Super-resolution Imaging of Live Bacteria Cells Using a Genetically Directed, Highly Photostable Fluoromodule

Abstract

The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG), and can be used to label proteins sparsely, enabling single protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit two-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro; and five-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion (STED) microscopy, yielding a four-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.

Octanoyl galactose ester-modified microemulsion system self-assembled by coix seed components to enhance tumor targeting and hepatoma therapy

“…Cy5, a commonly used near-infrared dye, was encapsulated into various microemulsions for visualization of distribution in vivo. 25,26 At 8 h post-intragastric administration of free Cy5, the fluorescence signal of HepG2 tumor-bearing mice mainly accumulated in the intestinal tract and was eliminated rapidly from 8 to 24 h. Due to nanoparticle-mediated EPR effect, Cy5/C-MEs presented a potential of tumor targeting after crossing the enterocytes, especially at the initial 4 h. In sharp contrast, Cy5/ Gal(oct)-C-MEs displayed the most obvious fluorescence signal in the tumor site, which was retained for 48 h ( Figure 6A). For further confirming the accumulation of the tumor, ex vivo imaging was observed immediately after harvest of the tumor tissues from HepG2 tumor-bearing mice.…”

Super‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule

“…First, replace the eYFP coding sequence with the dL5 coding sequence via any molecular cloning technique; we prefer Gibson assembly to generate the reported constructs (Saurabh et al 2016). Following this, insert ~500 base pairs of genomic DNA sequence immediately upstream of the gene and the gene’s ORF in frame with either the eYFP or dL5 sequence.…”

“…Specifically, STED requires a high quality donut achieved through precise optical engineering and alignment (Wu et al 2015). Practically, forming a high quality donut beam necessitates specific microscope design parameters (Lau et al 2012) and careful fine tuning of the alignment of the microscope, usually every time the microscope is used (Saurabh et al 2016)(Fig. 7).…”

“…Thus, all of the depletion intensity occurs in the short period after excitation, ideally before an excited fluorophore has a chance to spontaneously emit. Fast scanning is achieved by a resonant mirror that oscillates at a fixed frequency, described in our recent work (Saurabh et al 2016). Thus, the two scan parameters that are easily varied are the number of resonant mirror scans (summed per slow axis scan pixel) and the number of summed frames (i.e.…”

“…Structure of MG-ester is also shown. Relevant physical parameters of the complex are noted on the right(Saurabh et al 2016). Adapted with permission from Saurabh et al, J.…”