Label‑Free Spectral Imaging to Study Drug Distribution and Metabolism in Single Living Cells

During drug development, evaluation of drug and its metabolite is an essential process to understand drug activity, stability, toxicity and distribution. Liquid chromatography (LC) coupled with mass spectrometry (MS) has become the standard analytical tool for screening and identifying drug metabolites. Unlike LC/MS approach requiring liquifying the biological samples, we showed that spectral imaging (or spectral microscopy) could provide high-resolution images of doxorubicin (dox) and its metabolite doxorubicinol (dox’ol) in single living cells. Using this new method, we performed measurements without destroying the biological samples. We calculated the rate constant of dox translocating from extracellular moiety into the cell and the metabolism rate of dox to dox’ol in living cells. The translocation rate of dox into a single cell for spectral microscopy and LC/MS approaches was similar (~ 1.5 pM min−1 cell−1). When compared to spectral microscopy, the metabolism rate of dox was underestimated for about every 500 cells using LC/MS. The microscopy approach further showed that dox and dox’ol translocated to the nucleus at different rates of 0.8 and 0.3 pM min−1, respectively. LC/MS is not a practical approach to determine drug translocation from cytosol to nucleus. Using various methods, we confirmed that when combined with a high-resolution imaging, spectral characteristics of a molecule could be used as a powerful approach to analyze drug metabolism. We propose that spectral microscopy is a new method to study drug localization, translocation, transformation and identification with a resolution at a single cell level, while LC/MS is more appropriate for drug screening at an organ or tissue level

The Use of Advanced Spectral Imaging to Reveal Nanoparticle Identity in Biological Samples

Nanoparticles (NPs) have been used in drug delivery therapies, medical diagnostic strategies, and as current Covid-19 vaccine carriers. Many microscope-based imaging systems have been introduced to facilitate detection and visualization of NPs. Unfortunately, none can differentiate the core and the shell of NPs. Spectral imaging has been used to distinguish a drug molecule and its metabolite. We have recently integrated this technology to a resolution of 9 nm by using artificial intelligence-driven analyses. Such a resolution allowed us to collect many robust datapoints for each pixel of an image. Our analyses could recognize 45 spectral points within a pixel to detect unlabeled Ag-NPs and Au-NPs in single live cells and tissues (liver, heart, spleen and kidneys). The improved resolution and software provided a more specific fingerprinting for each single molecule, allowing simultaneous analyses of 990 complex interactions from the 45 points for each molecule within a pixel of an image. This in turn allowed us to detect surface-functionalization of Ag-NPs to distinguish the core from the shell of Ag-NPs for the first time. Our studies were validated using various laborious and time-consuming conventional techniques. We propose that spectral imaging has tremendous potential to study NP localization and identification in biological samples at a high temporal and spatial resolution, based primarily on spectral identity information

Voxel-wise comparisons of cellular microstructure and diffusion-MRI in mouse hippocampus using 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND)

A key challenge in medical imaging is determining a precise correspondence between image properties and tissue microstructure. This comparison is hindered by disparate scales and resolutions between medical imaging and histology. We present a new technique, 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND), for registering medical images with 3D histology to overcome these limitations. Ex vivo 120 × 120 × 200 μm resolution diffusion-MRI (dMRI) data was acquired at 7 T from adult C57Bl/6 mouse hippocampus. Tissue was then optically cleared using CLARITY and stained with cellular markers and confocal microscopy used to produce high-resolution images of the 3D-tissue microstructure. For each sample, a dense array of hippocampal landmarks was used to drive registration between upsampled dMRI data and the corresponding confocal images. The cell population in each MRI voxel was determined within hippocampal subregions and compared to MRI-derived metrics. 3D-BOND provided robust voxel-wise, cellular correlates of dMRI data. CA1 pyramidal and dentate gyrus granular layers had significantly different mean diffusivity (p > 0.001), which was related to microstructural features. Overall, mean and radial diffusivity correlated with cell and axon density and fractional anisotropy with astrocyte density, while apparent fibre density correlated negatively with axon density. Astrocytes, axons and blood vessels correlated to tensor orientation

