Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine. HSI has also shown great utility in fluorescence microscopy. However, traditional fluorescence microscopy HSI systems have suffered from limited signal strength due to the need to filter or disperse the emitted light across many spectral bands. We have previously demonstrated that sampling the fluorescence excitation spectrum may provide an alternative approach with improved signal strength. Here, we report on the use of excitation-scanning HSI for dynamic cell signaling studies—in this case, the study of the second messenger Ca2+. Time-lapse excitation-scanning HSI data of Ca2+ signals in human airway smooth muscle cells (HASMCs) were acquired and analyzed using four spectral analysis algorithms: linear unmixing (LU), spectral angle mapper (SAM), constrained energy minimization (CEM), and matched filter (MF), and the performances were compared. Results indicate that LU and MF provided similar linear responses to increasing Ca2+ and could both be effectively used for excitation-scanning HSI. A theoretical sensitivity framework was used to enable the filtering of analyzed images to reject pixels with signals below a minimum detectable limit. The results indicated that subtle kinetic features might be revealed through pixel filtering. Overall, the results suggest that excitation-scanning HSI can be employed for kinetic measurements of cell signals or other dynamic cellular events and that the selection of an appropriate analysis algorithm and pixel filtering may aid in the extraction of quantitative signal traces. These approaches may be especially helpful for cases where the signal of interest is masked by strong cellular autofluorescence or other competing signals

Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine. HSI has also shown great utility in fluorescence microscopy. However, traditional fluorescence microscopy HSI systems have suffered from limited signal strength due to the need to filter or disperse the emitted light across many spectral bands. We have previously demonstrated that sampling the fluorescence excitation spectrum may provide an alternative approach with improved signal strength. Here, we report on the use of excitation-scanning HSI for dynamic cell signaling studies—in this case, the study of the second messenger Ca2+. Time-lapse excitation-scanning HSI data of Ca2+ signals in human airway smooth muscle cells (HASMCs) were acquired and analyzed using four spectral analysis algorithms: linear unmixing (LU), spectral angle mapper (SAM), constrained energy minimization (CEM), and matched filter (MF), and the performances were compared. Results indicate that LU and MF provided similar linear responses to increasing Ca2+ and could both be effectively used for excitation-scanning HSI. A theoretical sensitivity framework was used to enable the filtering of analyzed images to reject pixels with signals below a minimum detectable limit. The results indicated that subtle kinetic features might be revealed through pixel filtering. Overall, the results suggest that excitation-scanning HSI can be employed for kinetic measurements of cell signals or other dynamic cellular events and that the selection of an appropriate analysis algorithm and pixel filtering may aid in the extraction of quantitative signal traces. These approaches may be especially helpful for cases where the signal of interest is masked by strong cellular autofluorescence or other competing signals

In the Absence of Effector Proteins, the <i>Pseudomonas aeruginosa</i> Type Three Secretion System Needle Tip Complex Contributes to Lung Injury and Systemic Inflammatory Responses

<div><p>Herein we describe a pathogenic role for the <i>Pseudomonas aeruginosa</i> type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which <i>P. aeruginosa</i> wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of <i>P. aeruginosa</i> pneumonia-induced lung endothelial barrier disruption.</p> </div

<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV associates with increased PMVEC permeability during early phase infection.

<p><b>A</b>. Isolated rat PMVECs were grown on transwells to confluence and permeability measured as a function of FITC-dextran flux across the monolayer. PMVECs were inoculated with medium alone, saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. Flux assays were initiated at 4-hours post-inoculation by the addition of FITC-dextran and flux monitored for 1-hour. Average flux rate data at 5-hours post-inoculation from the SS vehicle control transwells were used to normalized the flux rate from PA103 (ΔU/ΔT) or PA103 (ΔPcrV) (n=3 for each condition). Inoculation with PA103 (ΔU/ΔT) caused significant increases in PMVEC permeability compared to inoculation with PA103 (ΔPcrV). Error bars are the standard error of the mean. The asterisk indicates a significant difference by unpaired <i>t</i>-test (<i>P</i> < 0.05). <b>B</b>. Mock cell culture infection experiments were performed where ~2.5 x 10⁷ CFU/mL of each <i>P. aeruginosa</i> mutant strain was inoculated into DMEM culture medium (no PMVECs) and bacterial growth followed over time by direct plating. Error bars are the standard error of the mean of technical replicates from at least 3 independent experiments. There were no observable differences in growth rates between the two <i>P. aeruginosa</i> mutant strains. <b>C</b>. The ability of each of the P. <i>aeruginosa</i> mutant strains to adhere to PMVECs was assessed. Isolated rat PMVECs were grown in culture dishes to confluence and inoculated with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. At one hour post-inoculation, monolayers were washed copiously to remove planktonic bacteria, PMVECs permeabilized, and total associated bacteria determined by direct plating. Error bars are the standard error of the mean of technical replicates from at least 3 independent experiments. There were no observable differences in adherence to PMVECs between the two <i>P. aeruginosa</i> mutant strains.</p

<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV associates with lung injury.

