Methods of identifying, imaging and characterizing bacterial spores using lanthanide-beta-diones

Abstract

Methods, compositions and kits for staining, imaging, identifying and / or characterizing one or more bacterial spores in a sample by contacting the sample with a probe having the formula Ln [BD] n, where Ln is a member of the lanthanide series, BD is a beta-diketone, n is an integer, to stain one or more bacterial spores in the sample; and detecting a fluorescence signal of the one or more stained bacterial spores in the sample using a fluorescence microscope.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to Singapore patent application No. 10202260603W filed on December 27, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of imaging and detection. Specifically, a non-toxic lanthanide-β-diketone probe and related methods for in situ imaging and characterization of bacterial spores are disclosed. Background Art

[0004] Bacteria belonging to the genera Bacillus and Clostridium are ubiquitous in nature and have the ability to initiate endospore formation when they sense adverse environmental conditions. These spores can survive for many years, with the longest known survival period possibly reaching 250 million years. These long-lived dormant spores remain viable and can germinate into vegetative cells when favorable conditions return. Bacterial spores can cause many medically significant infections, particularly foodborne illnesses (e.g., Bacillus cereus, Clostridium perfringens, C. botulinum) and hospital-acquired infections (e.g., Clostridium difficile). It is speculated that some bacterial spores could be used as biological weapons without further modification, and could even be transported into space, thereby endangering life exploration experiments and inadvertently altering extraterrestrial ecosystems.

[0005] In addition to their threat as infectious pathogens, spore detection is also a concern for space exploration, as contamination of extraterrestrial planets by life of Earth origin could jeopardize life detection experiments and inadvertently alter extraterrestrial ecosystems. Similar spore contamination concerns exist in the food, pharmaceutical manufacturing, and medical device industries, where sterility is crucial. A long-standing and unmet need in all of these research areas is the rapid detection and sorting of suspected spore-containing materials.

[0006] Spore detection methods can be divided into direct and indirect methods. The earliest attempts at bacterial spore detection relied primarily on ultraviolet (UV) absorbance to reveal the presence of dipicolinic acid (DPA), a common component of bacterial spores. In contrast, more recent methods have shifted to using fluorescence spectroscopy to detect DPA. The current state-of-the-art technology is based on the detection of DPA released by spores in combination with terbium (III) cations (Tb 3+Visualization of this diffusible and non-spore-localized signal requires immobilizing the spores on a diffusion-retarding matrix (such as agarose) and using a DPA / Tb-compatible 3+ Due to these limitations, the DPA assay is primarily useful as an indirect indicator of germinating spores at the population level.

[0007] The detection limit of this indirect test is 10 5 CFU / ml, but can be increased to 10 if dodecylamine is used and heated at 60°C to maximize DPA release. 3 CFU / ml. Since only live spores with a complete DPA pool contribute to the fluorescent signal, pre-germinated spores that have released their DPA will not be detected. Therefore, DPA / Tb 3 Fluorescence cannot reveal whether a negative sample is completely devoid of spores or filled with germinated spores. In the latter case in particular, this can lead to an underestimation of the pathogenic threat, as germinated spores can easily transform into an infectious vegetative form. Colorimetric reagents have also been developed for DPA detection, such as erbium-pyrocatechol purple and betalain, but these reagents have poor detection limits (~10 6 CFU / ml), are also subject to similar restrictions.

[0008] Bacterial endospores have a rigid morphology that prevents many dyes from penetrating the spore structure. Therefore, conventional techniques require permeabilization of the spore structure to facilitate staining. For example, the Schaeffer-Fulton assay requires permeabilization of the spores by heat fixation and steam treatment to allow malachite green to enter the spores. Among fluorescent stains, some stain only dormant forms (ThT) or germinating forms (SYTO-16). The lack of staining for dormant or germinating forms makes visualization and counting of non-stained spores difficult or impossible. Other fluorescent stains stain dormant and germinating forms to the same degree and are visually indistinguishable (DAPI, AO), making it impossible to distinguish dormant from germinating spores. Finally, a problem unique to malachite green and ThT is that their associated protocols are inherently toxic to germinating spores, making these methods unsuitable for live imaging of germination events.

