Real‐time and non‐invasive monitoring of neuronal differentiation helps to increase understanding of neuronal development and develop stem cell therapies for neurodegenerative diseases. Conventional methods such as RT‐PCR, western blotting, and immunofluorescence (IF), lack single‐cell‐level resolution and require invasive procedures, fixation, and staining. These limitations hinder accurate monitoring progress of neural stem cell (NSC) differentiation and understanding its functions. Herein, a novel approach is reported to non‐invasively monitor neuronal differentiation in real‐time using cell‐based biosensors (CBBs) that detects hippocalcin, biomarker of neuronal differentiation. To construct the hippocalcin sensor proteins, two different hippocalcin bioreceptors are fused to each split‐intein, carrying split‐nuclear localization signal (NLS) peptides, respectively, and fluorescent protein is introduced as reporter. CBBs operated in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The NSC‐based biosensor shows fluorescence signal translocation only upon neuronal differentiation and not undifferentiated stem cells or glial cells. Furthermore, this approach allows monitoring of neural differentiation at earlier stages than detected using IF staining. It is believed that novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single‐cell‐level and providing a tool to evaluate how NSCs efficiently differentiate into neurons.
The imaging of real‐time fluxes of K+ions in live cell with high dynamic range (5–150 × 10−3
- NSF-PAR ID:
- 10049234
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Functional Materials
- Volume:
- 28
- Issue:
- 9
- ISSN:
- 1616-301X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
more » « less
Abstract The mouse subventricular zone (SVZ) produces neurons throughout life. It is useful for mechanism discovery and is relevant for regeneration. However, the SVZ is deep, significantly restricting live imaging since current methods do not extend beyond a few hundred microns. We developed and adapted three-photon microscopy (3PM) for non-invasive deep brain imaging in live mice, but its utility in imaging the SVZ niche was unknown. Here, with fluorescent dyes and genetic labeling, we show successful 3PM imaging in the whole SVZ, extending to a maximum depth of 1.5 mm ventral to the dura mater. 3PM imaging distinguished multiple SVZ cell types in postnatal and juvenile mice. We also detected fine processes on neural stem cells interacting with the vasculature. Previous live imaging removed overlying cortical tissue or lowered lenses into the brain, which could cause inflammation and alter neurogenesis. We found that neither astrocytes nor microglia become activated in the SVZ, suggesting 3PM does not induce major damage in the niche. Thus, we show for the first time 3PM imaging of the SVZ in live mice. This strategy could be useful for intravital visualization of cell dynamics, molecular, and pathological perturbation and regenerative events.
-
null (Ed.)Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10–15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU.more » « less
-
more » « less
Abstract Fluorescence imaging is a powerful tool for studying biologically relevant macromolecules, but its applicability is often limited by the fluorescent probe, which must demonstrate both high site‐specificity and emission efficiency. In this regard, M13 virus, a versatile biological scaffold, has previously been used to both assemble fluorophores on its viral capsid with molecular precision and to also target a variety of cells. Although M13‐fluorophore systems are highly selective, these complexes typically suffer from poor molecular detection limits due to low absorption cross‐sections and moderate quantum yields. To overcome these challenges, a coassembly of the M13 virus, cyanine 3 dye, and silver nanoparticles is developed to create a fluorescent tag capable of binding with molecular precision with high emissivity. Enhanced emission of cyanine 3 of up to 24‐fold is achieved by varying nanoparticle size and particle‐fluorophore separation. In addition, it is found that the fluorescence enhancement increases with increasing dye surface density on the viral capsid. Finally, this highly fluorescent probe is applied for in vitro staining of
E. coli . These results demonstrate an inexpensive framework for achieving tuned fluorescence enhancements. The methodology developed in this work is potentially amendable to fluorescent detection of a wide range of M13/cell combinations. -
more » « less
Abstract Nondestructive neurotransmitter detection and real‐time monitoring of stem cell differentiation are both of great significance in the field of neurodegenerative disease and regenerative medicine. Although luminescent biosensing nanoprobes have been developed to address this need, they have intrinsic limitations such as autofluorescence, scattering, and phototoxicity. Upconversion nanoparticles (UCNPs) have gained increasing attention for various biomedical applications due to their high photostability, low auto‐fluorescent background, and deep tissue penetration; however, UCNPs also suffer from low emission intensities due to undesirable energy migration pathways. To address the aforementioned issue, a single‐crystal core–shell–shell “sandwich” structured UCNP is developed that is designed to minimize deleterious energy back‐transfer to yield bright visible emissions using low power density excitations. These UCNPs show a remarkable enhancement of luminescent output relative to conventional β‐NaYF4:Yb,Er codoped UCNPs and β‐NaYF4:Yb,Er@NaYF4:Yb “active shell” alike. Moreover, this advanced core–shell–shell UCNP is subsequently used to develop a highly sensitive biosensor for the ultrasensitive detection of dopamine released from stem cell‐derived dopaminergic‐neurons. Given the challenges of in situ detection of neurotransmitters, the developed NIR‐based biosensing of neurotransmitters in stem cell‐derived neural interfaces present a unique tool for investigating single‐cell mechanisms associated with dopamine, or other neurotransmitters, and their roles in neurological processes.
