Optogenetic manipulation of cell migration with high spatiotemporal resolution using lattice lightsheet microscopy

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca2+ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca2+ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca2+ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.

Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

The data acquisition speed of point scanning microscopy techniques at sub-cellular resolution limits imaging of large samples, as sample stability and focus drift are becoming an issue. Therefore, light sheet fluorescent microscopy (LSFM) has become the method of choice for imaging cleared samples. However, this method still suffers from a trade-off between imaging depth and resolution, due to diffraction of the illuminating beam limiting the achievable field of view. After improvement of the latter with non-diffracting Bessel beams, lattice light sheet microscopy has substantially reduced the illumination point-spread-function (PSF). Here, we propose further improvement by generation of structured light sheets via phased arrays implemented as silicon nitride photonic integrated circuits (PICs). Beam generation with PICs results in much higher power efficiency than beam forming with conventional liquid crystal based spatial phase modulators, as it does not require the use of narrow blocking apertures. Moreover, this approach enables increased control over the generated field profile. Modeling of concrete PIC concepts indicates that sub-cellular resolution with mm scale imaging depths can be concomitantly achieved. Maintaining a small PSF along the axis perpendicular to the direction of light propagation is sacrificed in order to maintain it over increased imaging depth along the beam propagation axis. Rapid lateral scanning of the illumination beam inside the plane of the light sheet is then obtained by scanning of the laser wavelength within the excitation spectrum of the target fluorescent protein, allowing for a wide bidirectional field-of-view with high resolution.

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transient residence in the plasma membrane, Rhodamine-conjugated EGF (EGF-Rh) and Grb2-YFP were rapidly internalized and accumulated in endosomes. Quantitative image analysis revealed that on average two Grb2-YFP molecules were colocalized with one EGF-Rh in cells stimulated with 2 ng/ml EGF-Rh, and the excess of Grb2-YFP over EGF-Rh was even higher when a receptor-saturating concentration of EGF-Rh was used. Therefore, we hypothesize that a single EGFR molecule can be simultaneously associated with functionally distinct Grb2 interaction partners during and after endocytosis. Continuous presence of Grb2-YFP in endosomes was also observed when EGFR was activated by transforming growth factor-α and amphiregulin, suggesting that endosomal EGFRs remain ligand occupied and signaling competent, despite the fact that these growth factors are thought to dissociate from the receptor at acidic pH. The prolonged localization and activity of EGFR-Grb2 complexes in endosomes correlated with the sustained activation of extracellular stimulus-regulated kinase 1/2, suggesting that endosomal EGFRs contribute significantly to this signaling pathway. We propose that endosomal EGFRs function to extend signaling in time and space to compensate for rapid downregulation of surface EGFRs in cells with low receptor expression levels.

Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N-glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe(x)) in airway epithelial repair. To examine whether an sLe(x) decoration of EGFR can modulate receptor function during AEC repair. Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co-localization of sLe(x) and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT-PCR and Western blotting. The final step in the synthesis of sLe(x) was catalysed by a specific alpha-1,3-fucosyltransferase (FucT-IV). To evaluate the role of sLe(x) in EGFR activation, a knockdown of the FucT-IV gene with small interfering RNA (siRNA) and an inhibitory anti-sLe(x) antibody (KM-93) was used. We demonstrated a co-localization of sLe(x) with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe(x) after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT-IV demonstrates a temporal expression coordinate with epithelial repair. Down-regulation of FucT-IV expression in NHBE by specific siRNA suppressed sLe(x) expression. The use of FucT-IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe(x) and tyrosine-phosphorylated EGFR (pY(845)-EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. The present data demonstrate that sLe(x), in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC.

Hypoxia-Induced Autophagy Promotes EGFR Loss in Specific Cell Contexts, Which Leads to Cell Death and Enhanced Radiosensitivity