[0009] Therefore, there is a need for a non-destructive method that allows for the direct detection and characterization of bacterial spores. There is also a need for a method for transiently detecting bacterial spores and their germination states. There is also a need for live imaging of bacterial spores and their germination states. Summary of the Invention

[0010] The present disclosure provides compositions, methods, and kits for transient in situ fluorescent labeling of bacterial spores using lanthanide-β-diketones without further processing. Given their low toxicity to spores and the ability to distinguish between dormant and germinating forms, this method also allows for live imaging of individual germinating spores.

[0011] In one aspect, the present invention is a method for live imaging of one or more bacterial spores in a sample, comprising: contacting the sample with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain the one or more bacterial spores in the sample; and detecting a fluorescent signal from the one or more stained bacterial spores in the sample using a fluorescence microscope.

[0012] Another aspect of the present invention is a method for in situ distinguishing the germination state of one or more bacterial spores, comprising: contacting a sample containing one or more bacterial spores with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain the one or more bacterial spores in the sample; live imaging the sample using a fluorescence microscope for a fluorescent signal; and distinguishing between dormant bacterial spores and germinating or germinated bacterial spores in the sample. In some embodiments, the dormant bacterial spores have a fluorescent signal characterized by a circular fluorescent profile with a hollow center, while the germinating or germinated bacterial spores have a central core that is filled with fluorescence, wherein Ln is a member of the lanthanide series and BD is a β-diketone.

[0013] In other embodiments, the present invention is a method for real-time in vivo imaging of bacterial spore germination, comprising: contacting one or more bacterial spores with a germination probe solution comprising one or more germinants and a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample to form a mixture; placing the mixture on a slide; visualizing the morphology of the one or more bacterial spores on the slide over time using time-lapse phase contrast microscopy; and imaging the fluorescent signals of the one or more bacterial spores on the slide over time using time-lapse fluorescence microscopy.

[0014] The method for live imaging of one or more bacterial spores in a sample and the method for distinguishing the germination state of one or more bacterial spores in situ can further include using a USB-powered microscope, a digital microscope, a confocal microscope, a Raman microscope, or a bright-field microscope for signal detection. Thus, advantageous features of the method of the present invention include: (i) instantaneous nature, (ii) no special handling involved, (iii) no spore destruction—allowing live imaging of germinating spores, (iv) staining of spores to distinguish between dormant spores and germinating spores, and (v) the ability to use a low-cost USB-powered microscope (although details of the spores cannot be visualized), which can be used to provide at least preliminary on-site results of the sample.

[0015] In some aspects, the real-time live imaging of one or more bacterial spore germination comprises three phase contrast microscopy stages, including: 1) a bright phase of spore germination, 2) a gray phase of spore germination, associated with a most intense fluorescent signal localized in an outer region of the spore core, and 3) a dark phase of spore germination, associated with a most intense fluorescent signal localized in an area of the spore core, wherein the dark phase is indicative of bacterial spore germination.

[0016] In some embodiments, any of the above methods can be used for food pathogen detection, environmental monitoring, quality assurance, or microbiological research, for monitoring spore bioburden in food or pharmaceutical samples, for monitoring spore bioburden in the environment, or for monitoring spore bioburden as a quality assurance step in an industrial process. In some aspects, the industrial process is the production of self-healing concrete.

[0017] In some embodiments, the sample comprises a plurality of bacterial spores, and the method further comprises quantifying the plurality of bacterial spores in the sample. In some embodiments, the imaging and / or monitoring comprises generating a video of the sample to observe changes in germination over time.

[0018] The present invention also includes a kit comprising a germination probe having the formula Ln[BD]n in a first container, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, and a germination agent in a second container. In some aspects, the germination agent (or compound that supports germination) is L-cysteine, hypoxanthine, an oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some aspects, the kit comprises a plurality of germination agents, wherein each germination agent is provided in a separate container.

