The work was supported by Swiss National Science Foundation 310030_156818, 310030_182315, and NCCR_ 180541 AntiResist (to DB).

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

<ol>
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R., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biogeography+of+infection+revisited.">The biogeography of infection revisited.</a> Nat Rev Microbiol. 20 (10), 579-592 (2022).</li><li>Bumann, D., Cunrath, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+Salmonella-host+interactions+in+infected+host+tissues.">Heterogeneity of Salmonella-host interactions in infected host tissues.</a> Curr Opin Microbiol. 39, 57-63 (2017).</li><li>Hofmann, J., Keppler, S. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mesoscale+connectome+of+the+mouse+brain.">A mesoscale connectome of the mouse brain.</a> Nature. 508 (7495), 207-214 (2014).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-wide+maps+reveal+stereotyped+cell-type-based+cortical+architecture+and+subcortical+sexual+dimorphism.">Brain-wide maps reveal stereotyped cell-type-based cortical architecture and subcortical sexual dimorphism.</a> Cell. 171 (2), 456-469.e422 (2017).</li><li>Matho, K. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+optimization+of+fluorescent+proteins+for+intracellular+FRET.">Evolutionary optimization of fluorescent proteins for intracellular FRET.</a> Nat Biotechnol. 23 (3), 355-360 (2005).</li><li>Claudi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variation+of+Salmonella+in+host+tissues+delays+eradication+by+antimicrobial+chemotherapy.">Phenotypic variation of Salmonella in host tissues delays eradication by antimicrobial chemotherapy.</a> Cell. 158 (4), 722-733 (2014).</li><li>Ai, H. W., Olenych, S. G., Wong, P., Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hue-shifted+monomeric+variants+of+Clavularia+cyan+fluorescent+protein:+identification+of+the+molecular+determinants+of+color+and+applications+in+fluorescence+imaging.">Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging.</a> BMC Biol. 6, 13 (2008).</li><li>Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E., Rebane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+absorption+properties+of+fluorescent+proteins.">Two-photon absorption properties of fluorescent proteins.</a> Nat Methods. 8 (5), 393-399 (2011).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat Biotechnol. 22 (12), 1567-1572 (2004).</li><li>Burton, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disparate+impact+of+oxidative+host+defenses+determines+the+fate+of+Salmonella+during+systemic+infection+in+mice.">Disparate impact of oxidative host defenses determines the fate of Salmonella during systemic infection in mice.</a> Cell Host&amp;Microbe. 15 (1), 72-83 (2014).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardiac+perfusion+of+the+mouse+for+brain+tissue+dissection+and+fixation.">Transcardiac perfusion of the mouse for brain tissue dissection and fixation.</a> Bio Protoc. 11 (5), e3988 (2021).</li><li>de Olmos, J., Hardy, H., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+afferent+connections+of+the+main+and+the+accessory+olfactory+bulb+formations+in+the+rat:+an+experimental+HRP-study.">The afferent connections of the main and the accessory olfactory bulb formations in the rat: an experimental HRP-study.</a> J Comp Neurol. 181 (2), 213-244 (1978).</li><li>Smith, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIDRE:+an+illumination-correction+method+for+optical+microscopy.">CIDRE: an illumination-correction method for optical microscopy.</a> Nat Methods. 12 (5), 404-406 (2015).</li><li>Wendland, M., Bumann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+GFP+levels+for+analyzing+Salmonella+gene+expression+during+an+infection.">Optimization of GFP levels for analyzing Salmonella gene expression during an infection.</a> FEBS Lett. 521 (1-3), 105-108 (2002).</li><li>Barat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+intracellular+Salmonella+depends+on+surface-associated+antigens.">Immunity to intracellular Salmonella depends on surface-associated antigens.</a> PLoS Pathog. 8 (10), e1002966 (2012).</li><li>Rollenhagen, C., Sorensen, M., Rizos, K., Hurvitz, R., Bumann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+selection+based+on+expression+levels+during+infection+facilitates+vaccine+development+for+an+intracellular+pathogen.">Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen.</a> Proc Natl Acad Sci U S A. 101 (23), 8739-8744 (2004).</li><li>Chen, F., Cocaign-Bousquet, M., Girbal, L., Nouaille, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5'UTR+sequences+influence+protein+levels+in+Escherichia+coli+by+regulating+translation+initiation+and+mRNA+stability.">5'UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability.</a> Front Microbiol. 13, 1088941 (2022).</li><li>Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clearer+vision+for+in+vivo+imaging.">A clearer vision for in vivo imaging.</a> Nat Biotechnol. 19 (4), 316-317 (2001).</li><li>Bumann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Salmonella+gene+expression+in+an+infected+mammalian+host+using+the+green+fluorescent+protein+and+two-colour+flow+cytometry.">Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two-colour flow cytometry.</a> Mol.Microbiol. 43 (5), 1269-1283 (2002).</li><li>Nakayama, K., Kelly, S. 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The authors have nothing to disclose.