Noninvasive imaging of epidermal growth factor (EGF) receptor (EGFR) expression can provide valuable molecular information that could aid diagnostic and therapeutic decisions, particularly with targeted cancer therapies utilizing anti-EGFR antibodies. In this study we report on the development and validation of a nanoprobe for in-vivo imaging and discrimination of EGFR-overexpressing tumors from surrounding normal tissues that also expresses EGFR. Near-infrared quantum dots (QDs) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. These nanoprobes demonstrated excellent in-vitro and in-vivo binding affinity. In-vivo imaging demonstrated three distinct phases of tumor influx (~3min), clearance (~60min) and accumulation (1-6hrs) of EGF-QD nanoprobes. Both QD and EGF-QD demonstrated non-specific rapid tumor influx and clearance followed by an apparent dynamic equilibrium at ~60min. Subsequently (1-6hrs), while QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors. At 24hrs, tumor fluorescence decreased to near baseline levels for both QD and EGF-QD. Ex vivo whole-organ, tissue-homogenate fluorescence, confocal microscopy and immunofluorescence staining confirmed tumor-specific accumulation of EGF-QD nanoprobes at an early time-point (4hrs). The favorable pharmacokinetics, the ability to discriminate EGFR-overexpressing tumors from surrounding normal tissues using low concentration (10-pmol) of EGF-QD nanoprobe underscores the clinical relevance of this probe to evaluate therapeutic intervention.

Purpose: The present study was conducted to investigate the effect of a temporally incoherent (‘noise’) magnetic field (MF) on radiofrequency radiation (RFR)-induced epidermal growth factor (EGF) receptor clustering and phosporylation in cultured cells.Materials and methods: Human amniotic epithelial (FL) cells were exposed for 15 min to either a 1.8 GHz RFR (modulated at 217 Hz), a 2 μT incoherent MF, or concurrently to the RFR and incoherent MF. Epidermal growth factor treatment severed as the positive control. Epidermal growth factor receptor clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining, and phosphorylation of EGF receptors was measured by western blot technology.Results: Exposure of FL cells to the 1.8 GHz RFR at SAR (specific absorption rate) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min induced EGF receptor clustering and enhanced phosphorylation on tyrosine-1173 residue, whereas exposure to RFR at SAR of 0.1 W/kg for 15 min did not significantly cause these effects. Exposure to a 2 μT incoherent MF for 15 min did not significantly affect clustering and phosphorylation of EGF receptor in FL cells. When superimposed, the incoherent MF completely inhibited EGF receptor clustering and phosphorylation induced by RFR at SAR of 0.5, 1.0, and 2.0 W/kg, but did not inhibit the effects induced at SAR of 4.0 W/kg.Conclusion: Based on the data of the experiment, it is suggested that membrane receptors could be one of the main targets by which RFR interacts with cells. An incoherent MF could block the interaction to a certain extent.

Purpose: Many studies have shown that exposure to radiofrequency radiation (RFR) could activate cellular signal transduction pathways. In the present research, we investigated the effects of exposure to a 1.8-GHz RFR at different intensities on epidermal growth factor (EGF) receptor clustering and phosphorylation in human amniotic (FL) cells.Materials and methods: Receptor clustering on cellular membrane surface was analyzed using immunofluorescence assessed by confocal microscopy, and phosphorylation of EGF receptors was measured by western blot technology. EGF treatment served as a positive control.Results: The results showed that, compared with sham exposure, exposure to RFR at specific absorption rate (SAR) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min significantly induced EGF receptor clustering and enhanced phosphorylation on the tyrosine-1173 residue in FL cells, whereas exposure to a SAR 0.1 W/kg radiation for 15 min did not cause a significant effect.Conclusion: Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets that RFR interacts with cells, and the dose-rate threshold, in the case of EGF receptors, is between SAR of 0.1 and 0.5 W/kg. The results indicate a sigmoid dependence of RFR effects on intensity.

Purpose: N-cadherin (NCAD) and epidermal growth factor receptor (EGFR) have been found to be inducers of epithelial-to-mesenchymal transition that are known to play a role in invasion and metastasis of cancer. Over expression of NCAD and EGFR are associated with increased tumor invasiveness. However, their progressive expression in colorectal cancer (CRC) has not been studied. Therefore, in the present study, we investigated expression status of NCAD and EGFR in paired lesions of CRC. Experimental Design: We examined the expression of NCAD and EGFR in paired lesions of human CRC (hCRC) and human mice CRC (hmCRC) xenograft by immunofluorescence (IF). Images were acquired using the Olympus FluoView FV1000 (OFVFV1000) Confocal Microscope. The average FITC fluorescence intensity (aFFI) was measured by OFVFV1000 software. Western blot analysis was performed to validate results obtained from IF method. Results: Differential expression pattern of NCAD was observed in paired normal, primary and metastatic lesions of hCRC and hmCRC xenograft tissues. For NCAD, it was mainly localized in cytoplasm (diffused), nucleus (moderate levels), and membrane or cell junctions of CRC. For EGFR, normal colon tissue or primary CRC showing membranous EGFR-low positive staining. In contrast corresponding liver metastasis showing membranous EGFR-high positive staining. The NCAD and EGFR expressions were correlated with metastasis phenotype of hCRC. The expressions of NCAD ((aFFI; 812 vs. 261) and EGFR (aFFI 421 vs.131) were each found to be 3 times higher in liver metastasis of hCRC as compared to primary tumor. It has been observed that the expression of NCAD was substantially increased in metastatic lesion of hmCRC xenograft as compared to primary lesion of hmCRC xenograft. Conclusion: This study revealed that upregulation of NCAD and EGFR expressions is a mechanism by which CRC lesions acquire a more aggressive phenotype and, thereby, progress to a more advanced stage of CRC. Furthermore, our findings are likely to have implications in the design of clinical trials targeting the NCAD and EGFR proteins in CRC. Citation Format: Venkat R. Katkoori, Upender Manne, Marc D. Basson, Harvey L. Bumpers. Progressive expression of n-cadherin and epidermal growth factor receptor in colorectal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2620. doi:10.1158/1538-7445.AM2013-2620