[0019] The present invention also includes a composition comprising a germination probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, and a germination agent, wherein the germination agent (or a compound that supports germination) is L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some aspects, L-valine or D-valine can be used as a germination agent for C. novyi-NT strains.

[0020] Other features and characteristics of the disclosed subject matter, as well as the method of operation, the function of the related structural elements and combinations of parts, and the economy of manufacture, will become more apparent upon consideration of the following description and appended claims, all of which form a part of this specification.

[0021] In one aspect, the present invention is a device comprising a device for live imaging of one or more bacterial spores in a sample, wherein the one or more bacterial spores have been contacted with a probe having the formula Ln[BD]n. In some aspects, Ln can be a member of the lanthanide series, BD is a β-diketone, and n is an integer to stain the one or more bacterial spores in the sample. In some aspects, the device for live imaging is a camera, a microscope, an image capture device, a video capture device, or a combination thereof. One of ordinary skill in the art will appreciate that a camera, a microscope, an image capture device, a video capture device, or a combination thereof has a structure for performing imaging.

[0022] In some aspects, a device may include means for in situ distinguishing the germination state of one or more bacterial spores in a sample. In some aspects, the sample may include one or more bacterial spores stained with a probe having the formula Ln[BD]n. In some aspects, Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer. In some aspects, the device may be configured to acquire a live image fluorescence signal of the sample using a fluorescence microscope and distinguish between dormant bacterial spores and germinating or germinated bacterial spores in the sample.

[0023] In some aspects, the dormant bacterial spore has a fluorescent signal characterized by a circular fluorescent outline with a hollow center. In certain aspects, the germinating or germinated bacterial spore has a central core that is filled with fluorescence.

[0024] In some aspects, a device can comprise a device for real-time, in vivo imaging of germination of bacterial spores that have been contacted with a germination probe solution comprising one or more germinants and a probe having the formula Ln[BD]n.

[0025] In some aspects, Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer to stain one or more bacterial spore-forming mixtures in the sample. In some aspects, the device comprises a slide configured to hold the mixture.

[0026] In some aspects, the device comprises a time-lapse phase-contrast microscope configured to visualize bacterial spores in the mixture and image the fluorescent signal of the bacterial spores on a slide. In some aspects, the real-time live imaging of the germination of one or more bacterial spores comprises three phase-contrast microscopy phases, including: 1) a bright phase of spore germination, 2) a gray phase of spore germination, which is associated with the most intense fluorescent signal located in the outer region of the spore core, and 3) a dark phase of spore germination, which is associated with the most intense fluorescent signal located in the spore core region, wherein the dark phase is indicative of bacterial spore germination. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A Shown are fluorescent images of germinating C. novyi-NT spores stained with different ratios of Eu and TTA. Figure 1B Shown are fluorescent images of germinating C. novyi-NT spores stained with different concentrations of Eu[TTA]3. Figure 1C Shown are germinating C. novyi-NT spores stained with 1.4 mM Eu[TTA]3 (left), 1.4 mM EuCl3 (center), and 4.2 mM TTA (right). The top row represents widefield phase contrast images, and the bottom row represents fluorescence images.

[0028] Figure 2A Shown are dormant (left) and germinated (right) spores of Clostridium novyi-NT, Clostridium septicum, Bacillus subtilis, Clostridioides difficile, and Bacillus thuringiensis stained with 1.4 mM Eu[TTA] 3. Top represents widefield phase contrast images, bottom represents fluorescence images. Figure 2B Shown are the DPA (1017 cm) along the longest spore axis of dormant and germinated C. novyi-NT spores. -1 ) and europium (1531cm -1 ) Raman peak profiles. Average Eu Raman peak intensity distributions (top) for spores stained with Eu[TTA]3 (left) and EuCl3 (right), and average DPA Raman peak intensity distributions (bottom) for spores stained with Eu[TTA]3 (left) and EuCl3 (right), respectively (N = 6 for each sample).