The local tissue context of bacterial pathogens is crucial for determining local host attacks, bacterial adaptations, the local outcome of the host pathogen interactions and antimicrobial chemotherapy, and the individual contributions to overall disease outcome. Imaging micrometer sized bacteria in centimeters size organs has been challenging. Serial two-photon (STP) tomography provides sufficient spatial resolution to detect individual bacterial cells in entire organs, automatized sectioning and imaging, and sufficient throughput (~1 organ per day)11. While host antigens can be stained in vivo, pathogen cells should express suitable fluorescent proteins to ensure comprehensive detection of intracellular pathogen cells. The resulting data sets (0.5-1.5 TeraByte per organ) pose substantial challenges for IT infrastructures for data analysis and storage.
There are several critical steps in this method. First, a pathogen strain with detectable and homogeneous expression of fluorescent protein GFP or YFP is required. Ideally, a chromosomal expression cassette25 is used to minimize fluorescence heterogeneity due to plasmid copy-number variation. Sufficient fluorescence intensity is required but excessive levels of fluorescent protein should be avoided to avoid fitness impairments of the pathogen23. Appropriate expression levels can be obtained by selection of an appropriate promoter and fine-tuning of the ribosomal binding site25 or the entire 5&#39; untranslated region (UTR)26. Second, the perfusion fixation should involve an initial wash with buffer to remove as many erythrocytes as possible from the blood circulation. This is particularly critical for spleen and liver (although complete erythrocyte removal from these organs is difficult). Remaining erythrocytes absorb light in the visible part of the spectrum compromising imaging quality27. Third, storage of the fixed tissues in the cryoprotectant is critical to reduce tissue autofluorescence, which is particularly high in inflamed tissues and can overshadow the comparatively weak fluorescence of the pathogen cells11. Fourth, the effective cross-linking of the tissue to the surrounding agarose block is critical for smooth vibratome-cutting without the tissue jumping out of the agarose block. Fifth, fluorescent signals and their identification as pathogen cells must be independently verified using orthogonal approaches such as staining with antibodies to pathogen components (such as lipopolysaccharide for Gram-negative bacteria) and confocal microscopy of the sections retrieved from the tomograph11. Some infected tissues contain auto-fluorescent particles with similar shape and overlapping fluorescence spectra that can be easily misinterpreted as pathogen cells. Sixth, the quantity of pathogen cells within microcolonies should be compared to orthogonal approaches such as confocal microscopy to assess the accuracy. The overall bacterial loads based on these calculations should be verified by comparison to orthogonal approaches such as flow cytometry and plating.
Important modifications of the widely used STP protocol include placing a narrow bandpass filter 510/20 nm in front of photomultiplier 211, to reduce interference of green-yellow autofluorescence that is particularly strong in infected and inflamed liver, spleen, and Peyer&#39;s patches. The strong autofluorescence and increased light scattering of such organs compared to brain (which dominates other applications of STP) also generates a need for more effective correction for uneven illumination. As another modification, this protocol employs the CIDRE approach22 for this purpose (Figure 3) and AI-based segmentation of bacteria. Finally, tissue preprocessing was altered by including an incubation step in cryoprotectant at -20 &#176;C which reduces tissue autofluorescence and thus facilitates detection of small pathogen cells with relatively weak fluorescence11.
Troubleshooting might be necessary if no pathogen signals can be detected, or segmentation yields insufficient sensitivity (too many pathogen cells are missed) or insufficient precision (too many background particles are segmented as pathogen cells). If background tissue autofluorescence is detectable but there are too few pathogen signals, the pathogens might contain insufficient amounts of fluorescent proteins. This can be tested using confocal microscopy of tissue sections from the same infected tissue or flow cytometry of tissue homogenates19,28. Underlying reasons could be insufficient expression levels or instability of the expression cassette. Mitigation strategies could include alternative promoters to drive expression, codon-adaptation of the genes encoding the fluorescent protein for the pathogen species, employment of episomal constructs with higher copy number, or stabilization of expression cassettes by chromosomal integration or balanced-lethal complementation29. The choice of fluorescent protein is also important, but detection is possible with GFP.mut2, mWasabi, YPet, and TIMERbac. If segmentation is inaccurate, this might be caused by too weak pathogen fluorescence which could be addressed as described above, or too high tissue autofluorescence background. Extensive perfusion of wash solution or prolonged incubation in storage buffer immediately before embedding in the agarose block and tomography might resolve these issues. Finally, sufficient training of the neural network is required for precise classification, but excessive training can lead to overfitting that impairs performance for new samples.
Currently, no other method can image entire organs at sufficient spatial resolution in 3D for detecting individual bacteria. Future improvements in tissue clearing and light-sheet microscopy might achieve similar resolution. This might enable imaging at higher speed and with more fluorescent channels.
An important limitation of STP is the in-plane pixel resolution of ~0.5 &#181;m and vertical resolution of 5 to 10 &#181;m, which is insufficient for resolving closely located bacteria, e.g., within a densely packed microcolony. However, it is possible to retrieve tissue sections after tomography for secondary high-resolution confocal microscopy of selected tissue parts. Another limitation of STP is the availability of only three fluorescence channels, which restricts the number of fluorophores that can be imaged simultaneously. Again, secondary analysis of retrieved tissue sections with multiplexing methods can reveal the location and intensity of many more markers for selected tissue parts. This information could be integrated into the overall 3D structure of the surrounding tissue as determined with STP.
In conclusion, this protocol enables detailed investigations of host-pathogen interactions at the local and whole-organ level. The protocol should be easily adaptable to other pathogens (provided they can be obtained as fluorescent strains), other organs, and different host species.