The Descemet Membrane Endothelial Keratoplasty (DMEK) “Wave Maneuver”

A novel technique for Descemet membrane endothelial keratoplasty (DMEK) graft handling and centration without the endothelium touching the posterior part of the anterior chamber (AC), is presented here. It is particularly suitable for vitrectomized eyes, deep AC, and AC intraocular lenses (ACIOLs), potentially reducing surgery time and endothelial cell loss during surgery. This retrospective interventional case series includes 27 eyes with complex ocular pathology. All utilized a “Wave maneuver” to center an early elevated graft without completing graft centration on the bottom of the AC. Successful graft attachment and centration were evaluated intra and post-operatively. Best-corrected visual acuity (BCVA), central corneal thickness (CCT), and donor endothelial cell density (ECD) were measured pre-operatively, and three and six months post-operatively. DMEK grafts were successfully attached and centered in all cases. No maneuver-related complications were observed intraoperatively. BCVA improved from a pre-operative 0.2 ± 0.63, to 0.43 ± 0.49 and 0.76 ± 0.51 at the three- and six-month follow-ups, respectively (p < 0.01). CCT decreased from a pre-operative 742 ± 118, to 546 ± 87 and 512 ± 67 at three and six months, respectively (p < 0.01). ECD decreased from 2878 ± 419 cells/mm2 to 1153 ± 466 cells/mm2 at three and six months, respectively (p < 0.01). The “Wave maneuver” may be very beneficial in DMEK cases where the AC is either very deep or the bottom of the AC is compromised. The “Wave maneuver” learning curve was brief

Three-dimensional Nuclear Telomere Architecture Is Associated with Differential Time to Progression and Overall Survival in Glioblastoma Patients

The absence of biological markers allowing for the assessment of the evolution and prognosis of glioblastoma (GBM) is a major impediment to the clinical management of GBM patients. The observed variability in patients' treatment responses and in outcomes implies biological heterogeneity and the existence of unidentified patient categories. Here, we define for the first time three GBM patient categories with distinct and clinically predictive three-dimensional nuclear-telomeric architecture defined by telomere number, size, and frequency of telomeric aggregates. GBM patient samples were examined by three-dimensional fluorescent in situ hybridization of telomeres using two independent three-dimensional telomere-measurement tools (TeloView program [P1] and SpotScan system [P2]). These measurements identified three patients categories (categories 1–3), displaying significant differences in telomere numbers/nucleus (P1 = .0275; P2 ≤ .0001), telomere length (P1 and P2 = .0275), and number of telomeric aggregates (P1 = .0464; P2 ≤ .0001). These categories corresponded to patients with long-term, intermediate, and short-term survival, respectively (P = .0393). The time to progression analyses showed significant differences between the three categories (P = .0167). There was a correlation between time to progression, median survival, and nuclear telomere architecture. Our study suggests a link between patient outcome and three-dimensional nuclear-telomere organization and highlights the potential clinical power of telomere signatures as a new prognostic, predictive, and potentially pharmacodynamic biomarker in GBM. Furthermore, novel automated three-dimensional high-throughput scanning as developed here permits to obtain data from 300 nuclei in 20 minutes. This method is applicable to any cell type and scanning application

Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway

<p>RNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn <i>Macrobrachium rosenbergii</i> by knocking down the transcript level of an insulin-like androgenic gland hormone (<i>Mr-IAG</i>) through injections of dsRNA of the entire <i>Mr-IAG</i> ORF sequence (ds<i>Mr-IAG</i> – 518bp). Interestingly, <i>in-vivo</i> knockdown success and ds<i>Mr-IAG</i> lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in <i>M. rosenbergii</i>. We discovered the <i>Mr-Dicer</i> and <i>Mr-Argonaute</i> gene families, associated with the major knockdown pathways, in our <i>M. rosenbergii</i> transcriptomic library. In response to ds<i>Mr-IAG</i> administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a <i>SID1</i> gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing <i>Mr-SID1</i> specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments <i>in-vitro</i>, the case presented here demonstrates length dependency <i>in-vivo</i>, suggesting further complexity in the context of the entire organism.</p