[0029] Figure 3A Shown are pseudo-colored confocal images of dormant (left) and germinated (right) spores stained with Eu[TTA]3 (1.4 mM) - Clostridium novyi-NT (row 1), Clostridium septicum (row 2), Clostridium difficile (row 3), Bacillus subtilis (row 4), and Bacillus thuringiensis (row 5). Figures 3B-3D Pseudo-colored confocal images showing co-staining of Eu[TTA]3 (green) with the membrane dye Nile Red (red), co-staining of Eu[TTA]3 (green) with the DNA staining dye DAPI (blue), and co-staining of Eu[TTA]3 (green) with FITC-dextran 3k-5k (red), respectively.

[0030] Figure 4 Dormant and germinated C. novyi-NT spores treated with 1.4 mM of the corresponding lanthanide-TTA complex are shown. Images represent widefield phase contrast (rows 1 and 3) and fluorescence (rows 2 and 4) images of dormant spores (top) and germinated spores (bottom), stained with 1.4 mM Eu[TTA]3 (column 1), Sm[TTA]3 (column 2), Dy[TTA]3 (column 3), and Tb[TTA]3 (column 4), respectively.

[0031] Figure 5 Shown are dormant and germinating Clostridium novyi-NT spores stained with 1.4 mM of different europium β-diketone complexes. Images represent widefield phase contrast (rows 1 and 3) and fluorescence (rows 2 and 4) images of dormant spores (top) and germinating spores (bottom), stained with 1.4 mM Eu[BTFA]3, Eu[NTFA]3 (column 2), Eu[AA]3 (column 3), Eu[TriFAA]3 (column 4), and Eu[TetraFAA]3 (column 5), respectively.

[0032] Figure 6A Shown are pregerminated Clostridium novyi-NT and Bacillus subtilis spores treated with Eu[TTA]3 (1.4 mM) for 15 minutes and plated on BHI-FBS or LB agar plates, respectively. Data represent the mean of two independent experiments, and error bars represent standard deviations. Figure 6B and 6CShown are time-lapse images of Clostridium novyi-NT spores stained with Eu[TTA]3 during germination with L-cysteine, hypoxanthine, and oxygenase, and of Bacillus subtilis P1A1 spores stained with Eu[TTA]3 during germination with L-alanine. Each time panel contains the corresponding phase-contrast image (top) and fluorescence image (bottom). The inset below shows a magnified fluorescence image of a germinating spore and a 3D surface plot projection of the fluorescence of a single germinating spore using the Interactive3D Surface plot plugin in ImageJ.

[0033] Figure 7A Representative phase contrast and fluorescence images of individual light, gray, and dark phase spores and their Raman spectra are shown. Average Raman spectra of spores at three different stages of germination (N = 20 for each stage). DPA (1017 cm) of light, gray, and dark phase spores -1 ) and Eu(1531cm -1 ) Average Raman intensity distribution of the peak (N=20 in each stage). Figure 7B Eu[TTA]3 (1.4 mM) was added to pre-germinated wild-type P1A1 and cwlD mutant spores. The left panel shows wild-type spores, and the right panel shows cwlD mutant spores. The upper panel shows phase contrast images, and the lower panel shows fluorescence images. Figure 7C Dormant Clostridium novyi-NT spores treated with decoating buffer, subsequently treated with cortex lysis buffer containing lysozyme, and stained with Eu[TTA]3 (1.4 mM) are shown. Column 1 depicts untreated dormant spores stained with Eu[TTA]3, column 2 represents decoated spores stained with Eu[TTA]3, and column 3 represents dormant spores treated with lysozyme. Column 4 represents decoated spores treated with lysozyme. The upper panel depicts phase contrast images, and the lower panel depicts fluorescence images.

[0034] Figure 8 (Top) RGB images of dormant and germinating C. novyi-NT and B. subtilis spores stained with malachite green for 15 minutes and counterstained with safranin. (Bottom) Phase contrast (rows 1 and 3) and fluorescence (rows 2 and 4) images of dormant (left) and germinating (right) C. novyi-NT and B. subtilis spores stained with DAPI (5 μM) and acridine orange (9.3 mg / ml), respectively, for 30 minutes.

[0035] Figure 9 Fluorescence images represent dormant (left) and germinated (right) spores of Clostridium novyi-NT, Clostridium septicum, Clostridium difficile, Bacillus subtilis, and Bacillus thuringiensis stained with 1.4 mM Eu[TTA]3.

