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Article ; Online: Axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy.

Gao, Peng / Nienhaus, G Ulrich

Optics letters

2021 Volume 46, Issue 9, Page(s) 2184–2187

Abstract: Investigating the dynamics and interactions of biomolecules within or attached to membranes of living cells is crucial for understanding biology at the molecular level. In this pursuit, classical, diffraction-limited optical fluorescence microscopy is ... ...

Abstract	Investigating the dynamics and interactions of biomolecules within or attached to membranes of living cells is crucial for understanding biology at the molecular level. In this pursuit, classical, diffraction-limited optical fluorescence microscopy is widely used, but it faces limitations due to (1) the heterogeneity of biomembranes on the nanoscale and (2) the intrinsic motion of membranes with respect to the focus. Here we introduce a new confocal microscopy-based fluctuation spectroscopy technique aimed at alleviating these two problems, called axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy (axial ls-STED-FCS). Axial line scanning by means of a tunable acoustic gradient index of refraction lens provides a time resolution of a few microseconds, which is more than two orders of magnitude greater than that of conventional, lateral line-scanning fluorescence correlation spectroscopy (typically around 1 ms). Using STED excitation, the observation area on the membrane can be reduced 10-100 fold, resulting in sub-diffraction spatial resolution and the ability to study samples with densely labeled membranes. Due to these attractive properties, we expect that the axial ls-STED-FCS will find wide application, especially in the biomolecular sciences.
MeSH term(s)	Diffusion ; Spectrometry, Fluorescence
Language	English
Publishing date	2021-04-30
Publishing country	United States
Document type	Journal Article
ISSN	1539-4794
ISSN (online)	1539-4794
DOI	10.1364/OL.420765
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Article: Different Mechanisms of Catalytic Complex Formation in Two L-Tryptophan Processing Dioxygenases.

Nienhaus, Karin / Nienhaus, G Ulrich

Frontiers in molecular biosciences

2018 Volume 4, Page(s) 94

Abstract: The human heme enzymes tryptophan 2,3-dioxygenase (hTDO) and indoleamine 2,3 dioxygenase (hIDO) catalyze the initial step in L-tryptophan (L-Trp) catabolism, the insertion of dioxygen into L-Trp. Overexpression of these enzymes causes depletion of L-Trp ... ...

Abstract	The human heme enzymes tryptophan 2,3-dioxygenase (hTDO) and indoleamine 2,3 dioxygenase (hIDO) catalyze the initial step in L-tryptophan (L-Trp) catabolism, the insertion of dioxygen into L-Trp. Overexpression of these enzymes causes depletion of L-Trp and accumulation of metabolic products, and thereby contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. Understanding the assembly of the catalytically active, ternary enzyme-substrate-ligand complexes is not yet fully resolved, but an essential prerequisite for designing efficient and selective de novo inhibitors. Evidence is mounting that the ternary complex forms by sequential binding of ligand and substrate in a specific order. In hTDO, the apolar L-Trp binds first, decreasing active-site solvation and, as a result, reducing non-productive oxidation of the heme iron by the dioxygen ligand, which may leave the substrate bound to a ferric heme iron. In hIDO, by contrast, dioxygen must first coordinate to the heme iron because a bound substrate would occlude ligand access to the heme iron, so the ternary complex can no longer form. Consequently, faster association of L-Trp at high concentrations results in substrate inhibition. Here, we summarize our present knowledge of ternary complex formation in hTDO and hIDO and relate these findings to structural peculiarities of their active sites.
Language	English
Publishing date	2018-01-04
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2814330-9
ISSN	2296-889X
ISSN	2296-889X
DOI	10.3389/fmolb.2017.00094
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Article: Wavelet-based background and noise subtraction for fluorescence microscopy images.

Hüpfel, Manuel / Yu Kobitski, Andrei / Zhang, Weichun / Nienhaus, G Ulrich

Biomedical optics express

2021 Volume 12, Issue 2, Page(s) 969–980

Abstract: Fluorescence microscopy images are inevitably contaminated by background intensity contributions. Fluorescence from out-of-focus planes and scattered light are important sources of slowly varying, low spatial frequency background, whereas background ... ...

