Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes.

We demonstrate stimulated emission depletion (STED) microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multicolor, and live-cell STED microscopy.

In spite of their relatively low fluorescence quantum yield, cyanine dyes such as Cy3, Cy5, or Cy7 are widely used in single-molecule fluorescence applications due to their high extinction coefficients and excellent photon yields. We show that the fluorescence quantum yield and lifetime of red-emitting cyanine dyes can be substantially increased in heavy water (D2 O) compared with water (H2 O). We find that the magnitude of the quantum yield increase in D2 O scales with the emission wavelength, reaching a particularly high value of 2.6-fold for the most red-emitting dye investigated, Cy7. We further demonstrate a higher photon yield in single-molecule superresolution experiments in D2 O compared to H2 O, which leads to an improved localization precision and hence better spatial resolution. This finding is especially beneficial for biological applications of fluorescence microscopy, which are typically carried out in aqueous media and which greatly profit from the red spectral range due to reduced cellular auto-fluorescence.

Endothelial cells of the blood-brain barrier form a structural and functional barrier maintaining brain homeostasis via paracellular tight junctions and specific transporters such as P-glycoprotein. The blood-brain barrier is responsible for negligible bioavailability of many neuroprotective drugs. In Alzheimer's disease, current treatment approaches include inhibitors of BACE-1 (b-site of amyloid precursor protein cleaving enzyme), a proteinase generating neurotoxic b-amyloid. It is known that BACE-1 is highly expressed in endosomes and membranes of neurons and glia. We now provide evidence that BACE-1 is expressed in blood-brain barrier endothelial cells of human, mouse, and bovine origin. We further show its predominant membrane localization by 3D-dSTORM super-resolution microscopy, and by biochemical fractionation that further shows an abluminal distribution of BACE-1 in brain microvessels. We confirm its functionality in processing APP in primary mouse brain endothelial cells. In an Alzheimer's disease mouse model we show that BACE-1 is upregulated at the blood-brain barrier compared to healthy controls. We therefore suggest a critical role for BACE-1 at the blood-brain barrier in b-amyloid generation and in vascular aspects of Alzheimer's disease, particularly in the development of cerebral amyloid angiopathy.

Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.

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We demonstrate correlative super-resolution PALM, PAINT and dSTORM imaging of RNA polymerase, membrane and chromosomal DNA in fixed E. coli. This protocol allows the combination of precise structural information of the nucleoid (dSTORM) with quantitative super-resolution imaging (PALM) of interacting proteins. The spatial distribution and organization of RNA polymerase and DNA are visualized in bacterial cells grown at doubling times of 25 or 44 min. We observe that RNA polymerase is concentrated at the edge of the highly structured nucleoid during fast growth, whereas it is found more evenly distributed during medium-fast growth. In both conditions, the nucleoid shows densely packed areas which appear to be inaccessible to RNA polymerase. This finding is confirmed by live-cell tracking of RNA polymerase and subsequent imaging of the respective nucleoids using a protocol for fast fixation on-the-slide.

Two-component signaling systems are a major strategy employed by bacteria, and to some extent, yeast and plants, to respond to environmental stress. The EnvZ/OmpR system in E. coli responds to osmotic and acid stress and is responsible for regulating the protein composition of the outer membrane. EnvZ is a histidine kinase located in the inner membrane. Upon activation, it is autophosphorylated by ATP and subsequently, it activates OmpR. Phosphorylated OmpR binds with high affinity to the regulatory regions of the ompF and ompC porin genes to regulate their transcription. We set out to visualize these two-components in single bacterial cells during different environmental stress conditions and to examine the subsequent modifications to the bacterial nucleoid as a result. We created a chromosomally-encoded, active, fluorescent OmpR-PAmCherry fusion protein and compared its expression levels with RNA polymerase. Quantitative western blotting had indicated that these two proteins were expressed at similar levels. From our images, it is evident that OmpR is significantly less abundant compared to RNA polymerase. In cross-sectional axial images, we observed OmpR molecules closely juxtaposed near the inner membrane during acidic and hyposomotic growth. In acidic conditions, the chromosome was compacted. Surprisingly, under acidic conditions, we also observed evidence of a spatial correlation between the DNA and the inner membrane, suggesting a mechanical link through an active DNA-OmpR-EnvZ complex. This work represents the first direct visualization of a response regulator with respect to the bacterial chromosome.