Accurate Detection of Low Levels of Fluorescence Emission in Autofluorescent Background: Francisella-Infected Macrophage Cells

Abstract

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence.

Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

“…While the above-mentioned experiments clearly show that unencapsulated strains adhere more efficiently to human cells, it was previously shown using elegant biochemical and immunological experiments that the capsule protects the pneumococci from recognition of the human immune system (3,4,38). To examine this conundrum in more detail, we tested whether our reporter strains could be used to directly visualize encapsulated and unencapsulated pneumococci in the presence of human neutrophils in a dual-color experiment.…”

“…While these reporters generated relatively good fluorescent signals when present as single copies stably integrated in the chromosome and driven by strong promoters, they were still not bright enough to be used in complex host-pathogen experiments in which high levels of experimental autofluorescence are present (data not shown). In general, untagged GFP is difficult to image because the fluorescence signal spreads through the entire cytoplasm by fast diffusion during the image acquisition time and is overwhelmed by cellular autofluorescence (37,38). To overcome this problem, we fused sfGFP(Bs) and mKate2 (here called gfp and rfp, respectively) to the 3= end of hlpA (SPD_0997), encoding the only known nucleoid binding protein in Streptococcus (39), and stably integrated the fusions by double crossover in the pneumococcal chromosome at the hlpA locus (Fig.…”

Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

“…The most important endogenous fluorophores are pyridinic (NAD(P)H) and flavins coenzymes, lipofuscins, advanced glycation end products (AGEs), collagen, and elastin (of the extracellular matrix) [ 29 , 30 ]. The autofluorescence of microglia/macrophage was identified to originate from the redox cofactor, flavin adenine dinucleotide, on the basis of spectral properties [ 31 ]. Other authors assigned the endogenous fluorescence to lipofuscin granules by ultrastructural analysis [ 22 ].…”

Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans

“…Hyperspectral confocal fluorescence imaging (Davis et al, 2010; Sinclair et al, 2006; Vermaas et al, 2008) was employed to facilitate isolation and assignment of the distinct spectral components present in images and bulk fluorometry data. For imaging, N. oleoabundans cells were collected following 12 days in culture, dark adapted, stained with Nile Red as described above, and delivered to a glass slide and coverslip assembly for imaging.…”