Abstract	Fluorescence microscopy images are inevitably contaminated by background intensity contributions. Fluorescence from out-of-focus planes and scattered light are important sources of slowly varying, low spatial frequency background, whereas background varying from pixel to pixel (high frequency noise) is introduced by the detection system. Here we present a powerful, easy-to-use software, wavelet-based background and noise subtraction (WBNS), which effectively removes both of these components. To assess its performance, we apply WBNS to synthetic images and compare the results quantitatively with the ground truth and with images processed by other background removal algorithms. We further evaluate WBNS on real images taken with a light-sheet microscope and a super-resolution stimulated emission depletion microscope. For both cases, we compare the WBNS algorithm with hardware-based background removal techniques and present a quantitative assessment of the results. WBNS shows an excellent performance in all these applications and significantly enhances the visual appearance of fluorescence images. Moreover, it may serve as a pre-processing step for further quantitative analysis.
Language	English
Publishing date	2021-01-22
Publishing country	United States
Document type	Journal Article
ZDB-ID	2572216-5
ISSN	2156-7085
ISSN	2156-7085
DOI	10.1364/BOE.413181
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Article ; Online: Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells.

Ambrosi, Giulia / Voloshanenko, Oksana / Eckert, Antonia F / Kranz, Dominique / Nienhaus, G Ulrich / Boutros, Michael

eLife

2022 Volume 11

Abstract: Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β- ... ...

Abstract	Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous β-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both β-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or a truncating
MeSH term(s)	Alleles ; Carcinogenesis/genetics ; Carcinoma, Hepatocellular/genetics ; Colorectal Neoplasms/genetics ; Genetic Engineering/methods ; Genetic Variation ; Genome ; HCT116 Cells ; Humans ; Liver Neoplasms/genetics ; Mutation ; Wnt Signaling Pathway/physiology ; beta Catenin/analysis ; beta Catenin/genetics
Chemical Substances	CTNNB1 protein, human ; beta Catenin
Language	English
Publishing date	2022-01-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.64498
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Article ; Online: Precise background subtraction in stimulated emission double depletion nanoscopy.

Gao, Peng / Nienhaus, G Ulrich

Optics letters

2017 Volume 42, Issue 4, Page(s) 831–834

Abstract: Low-resolution background in stimulated emission depletion (STED) nanoscopy can arise from incomplete depletion or re-excitation by the STED beam. We have recently introduced stimulated emission double depletion (STEDD), a technique to efficiently ... ...

Abstract	Low-resolution background in stimulated emission depletion (STED) nanoscopy can arise from incomplete depletion or re-excitation by the STED beam. We have recently introduced stimulated emission double depletion (STEDD), a technique to efficiently suppress this background. In STEDD, the conventional, doughnut-shaped STED pulse, which depletes excited fluorophores outside the center of the focal region, is followed by a second Gaussian STED pulse, which specifically depletes the central region. The background is removed by calculating a weighted difference of photon events collected before and after the second STED pulse. Here, we present a simple, yet powerful, method to determine the weight factor, which depends on the fluorescence decay, from a direct analysis of the acquired data. We vary the weight factor to identify its optimal value as the one for which the weight of high-frequency components in the spectrum of the acquired STEDD image is maximized. This strategy is also applicable to other differential approaches for background suppression in imaging.
Language	English
Publishing date	2017--15
Publishing country	United States
Document type	Journal Article
ISSN	1539-4794
ISSN (online)	1539-4794
DOI	10.1364/OL.42.000831
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Article ; Online: Stimulated emission double depletion nanoscopy with background correction at the single-pixel level.

Sedeh, Amirhossein Barati / Kobitski, Andrei / Dai, Siqing / Eroğlu-Kayıkçı, Süheyla / Nienhaus, Karin / Hilbert, Lennart / Nienhaus, G Ulrich

Optics letters

2023 Volume 48, Issue 21, Page(s) 5791–5794

Abstract: Fluorescence microscopy images are inevitably tainted by background contributions including emission from out-of-focus planes, scattered light, and detector noise. In stimulated emission depletion (STED) nanoscopy, an additional, method-specific ... ...

Abstract	Fluorescence microscopy images are inevitably tainted by background contributions including emission from out-of-focus planes, scattered light, and detector noise. In stimulated emission depletion (STED) nanoscopy, an additional, method-specific background arises from incomplete depletion and re-excitation by the depletion beam. Various approaches have been proposed to remove the background from a STED image, some of which rely on the acquisition of a separate background image that is subtracted from the STED image with a weighting factor. Using stimulated emission double depletion (STEDD) nanoscopy, we observed that the weighting factor varies locally in densely labeled samples, so that background removal with a single (global) weighting factor generates local image artifacts due to incorrect background subtraction. Here we present an algorithm that computes the optimal weighting factor at the single-pixel level, yielding a difference image with excellent suppression of low-frequency background.
Language	English
Publishing date	2023-10-31
Publishing country	United States
Document type	Journal Article
ISSN	1539-4794
ISSN (online)	1539-4794
DOI	10.1364/OL.502001
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Article ; Online: In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy.

Shang, Li / Nienhaus, G Ulrich

Accounts of chemical research

2017 Volume 50, Issue 2, Page(s) 387–395

Abstract: Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which ...

