.

A hyperspectral fluorescence lifetime probe for skin cancer diagnosis

The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.

Detection of single nucleotide polymorphisms through the application of lifetime fluorescent probes

Fluorescence-based approaches for genotyping single nucleotide polymorphisms (SNPs) commonly involve enzymatic cleavage, stringent control of hybridization temperature, or extensive post amplification clean up. Our research to SNP genotyping employs fluorescence lifetime probes, which consist of a single fluorophore that is covalently bound to a oligonucleotide complementary to a SNP allele. We require that the matched and mismatched forms correspond to different lifetimes of the fluorophore. In other words, fluorophore lifetime reports whether the base has entered into the double strand or is mismatched. The constraints on hybridization conditions are greatly relaxed and enzymatic cleavage is not required. This fluorescent lifetime-based technology allows resolving SNP alleles in both the homozygous and heterozygous states using a single lifetime probe. Initial studies have been conducted on the human β-globin gene, known to cause sickle cell anemia. Our results show the fluorophore lifetime is measurably different for the mutant and wild forms. We are currently characterizing the effects of probe/target base pair mismatch beyond the initial SNP. Preliminary studies suggest fluorescence polarization data likely provides additional specificity. This research was supported by the North Dakota INBRE grant.

All-Solid-State Diode-Pumped Fluorescence Lifetime Imaging System for Biomedicaine and Microscopy

The determination of fluorescence lifetime requires only relative measurements of intensity and so is especially useful for biomedical samples in which the heterogeneous nature of tissue and autofluorescence cause significant problems. Since fluorescence lifetime is dependent upon both radiative and non-radiative decay rates, it may be used to distinguish between different fluorophore molecules (with different radiative decay rates) and to monitor local environmental perturbations that affect the non-radiative decay rate. Fluorescence lifetime probes have been demonstrated for many biologically significant analytes including [O2], [Ca2+] and pH. Fluorescence lifetime imaging (FLIM) can be applied to almost any optical imaging modality, including microscopy and potentially to non-invasive optical biopsy. Fluorescence lifetime data may be acquired in the frequency or time domain. The recent development of user-friendly and relatively portable ultrafast laser technology and the availability of ultrafast gated optical image intensifiers (GOI’s) enable the development of potentially inexpensive time domain FLIM instruments that may be deployed outside specialist laser laboratories.