Architecting a transcriptional repressor-based genetic inverter for tryptophan derived pathway regulation in Escherichia coli.

Efficient microbial cell factories require intricate and precise metabolic regulations for optimized production, which can be significantly aided by implementing regulatory genetic circuits with versatile functions. However, constructing functionally diverse genetic circuits in host strains is challenging. Especially, functional diversification based on transcriptional repressors has been rarely explored due to the difficulty in inverting their repression properties. To address this, we proposed a design logic to create transcriptional repressor-based genetic inverters for functional enrichment. As proof of concept, a tryptophan-inducible genetic inverter was constructed by integrating two sets of transcriptional repressors, PtrpO1-TrpR1 and PtetO1-TetR. In this genetic inverter, the repression of TetR towards PtetO1 could be alleviated by the tryptophan-TrpR1 complex in the presence of tryptophan, leading to the activated output. Subsequently, we optimized the dynamic performance of the inverter and constructed tryptophan-triggered dynamic activation systems. Further coupling of the original repression function of PtrpO1-TrpR1 with inverter variants realized the tryptophan-triggered bifunctional regulation system. Finally, the dynamic regulation systems enabled tryptophan production monitoring. These systems also remarkably increased the titers of the tryptophan derivatives tryptamine and violacein by 2.0-fold and 7.4-fold, respectively. The successful design and application of the genetic inverter enhanced the applicability of transcriptional repressors.

Roadmap on computational methods in optical imaging and holography [invited].

Computational methods have been established as cornerstones in optical imaging and holography in recent years. Every year, the dependence of optical imaging and holography on computational methods is increasing significantly to the extent that optical methods and components are being completely and efficiently replaced with computational methods at low cost. This roadmap reviews the current scenario in four major areas namely incoherent digital holography, quantitative phase imaging, imaging through scattering layers, and super-resolution imaging. In addition to registering the perspectives of the modern-day architects of the above research areas, the roadmap also reports some of the latest studies on the topic. Computational codes and pseudocodes are presented for computational methods in a plug-and-play fashion for readers to not only read and understand but also practice the latest algorithms with their data. We believe that this roadmap will be a valuable tool for analyzing the current trends in computational methods to predict and prepare the future of computational methods in optical imaging and holography. Supplementary Information: The online version contains supplementary material available at 10.1007/s00340-024-08280-3.

Optimizing self-interference digital holography for single-molecule localization.

Self-interference digital holography (SIDH) can image incoherently emitting objects over large axial ranges from three two-dimensional images. By combining SIDH with single-molecule localization microscopy (SMLM), incoherently emitting objects can be localized with nanometer precision over a wide axial range without mechanical refocusing. However, background light substantially degrades the performance of SIDH due to the relatively large size of the hologram. To optimize the performance of SIDH, we performed simulations to study the optimal hologram radius (Rh) for different levels of background photons. The results show that by reducing the size of the hologram, we can achieve a localization precision of better than 60 nm laterally and 80 nm axially over a 10 µm axial range under the conditions of low signal level (6000 photons) with 10 photons/pixel of background noise. We then performed experiments to demonstrate our optimized SIDH system. The results show that point sources emitting as few as 2120 photons can be successfully detected. We further demonstrated that we can successfully reconstruct point-like sources emitting 4200 photons over a 10 µm axial range by light-sheet SIDH.

Three-dimensional nanoscale localization of point-like objects using self-interference digital holography.

We propose localizing point-like fluorescent emitters in three dimensions with nanometer precision throughout large volumes using self-interference digital holography (SIDH). SIDH enables imaging of incoherently emitting objects over large axial ranges without refocusing, and single molecule localization techniques allow sub-50 nm resolution in the lateral and axial dimensions. We demonstrate three-dimensional localization with SIDH by imaging 100 and 40 nm fluorescent nanospheres. With 49,000 photons detected, SIDH achieves a localization precision of 5 nm laterally and 40 nm axially. We are able to detect the nanospheres from as few as 13,000 detected photons.