Abstract	Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the "protein corona", forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with negatively charged NP surfaces, and the observed protein layer thickness always matched the known molecular dimensions of the proteins binding in certain orientations. Temperature and NP surface functionalization have also been identified as important parameters controlling protein corona formation. Notably, while the corona formed from a single type of serum protein was reversible, protein adsorption from complex biological media such as blood serum was entirely irreversible. These quantitative in vitro studies are of great relevance to the bio-nano community and especially to researchers developing engineered nanomaterials for biological and biomedical applications. Future efforts will be directed toward elucidating kinetic aspects of protein corona formation and the detailed structure of the adsorbed proteins at the molecular level. To better appreciate the biological responses triggered by NP exposure, more efforts will be devoted to the exploration of the biomolecular corona as it forms on NPs in contact with living cells, tissues, and even entire model organisms. These studies are challenging when performed in a well-controlled and quantitative fashion and rely on the availability of sophisticated analytical tools, particularly, quantitative optical imaging techniques including FCS and related fluctuation methods.
MeSH term(s)	Adsorption ; Calorimetry ; Humans ; Nanoparticles/chemistry ; Nanoparticles/metabolism ; Protein Binding ; Protein Corona/chemistry ; Proteins/chemistry ; Proteins/metabolism ; Serum Albumin/chemistry ; Serum Albumin/metabolism ; Spectrometry, Fluorescence ; Static Electricity ; Surface Properties ; Temperature
Chemical Substances	Protein Corona ; Proteins ; Serum Albumin
Language	English
Publishing date	2017-02-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1483291-4
ISSN	1520-4898 ; 0001-4842
ISSN (online)	1520-4898
ISSN	0001-4842
DOI	10.1021/acs.accounts.6b00579
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Article ; Online: A fatigue-resistant photoswitchable fluorescent protein for optical nanoscopy.

Nienhaus, G Ulrich

Angewandte Chemie (International ed. in English)

2012 Volume 51, Issue 6, Page(s) 1312–1314

MeSH term(s)	Fluorescent Dyes/chemistry ; Green Fluorescent Proteins/chemistry ; Microscopy, Confocal ; Microscopy, Fluorescence/methods ; Nanotechnology/instrumentation ; Nanotechnology/methods ; Photochemical Processes
Chemical Substances	Fluorescent Dyes ; Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2012-02-06
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2011836-3
ISSN	1521-3773 ; 1433-7851
ISSN (online)	1521-3773
ISSN	1433-7851
DOI	10.1002/anie.201108036
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Article ; Online: Chromophore photophysics and dynamics in fluorescent proteins of the GFP family.

Nienhaus, Karin / Nienhaus, G Ulrich

Journal of physics. Condensed matter : an Institute of Physics journal

2016 Volume 28, Issue 44, Page(s) 443001

Abstract: Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many ... ...

Abstract	Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.
Language	English
Publishing date	2016-11-09
Publishing country	England
Document type	Journal Article
ZDB-ID	1472968-4
ISSN	1361-648X ; 0953-8984
ISSN (online)	1361-648X
ISSN	0953-8984
DOI	10.1088/0953-8984/28/44/443001
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Article ; Online: Confocal laser scanning microscopy with spatiotemporal structured illumination.

Gao, Peng / Nienhaus, G Ulrich

Optics letters

2016 Volume 41, Issue 6, Page(s) 1193–1196

Abstract: Confocal laser scanning microscopy (CLSM), which is widely utilized in the biological and biomedical sciences, is limited in spatial resolution due to diffraction to about half the light wavelength. Here we have combined structured illumination with CLSM ...

Abstract	Confocal laser scanning microscopy (CLSM), which is widely utilized in the biological and biomedical sciences, is limited in spatial resolution due to diffraction to about half the light wavelength. Here we have combined structured illumination with CLSM to enhance its spatial resolution. To this end, we have used a spatial light modulator (SLM) to generate fringe patterns of different orientations and phase shifts in the excitation spot without any mechanical movement. We have achieved 1.8 and 1.7 times enhanced lateral and axial resolutions, respectively, by synthesizing the object spectrum along different illumination directions. This technique is thus a promising tool for high-resolution morphological or fluorescence imaging, especially in deep tissue.
MeSH term(s)	Gold/chemistry ; Lighting/methods ; Mechanical Phenomena ; Metal Nanoparticles ; Microscopy, Confocal/methods ; Spatio-Temporal Analysis
Chemical Substances	Gold (7440-57-5)
Language	English
Publishing date	2016-03-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1539-4794
ISSN (online)	1539-4794
DOI	10.1364/OL.41.001193
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