Infections occur in tissues with complex anatomy and compartmentalized physiology. The diverse microenvironments that co-exist in the infected tissue can determine the fates of local pathogen subsets and their contributions to overall disease outcome1,2,3. However, comprehensive 3D mapping of microbial pathogens in cm-sized tissues remains challenging4. Imaging of the brain and other organs is a highly active research field with constantly improving experimental strategies5, but many methods still lack the sub-&#181;m resolution that would be required to identify &#181;m-sized bacterial pathogens confidently. In contrast, serial two-photon (STP) tomography6 enables automated multi-color, deformation-free imaging of entire tissues with sub-&#181;m in-plane resolution, yielding full volumetric datasets. This method combines repeated physical sectioning of the tissue using a vibratome with intermittent two-photon imaging of the emerging block faces with infrared light. STP tomography has been widely used for mapping thin axons in the brain to establish connectivity maps7,8,9,10.
STP tomography also enables 3D mapping of individual microbial pathogen cells (Salmonella, Toxoplasma) in the entire infected tissues11,12 using a tomograph. Second-harmonic generation reveals collagen sheaths around arteries and in fibrous bands such as the trabeculae of spleen, thus providing anatomical context. In vivo injected fluorescent antibodies can be used to stain host cells to reveal interactions between individual pathogen cells and infiltrating immune cells such as neutrophils. Here, the pipeline involving processing of the tissue, imaging, stitching of imaging tiles with illumination correction, stacking of images in three dimensions, and segmentation using machine-learning tools, is described. This pipeline yields 3D positions of individual pathogens cells and microcolonies within their host context. Counting the number of individual cells within microcolonies remains difficult because of resolution limits, but such numbers can be estimated based on the integrated brightness of the microcolony. The pipeline can be readily adapted to other infection models if recombinant GFP- or YFP-expressing pathogens are available.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose Low Melt</td><td>Roth</td><td>Art. 6351.5 25g</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>6768-500G</td><td></td></tr><tr><td>Instant adhesive Loctite 435</td><td>Henkel</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Poly(ethylene glycol)</td><td>Sigma-Aldrich</td><td>P5413-1kg</td><td></td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma-Aldrich</td><td>PVP-100G</td><td></td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>71321-25g</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106453</td><td></td></tr><tr><td>Sodium periodate</td><td>Sigma-Aldrich</td><td>311448-100G</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>71640-250G</td><td></td></tr><tr><td>Sodium phosphate monobasic dihydrate</td><td>Sigma-Aldrich</td><td>71500-1KG</td><td></td></tr><tr><td>Sodium tetraborate</td><td>Sigma-Aldrich</td><td>221732-100g</td><td></td></tr><tr><td>Sucrose</td><td>AppliChem</td><td>A4734,1000</td><td></td></tr><tr><td>Tris-buffered saline (TBS)</td><td>Merck</td><td>T5912-1L</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td></td></tr><tr><td>Vacuum filtration 500</td><td>TPP</td><td>TPP99250</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Blade</td><td>Campden Instruments Limited&amp;nbsp;</td><td>01-01-4692</td><td></td></tr><tr><td>MAITAI Laser</td><td>Spectra-Physics</td><td></td><td></td></tr><tr><td>Peel away plastic mold</td><td>Sigma-Aldrich</td><td>E6032-1CS</td><td></td></tr><tr><td>TissueCyte 1000 tomograph</td><td>TissueVision</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibody/dyes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Merck</td><td>D9542-5MG</td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-LPS &lt;em&gt;Salmonella&lt;/em&gt;, rabbit</td><td>Sifin</td><td>REF TS 1624</td><td></td></tr><tr><td>anti-CD169-PE, clone 3D6.112</td><td>Biolegend&amp;nbsp;</td><td>142403</td><td></td></tr><tr><td>anti-Ly-6G-PE, clone 1A8</td><td>Biolegend&amp;nbsp;</td><td>127608</td><td></td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>chicken anti-rabbit Alexa 647</td><td>Invitrogen</td><td>A-21443</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td>Company</td><td>Version</td><td></td></tr><tr><td>Fiji</td><td>Image J</td><td>1.54g or later</td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>2017b/2018b or later</td><td></td></tr><tr><td>Orchestrator (tomograph)</td><td>TissueVision</td><td></td><td></td></tr><tr><td>Visualization software Imaris</td><td>Oxford Instruments</td><td>9.9.0 or later</td><td></td></tr></tbody></table>