Stripe artifact reduction for digital scanned structured illumination light sheet microscopy.

Light sheet microscopy is an important and widely used method for studying large and semi-opaque biological specimens. One drawback of the approach is that it often results in stripe artifacts due to absorption and scattering in the illumination path. Here we describe a new approach which will effectively mitigate the artifacts in digital scanned light sheet microscopy (DSLM) and digital scanned structured illumination light sheet microscopy (DSLM-SI). We further improve the results of DSLM-SI through a new reconstruction method which achieves clearer reconstructed images. We demonstrate the reduction of stripe artifacts by imaging 156 microns deep into the larval zebrafish central nervous system. The magnitude of stripe artifacts is reduced by an average of 20% across three datasets.

Charge-Directed Immobilization of Bacteriophage on Nanostructured Electrode for Whole-Cell Electrochemical Biosensors.

A new type of carbon nanotube (CNT)-based impedimetric biosensing method has been developed for rapid and selective detection of live bacterial cells. A proof-of-concept study was conducted using T2 bacteriophage-based biosensors for electrochemical detection of Escherichia coli B. The T2 bacteriophage (virus) served as the biorecognition element, which was immobilized on polyethylenimine (PEI)-functionalized carbon nanotube transducer on glassy carbon electrode. Charge-directed, orientated immobilization of bacteriophage particles on carbon nanotubes was achieved through covalent linkage of phage capsid onto the carbon nanotubes. The presence of the immobilized phage on carbon nanotube-modified electrode was confirmed by fluorescence microscopy. Electrochemical impedance spectroscopy (EIS) was used to monitor the changes in the interfacial impedance due to the binding of E. coli B to T2 phage on the CNT-modified electrode. The detection was highly selective toward the B strain of E. coli as no signal was observed for the nonhost K strain of E. coli. The present achievable detection limit of the biosensor is 103 CFU/mL.

Interferometric imaging with three objectives.

We propose a microscope that utilizes three high numerical aperture objectives to image a sample from multiple angles, interferometrically combining the fields from each objective to obtain a higher resolution and more isotropic point-spread function than standard wide-field and confocal techniques. The proposed microscope utilizes three 0.9 NA objectives with large working distance and, therefore, has both a large imaging volume and an effective 2.7 NA. The FWHM of the point-spread function is 135 nm in the plane of the objectives, a factor of 2 better than the lateral resolution with a single objective. Out of plane, the PSF is equivalent to that of a conventional microscope. Here, we introduce the theoretical framework for evaluating the performance of the microscope and use that framework to quantitatively compare our proposed microscope to existing techniques in confocal and wide-field implementations.

Adaptive optics stochastic optical reconstruction microscopy (AO-STORM) using a genetic algorithm.

The resolution of Single Molecule Localization Microscopy (SML) is dependent on the width of the Point Spread Function (PSF) and the number of photons collected. However, biological samples tend to degrade the shape of the PSF due to the heterogeneity of the index of refraction. In addition, there are aberrations caused by imperfections in the optical components and alignment, and the refractive index mismatch between the coverslip and the sample, all of which directly reduce the accuracy of SML. Adaptive Optics (AO) can play a critical role in compensating for aberrations in order to increase the resolution. However the stochastic nature of single molecule emission presents a challenge for wavefront optimization because the large fluctuations in photon emission do not permit many traditional optimization techniques to be used. Here we present an approach that optimizes the wavefront during SML acquisition by combining an intensity independent merit function with a Genetic algorithm (GA) to optimize the PSF despite the fluctuating intensity. We demonstrate the use of AO with GA in tissue culture cells and through ~50µm of tissue in the Drosophila Central Nervous System (CNS) to achieve a 4-fold increase in the localization precision.

Super-resolution 3D microscopy of live whole cells using structured illumination.

Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.

Micelle-templated composite quantum dots for super-resolution imaging.

