Julia Roberti

Bio: Julia Roberti is an academic researcher from Leica Microsystems. The author has contributed to research in topics: Fluorophore & Rhodamines. The author has an hindex of 1, co-authored 4 publications receiving 93 citations.

A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy

Abstract: Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. It is difficult to develop suitable fluorescent probes for live-cell nanoscopy, but a general strategy is now reported that can transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signals. Using this approach, probes in a variety of colours were developed for different cellular targets and used for wash-free, multicolour, live-cell confocal and STED microscopy.

A general strategy to develop cell permeable and fluorogenic probes for multi-colour nanoscopy

Abstract: Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from low cell permeability and unspecific background signal. We report a general strategy to transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signal. The strategy is based on the conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide. This conversion does not affect the spectroscopic properties of the fluorophore but permits it to exist in a dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell permeable spirolactam. Probes based on such fluorophores generally are fluorogenic as the equilibrium shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1000-fold. Using this simple design principle we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. The work establishes a general strategy to develop fluorogenic probes for live-cell bioimaging.

HaloTag9: an engineered protein tag to improve fluorophore performance

Abstract: HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

HaloTag8: an engineered protein tag to improve fluorophore performance

Abstract: HaloTag8 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag8 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag8 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

Tutorial: guidance for quantitative confocal microscopy.

Abstract: When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, choosing the most suitable microscope for a given application and configuring the microscope parameters. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. Common pitfalls such as photobleaching and cross-talk are addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. Finally, guidelines for analyzing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. A visual summary of this tutorial is available as a poster (https://doi.org/10.1038/s41596-020-0307-7).

Molecular Fluorophores for Deep-Tissue Bioimaging.

Abstract: Fluorescence imaging has made tremendous inroads toward understanding the complexity of biological systems, but in vivo deep-tissue imaging remains a great challenge due to the optical opacity of biological tissue. Recent improvements in laser and detector manufacturing have allowed the expansion of nonlinear and linear fluorescence imaging to the underexplored "tissue-transparent" second near-infrared (NIR-II; 1000-1700 nm) window, opening up new opportunities for optical access deep inside opaque tissue. Molecular fluorophores have historically played a major role in fluorescence bioimaging. It is increasingly important to design new molecular fluorophores to fully unlock the potential of NIR-II imaging techniques. In this outlook, we give an overview of the novel molecular fluorophores developed for deep-tissue bioimaging in the past five years and discuss their pros and cons in applications. Guidelines for designing new molecular fluorophores with the desirable properties are also provided.

A general method to optimize and functionalize red-shifted rhodamine dyes.

Abstract: Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted ‘Janelia Fluor’ (JF) dyes useful for biological imaging experiments in cells and in vivo. A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.

Rational design of small molecule fluorescent probes for biological applications

Abstract: Fluorescent small molecules are powerful tools for visualizing biological events, embodying an essential facet of chemical biology. Since the discovery of the first organic fluorophore, quinine, in 1845, both synthetic and theoretical efforts have endeavored to "modulate" fluorescent compounds. An advantage of synthetic dyes is the ability to employ modern organic chemistry strategies to tailor chemical structures and thereby rationally tune photophysical properties and functionality of the fluorophore. This review explores general factors affecting fluorophore excitation and emission spectra, molar absorption, Stokes shift, and quantum efficiency; and provides guidelines for chemist to create novel probes. Structure-property relationships concerning the substituents are discussed in detail with examples for several dye families. We also present a survey of functional probes based on PeT, FRET, and environmental or photo-sensitivity, focusing on representative recent work in each category. We believe that a full understanding of dyes with diverse chemical moieties enables the rational design of probes for the precise interrogation of biochemical and biological phenomena.

Rational Design of a Highly Selective Near-Infrared Two-Photon Fluorogenic Probe for Imaging Orthotopic Hepatocellular Carcinoma Chemotherapy.

Abstract: Selective fluorescence imaging of biomarkers in vivo and in situ for evaluating orthotopic hepatocellular carcinoma (HCC) chemotherapy remains a great challenge due to current imaging agents suffering from the potential interferences of other hydrolases. Herein, we observed that carbamate unit showed a high selectivity toward the HCC-related biomarker carboxylesterase (CE) for evaluation of treatment. A near-infrared two-photon fluorescent probe was developed to not only specially image CE activity in vivo and in situ but also target orthotopic liver tumor after systemic administration. The in vivo signals of the probe correlate well with tumor apoptosis, making it possible to evaluate the status of treatment. The probe enables the imaging of CE activity in situ with a high-resolution three-dimensional view for the first time. This study may promote advances in optical imaging approaches for precise imaging-guided diagnosis of HCC in situ and its evaluation of treatment.