high&#45;resolution three&#45;dimensional whole&#45;organ tomography of microbial infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

The described procedure enables detection of individual Salmonella cells in entire mouse organs such as spleen, liver, mesenteric lymph nodes, and Peyer&#39;s patches11 (Figure 5 and Figure 6). It also detects Toxoplasma gondii parasites in mouse brain12. Some infected tissues including liver, Peyer&#39;s patches, and spleen emit substantial autofluorescence in the green-yellow range. Autofluorescence is further enhanced by the fixation with paraformaldehyde, which is necessary to preserve the tissue structure. Detection of green fluorescence from GFP, mWasabi, and the green component of TIMERbac against this autofluorescence background is improved by putting a narrow bandpass filter 510/20 nm (transmitting most GFP emissions but blocking a large part of the autofluorescence spectrum) in front of photomultiplier 2 (which collects green emissions) and by reducing tissue autofluorescence by storing fixed tissues for 3 or more days in cryoprotectant11. However, bacteria should still express at least a few thousand copies per cell of GFP or other fluorescent proteins. On the other hand, excessive fluorescent protein levels should be avoided to minimize fitness costs that could lead to attenuated virulence23.
Correct segmentation of fluorescent bacteria in the tissues can be confirmed by immunohistochemistry of retrieved tissue sections after imaging. Specifically, objects stained with an antibody to bacterial surface components such as lipopolysaccharide can be aligned with the fluorescence image obtained by STP tomography (Figure 5). It is important to note that some stained Salmonella cells lack fluorescent proteins and thus detectable fluorescence in both confocal microscopy and STP tomography. These cells are Salmonella that have been killed by the host immune system as demonstrated by flow cytometric sorting and growth cultures from single sorted cells as well as the close correlation between the number of colony-forming units on agar plates and the number of fluorescent Salmonella cells as determined by flow cytometry24. In addition, plasmid loss can result in non-fluorescent viable cells and this needs to be tested by plating on media with and without appropriate antibiotics corresponding to the selection marker on the plasmid. For pSC101-derived plasmids, plasmid loss in vivo is rare19. For most chromosomally integrated expression cassettes such as sifB::gfp used in11, loss of expression is undetectable in vivo. If the segmentation is inconsistent with immunohistochemistry data, the segmentation pipeline needs to be modified.
The resolution of STP tomography is insufficient to resolve individual bacterial cells within densely packed microcolonies. However, the total fluorescence intensity of the microcolony enables estimation of the number of Salmonella cells. This requires a fluorescent strain with highly homogenous fluorescence levels such as Salmonella sifB::gfp11. Combining the estimated number of Salmonella cells for all microcolonies and single cells yields total bacterial tissue loads that are consistent with alternative methods such as plating or flow cytometry of tissue homogenates11. Plating and flow cytometry cannot be done directly from the same tissues because they need to be perfusion-fixed for STP tomography. Instead, they have to be done with additional animals that are not fixed. If the median bacterial loads as determined by the various approaches differ by more than 3-fold, the viability of fluorescent bacteria might be compromised (in case of lower colony-forming units) or some bacteria might have lost the fluorescent reporter construct (in case of higher colony-forming units). Control experiment will be required to identify the source of such discrepancies.
STP tomography provides the localization of bacterial cells within the 3D structure of the infected tissues. The second harmonic signals of collagen provide anatomical landmarks such as arteries and trabeculae. In addition, host cells can be stained in vivo by injecting an antibody to surface markers prior to perfusion (Figure 5 and Figure 6). This staining provides additional landmarks for tissue compartments and specific microenvironments including inflammation foci (intracellular markers and some compartments with diffusional barriers such as the splenic white pulp or the brain are not easily accessible suitable for this in vivo staining). This approach revealed the splenic white pulp as a tissue compartment that enables long-term survival of Salmonella during antimicrobial chemotherapy11.