Quantum dots (QDs) have tremendous potential for biomedical imaging, including super-resolution techniques that permit imaging below the diffraction limit. However, most QDs are produced via organic methods, and hence require surface treatment to render them water-soluble for biological applications. Previously, we reported a micelle-templating method that yields nanocomposites containing multiple core/shell ZnS-CdSe QDs within the same nanocarrier, increasing overall particle brightness and virtually eliminating QD blinking. Here, this technique is extended to the encapsulation of Mn-doped ZnSe QDs (Mn-ZnSe QDs), which have potential applications in super-resolution imaging as a result of the introduction of Mn(2+) dopant energy levels. The size, shape and fluorescence characteristics of these doped QD-micelles were compared to those of micelles created using core/shell ZnS-CdSe QDs (ZnS-CdSe QD-micelles). Additionally, the stability of both types of particles to photo-oxidation was investigated. Compared to commercial QDs, micelle-templated QDs demonstrated superior fluorescence intensity, higher signal-to-noise ratios, and greater stability against photo-oxidization,while reducing blinking. Additionally, the fluorescence of doped QD-micelles could be modulated from a bright 'on' state to a dark 'off' state, with a modulation depth of up to 76%, suggesting the potential of doped QD-micelles for applications in super-resolution imaging.

A modular platform to generate functional sympathetic neuron-innervated heart assembloids.

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.

Decreased GABA levels during development result in increased connectivity in the larval zebrafish tectum.

γ-aminobutyric acid (GABA) is an abundant neurotransmitter that plays multiple roles in the vertebrate central nervous system (CNS). In the early developing CNS, GABAergic signaling acts to depolarize cells. It mediates several aspects of neural development, including cell proliferation, neuronal migration, neurite growth, and synapse formation, as well as the development of critical periods. Later in CNS development, GABAergic signaling acts in an inhibitory manner when it becomes the predominant inhibitory neurotransmitter in the brain. This behavior switch occurs due to changes in chloride/cation transporter expression. Abnormalities of GABAergic signaling appear to underlie several human neurological conditions, including seizure disorders. However, the impact of reduced GABAergic signaling on brain development has been challenging to study in mammals. Here we take advantage of zebrafish and light sheet imaging to assess the impact of reduced GABAergic signaling on the functional circuitry in the larval zebrafish optic tectum. Zebrafish have three gad genes: two gad1 paralogs known as gad1a and gad1b, and gad2. The gad1b and gad2 genes are expressed in the developing optic tectum. Null mutations in gad1b significantly reduce GABA levels in the brain and increase electrophysiological activity in the optic tectum. Fast light sheet imaging of genetically encoded calcium indicator (GCaMP)-expressing gab1b null larval zebrafish revealed patterns of neural activity that were different than either gad1b-normal larvae or gad1b-normal larvae acutely exposed to pentylenetetrazole (PTZ). These results demonstrate that reduced GABAergic signaling during development increases functional connectivity and concomitantly hyper-synchronization of neuronal networks.

Phase diversity for three-dimensional imaging.

Phase diversity (PD) is a powerful technique for estimating wavefront aberrations from two-dimensional images of extended scenes. PD can work with extended incoherent images and, in an adaptive optics system, does not need extra hardware in addition to the deformable mirror. For these reasons, PD should be well suited to aberration measurement in microscopy applications. But, in biological widefield microscopy, the objects being imaged are frequently three-dimensional, and the images contain out-of-focus light. In this paper, we introduce multiplane PD and show that PD can be extended to widefield imaging of three-dimensional objects. This should be particularly useful in the field of biological fluorescence microscopy where the objects are very light sensitive and the aberrations cannot easily be determined.

Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

Adaptive optics for optical microscopy [Invited].

Optical microscopy is widely used to visualize fine structures. When applied to bioimaging, its performance is often degraded by sample-induced aberrations. In recent years, adaptive optics (AO), originally developed to correct for atmosphere-associated aberrations, has been applied to a wide range of microscopy modalities, enabling high- or super-resolution imaging of biological structure and function in complex tissues. Here, we review classic and recently developed AO techniques and their applications in optical microscopy.