We introduce a compact method for generating guide stars to enable direct wavefront sensing in Bessel two-photon microscopy. By sequentially modulating an axicon phase and a thin lens phase using a spatial light modulator (SLM), our approach achieves seamless switching between Bessel and Gaussian illumination beams without requiring mechanical movement or additional optical components. The Bessel beam provides an expanded field of view for imaging, while the Gaussian beam serves as a point-like guide star for wavefront sensing. Experimental validation in a Bessel two-photon light sheet microscope demonstrates that wavefront correction based on our method enhances the signal-to-noise ratio (SNR) by 7.3 times and improves axial resolution by 4.4 times.

IntoductionIdentification of molecular alterations associated with tumor behavior is necessary to guide clinical management. The 2022 WHO classification has organized the thyroid follicular cell-derived neoplasms into benign, low-risk and high-risk neoplasms, and emphasized the value of biomarkers that may provide differential diagnostic and prognostic information to avoid overtreatment of low risk neoplasms. This work aims to study the epidermal growth factor receptor (EGFR) expression, functional and spatial dynamics in relation to specific miRNAs alterations in papillary thyroid cancer (PTC) and in non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) considered as models of high-risk and low-risk thyroid tumors respectively.MethodsPrimary thyroid cultured cells were used for miRNA gain/loss of function and luciferase reporter assays. Paraffin embedded tissues were used for real time PCR, immuno-fluorescence stain and confocal microscopy experiments.ResultsOur results showed that in PTC, EGFR mRNA is reduced as an effect of miR-146b-5p upregulation. The EGF expression is low and the ERK pathway is inhibited. The EGFR protein high cytoplasmic expression and colocalization with the endosomal/exosomal markers, ALIX and CD63, suggest the occurrence of stress-induced EGFR internalization, accumulation in endosomal vesicles and secretion via exosomes. In NIFTP EGFR transcription is increased in association with downregulation of miR-7-5p and the EGFR/ERK pathway is active indicating dependence on the canonical EGFR pathway for growth.ConclusionDownregulation of transcript level along with cytoplasmic accumulation of undegraded protein is a new pattern of EGFR regulation associated with malignancy in thyroid. Further research is needed to elucidate the intracellular trafficking defects responsible for this specific EGFR dynamic in PTC.

A novel z‐scan‐free stimulated Raman scattering tomography (SRST) is presented which is enabled by using optical beating technique (OBT) associated with Bessel beams to achieve deeper penetration for label‐free volumetric chemical imaging with subcellular resolution. In SRST, the pump beam is modulated with a spatial light modulator to convert to Bessel beam with optical beating, which is overlapped with the Bessel Stokes beam in the sample. By electronically varying the beating frequency, the depth‐resolved SRS signals about the volumetric tissue are encoded in the spatial frequency domain and thus, SRST can be rapidly retrieved by implementing inverse fast Fourier transform without a need of mechanical depth‐scan. It is demonstrated that SRST imaging using Bessel beams with self‐reconstructing properties provides at least twofold improvement in imaging depth in highly scattering polymer beads phantom as compared to conventional SRS microscopy. The capability of SRST for label‐free volumetric deeper imaging on a variety of imaging targets (e.g., Raman‐active crystals, plant cells, and biological tissue) is also proved. The generality of optical sectioning ability of Bessel beam‐OBT in SRST can be readily extended to practically any other nonlinear optical imaging modalities for deep tissue 3D imaging in biological and biomedical systems.

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.