<p><b>A</b>. Injured areas of the lung were prepared for histological hematoxylin/eosin analysis at 48-hours post-inoculation. Animals were inoculated with saline solution (vehicle control), with ~1 x 10⁷ CFUs of PA103 (ΔU/ΔT), or with ~1 x 10⁷ CFUs of PA103 (ΔPcrV). Lung sections imaged at 10X (top panels) and 20X (bottom panels) revealed that intratracheal instillation of PA103 (ΔU/ΔT) or PA103 (ΔPcrV) caused lung injury. Inoculation with PA103 (ΔU/ΔT) resulted in fluid accumulation around the bronchovascular bundles (vascular cuffs, arrow heads, upper panel) and lesions reminiscent of diffuse alveolar damage (arrow heads, lower panel), characterized by fluid accumulation and inflammatory cell infiltration within the air space. Inoculation with PA103 (ΔPcrV) caused collapsed air spaces, resembling atelectasis with recruitment of inflammatory cells. These histological patterns suggest that the two PA103 mutants are capable to cause lung damage/injury, although the lesions appear distinct from one another. <b>B</b>. Injured areas of the lung were prepared for histological myeloperoxidase (MPO) staining analysis to reveal the extent of neutrophil infiltration. Inoculation with PA103 (ΔU/ΔT) caused significant neutrophil recruitment compared to either vehicle control or to inoculation with PA103 (ΔPcrV). The average numbers of MPO positive cells from at least 5 independent fields of view from three different animals per condition are presented. Error bars are the standard error of the mean. The asterisks indicate a statistically significant difference (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test. <b>C</b>. Lungs from vehicle control (saline solution) and <i>P. aeruginosa</i>-inoculated animals were isolated <i>en </i><i>bloc</i> at 48-hours post-inoculation for analysis of hydraulic permeability (K_f). Inoculation with PA103 (ΔU/ΔT) caused significant permeability increases compared to either vehicle control or to inoculation with PA103 (ΔPcrV). The average K_f values from 5 different animals per group are shown. Error bars are the standard error of the mean. The asterisks indicate a statistically significant difference (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test.</p

<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV elicits increased PMVEC permeability during early phase infection via cytoskeletal perturbation in a host cell death-independent manner.

<p><b>A</b>. Isolated rat PMVECs were grown in culture dishes to confluence and viability assessed by Trypan Blue exclusion assay. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVEC viability was assessed at 5-hours post-inoculation, and data are expressed as percent total viable cells. Neither PA103 mutant caused significant cell death compared to vehicle control-treated PMVECs. The average viable PMVEC numbers from at least 3 independent experiments per condition are presented. Error bars are the standard error of the mean. <b>B</b>. Isolated rat PMVECs were grown in culture dishes to confluence and damage assessed by LDH extracellular release assay. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVEC damage was assessed over a time course post-inoculation. Culture medium was sampled to measure extracellular LDH and data normalized to the total monolayer (intracellular) LDH. While measurable, compared to vehicle control-treated PMVECs, both PA103 mutants caused only negligible rates of LDH release. The average amounts of extracellular LDH from at least 3 independent experiments are presented. <b>C</b>. Isolated rat PMVECs were grown on cover slips to confluence and actin cytoskeleton was visualized by FITC-phalloidin staining. Z-stacks from whole cellular volume images were acquired via confocal fluorescence microscopy. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. Inoculation with PA103 (ΔU/ΔT) mutant caused evident cytoskeletal rearrangement (stress fibers) compared to vehicle control and PA103 (ΔPcrV) inoculation.</p

<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV associates with mortality in a rat model of lung infection.

<p><b>A</b>. Intratracheal instillation of the P. <i>aeruginosa</i> wild type PA103 and mutant PA103 (ΔU/ΔT) strains into rats resulted in dose-dependent mortality compared to the mutant PA103 (ΔPcrV) which caused no mortality at 1-week post-infection. Doses (CFUs/mL) are listed in the figure. Data represent at least n=20 per group. <b>B</b>. One week survival analysis (Kaplan Meier curve) at a sub-LD₅₀ dose (~1 x 10⁷ CFUs). Mortality in the PA103 (ΔU/ΔT) cohort was observed within 48-hours post-infection. Black dotted line represents vehicle control (saline solution), red dotted line represents infection with PA103 (ΔPcrV), and blue dotted line represents Infection with PA103 (ΔU/ΔT). Data represent at least n=20 per group.</p