Volumetric light sheet imaging with adaptive optics correction.

Light sheet microscopy has developed quickly over the past decades and become a popular method for imaging live model organisms and other thick biological tissues. For rapid volumetric imaging, an electrically tunable lens can be used to rapidly change the imaging plane in the sample. For larger fields of view and higher NA objectives, the electrically tunable lens introduces aberrations in the system, particularly away from the nominal focus and off-axis. Here, we describe a system that employs an electrically tunable lens and adaptive optics to image over a volume of 499 × 499 × 192 µm3 with close to diffraction-limited resolution. Compared to the system without adaptive optics, the performance shows an increase in signal to background ratio by a factor of 3.5. While the system currently requires 7s/volume, it should be straightforward to increase the imaging speed to under 1s per volume.

Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics.

Introduction: Mitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging. Methods: In this study, we developed a two-photon fluorescence microscope with adaptive optics (TPFM-AO) for high-resolution imaging, which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensorless approach for correcting low order tissue aberrations. Results: Using AO increases the fluorescence intensity of the point spread function (PSF) and achieves fast imaging of subcellular organelles with 400 nm resolution through 85 µm of highly scattering tissue. We achieved ~1.55×, ~3.58×, and ~1.77× intensity increases using AO, and a reduction of the PSF width by ~0.83×, ~0.74×, and ~0.9× at the depths of 0, 50 µm and 85 µm in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5× 67.5 µm. We also investigate the role of initial signal and background levels in sample correction quality by varying the laser power and camera exposure time and develop an intensity-based criteria for sample correction. Discussion: This study demonstrates a promising tool for imaging of mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.

Super-resolution video microscopy of live cells by structured illumination.

Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

Fundamental precision bounds for three-dimensional optical localization microscopy using self-interference digital holography.

Localization based microscopy using self-interference digital holography (SIDH) provides three-dimensional (3D) positional information about point sources with nanometer scale precision. To understand the performance limits of SIDH, here we calculate the theoretical limit to localization precision for SIDH when designed with two different configurations. One configuration creates the hologram using a plane wave and a spherical wave while the second configuration creates the hologram using two spherical waves. We further compare the calculated precision bounds to the 3D single molecule localization precision from different Point Spread Functions. SIDH results in almost constant localization precision in all three dimensions for a 20 µm thick depth of field. For high signal-to-background ratio (SBR), SIDH on average achieves better localization precision. For lower SBR values, the large size of the hologram on the detector becomes a problem, and PSF models perform better.

Gd3+-activated narrowband ultraviolet-B persistent luminescence through persistent energy transfer.

This work reports the realization of Gd3+ persistent luminescence in the narrowband ultraviolet-B (NB-UVB; 310-313 nm) through persistent energy transfer from a sensitizer of Pr3+, Pb2+ or Bi3+. We propose a general design concept to develop Gd3+-activated NB-UVB persistent phosphors from Pr3+-, Pb2+- or Bi3+-activated ultraviolet-C (200-280 nm) or ultraviolet-B (280-315 nm) persistent phosphors, leading to the discovery of ten Gd3+ NB-UVB persistent phosphors such as Sr3Gd2Si6O18:Pr3+, Sr3Gd2Si6O18:Pb2+ and Y2GdAl2Ga3O12:Bi3+ as well as five ultraviolet-B persistent phosphors such as Y3Al2Ga3O12:Pr3+, Sr3Y2Si6O18:Pb2+ and Y3Al2Ga3O12:Bi3+. The persistent energy transfer from the sensitizers to Gd3+ is very efficient and the Gd3+ NB-UVB afterglow can last for more than 10 hours. This study expands the persistent luminescence research to the NB-UVB as well as the broader ultraviolet-B spectral regions. The NB-UVB persistent phosphors may act as self-sustained glowing NB-UVB radiation sources for dermatological therapy.