Finally, Salmonella replicating at moderate or slow rates can be identified and localized within the 3D structure of tissues using strains that express timerbac, a single cell reporter for replication rate11,15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66469/66469fig01.jpg" /> 
Figure 1: Procedure for whole-organ imaging using serial two-photon (STP) tomography. (A-B) The organ is harvested after transcardial perfusion and stored overnight in 4% paraformaldehyde (PFA) at 4 &#176;C. (C-E) The organ is embedded in oxidized agarose and cross-linked, then the tissue is scanned and sliced using STP tomography. The slices are collected for subsequent immunohistochemistry. (F-J) Computational analysis pipelines for quantifying bacterial numbers, confirmation of fluorescent bacteria, and 3D reconstruction of bacterial positions. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66469/66469fig02.jpg" /> 
Figure 2: Serial two-photon (STP) tomography setup. (A) Tomograph which incorporates (B) 2-photon imaging with (C) automated serial tissue sectioning. (D) Collected tissue slices for follow-up investigations. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66469/66469fig03.jpg" /> 
Figure 3: Tile stitching and illumination correction. Tiles are stitched and uneven illumination is corrected for using corrected intensity distributions using regularized energy minimization (CIDRE). <a href="https://www.jove.com/files/ftp_upload/66469/66469fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66469/66469fig04.jpg" /> 
Figure 4: Segmentation of Salmonella expressing the green fluorescent protein (GFP) using a Support Vector Machine (SVM) and a Convolutional Neural Network (CNN). (A) Representative images of GFP objects segmented by SVM (left) and corresponding images (right) with regions segmented by SVM (Scale bar: 10 &#181;m). (B) Clustered red-green-blue (RGB) values distribution for the segmented regions. (C)&#160;Representative images of non-GFP objects falsely identified by the SVM as bacteria (left). CNN correctly dismisses them as background (right, scale bar: 10 &#181;m). <a href="https://www.jove.com/files/ftp_upload/66469/66469fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66469/66469fig05.jpg" /> 
Figure 5: Detection of Salmonella expressing the green fluorescent protein (GFP) by tomography and confirmation by immunohistochemistry. Images of the same section acquired by tomography (left) or confocal microscopy after staining with an antibody to Salmonella lipopolysaccharide (right). Neutrophils (red) were stained by in vivo injection of a PE-labeled anti-Ly-6G antibody prior to perfusion. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66469/66469fig06.jpg" /> 
Figure 6: 3D reconstruction and localization of Salmonella in infected mouse spleen. (A) Three-dimensional (3D) reconstruction of a 5 mm thick spleen slice and stained in vivo before perfusion with anti-CD169 antibody (red). The blue signal represents collagen detected by second harmonics. (B) One optical plane of the 3D stack shown in (A). The positions of Salmonella cells or microcolonies are indicated by stars. (C) 3D reconstruction of Salmonella positions&#160;(stars) in infected liver. The arteries are visible based on their collagen sheaths (blue). Scale bar: 1 mm. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues at JoVE.com

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

high-resolution-three-dimensional-whole-organ-tomography-microbial

Research

JoVE Journal

Immunology and Infection

825 Views.  University of Basel. Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Video: Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bioengineering

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Biology

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Environment

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Developmental Biology

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.

High-Resolution Three-Dimensional Whole-Organ Tomography of Microbial Infections

Summary

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High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

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Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Visualization of Organelles In Situ by Cryo-STEM Tomography