[0036] Figure 10An aliquot of a 2.5% (w / v) milk sample spiked with dormant C. novyi-NT spores was stained with Eu[TTA]3 (1.4 mM). Images represent phase contrast (top) and fluorescence (bottom) images of C. novyi-NT spores in a milk matrix with (left) and without (right) the addition of Eu[TTA]3.

[0037] Figure 11 Shown are 3D maximum intensity projection images of Z-stack confocal images of dormant (left) and germinated (right) C. novyi-NT (top) and C. septicum (bottom) spores from Figure 3.

[0038] Figure 12 Table showing a comparison of the performance of various spore stains.

[0039] Figure 13A Shown are Z-average intensity projection images of Z-stack confocal images of Clostridium novyi-NT (left) and Bacillus subtilis (right) cultures germinated in BHI-FBS oxygenase medium and LB medium, respectively, both stained with Eu[TTA]3 (green). Figure 13B Shown are single Z-plane confocal images of an overnight culture of early-sporulating B. cereus in 2xSG sporulation medium, stained with Eu[TTA]3 (green). Scale bar 2 μm. Figure 13C Phase contrast (top) and fluorescence (bottom) images of Bacillus cereus spores in a 2.5% (v / v) milk matrix stained (left) and unstained (right) with Eu[TTA] 3 are shown. Scale bar: 2 μm. DETAILED DESCRIPTION

[0040] Although aspects of the disclosed subject matter can be embodied in a variety of forms, the following description is intended to disclose only some of these forms as specific examples of the subject matter encompassed by the disclosed subject matter. Therefore, the disclosed subject matter is not intended to be limited to the forms or embodiments described above.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, the present specification (including definitions) will prevail.

[0042] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0043] Concentration, amount and other numerical data herein can be expressed or presented in a range format. It should be understood that this range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly recorded as range limits, but also including all individual numerical values or subranges included within the range, as if each numerical value and subrange were clearly recorded. For example, the numerical range of "about 0.01 to 2.0" should be interpreted as not only including the value of about 0.01 to about 2.0 clearly recorded, but also including individual values and subranges within the range. Therefore, included in this numerical range are individual values such as 0.5, 0.7 and 1.5, and subranges such as from 0.5 to 1.7, 0.7 to 1.5 and from 1.0 to 1.5. In addition, no matter the breadth of the scope or the described features, this explanation should be applied. In addition, it should be noted that, unless otherwise stated, all percentages are weight percentages.

[0044] In understanding the scope of the present disclosure, the terms "including" or "comprising" and their derivatives as used herein are intended to be open terms, which indicate the presence of described features, elements, components, groups, integers and / or steps, but do not exclude the presence of other unmentioned features, elements, components, groups, integers and / or steps. The foregoing also applies to words with similar meanings, such as the terms "including", "having" and their derivatives. The terms "consisting" and their derivatives as used herein are intended to be closed terms, which indicate the presence of described features, elements, components, groups, integers and / or steps, but exclude the presence of other unmentioned features, elements, components, groups, integers and / or steps. The term "consisting essentially of" as used herein is intended to indicate the presence of described features, elements, components, groups, integers and / or steps and those features, elements, components, groups, integers and / or steps that do not substantially affect the basic and novel properties of the features, elements, components, groups, integers and / or steps. It is understood that reference to any of these transitional terms (i.e., "comprising," "consisting of," or "consisting essentially of") provides direct support for substitution with any other transitional term not specifically used. For example, modifying the term from "comprising" to "consisting essentially of" or "consisting of" will find direct support for any element disclosed throughout this disclosure as a result of this definition. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0045] As used herein, for convenience, multiple compounds, elements, or steps may be presented in a common list. However, these lists should be interpreted as if each member of the list is individually identified as a separate and unique member. Therefore, any individual member of such a list should not be considered a de facto equivalent of any other member of the same list based solely on presentation in a common group without an indication to the contrary.

[0046] The present disclosure encompasses the detection of any bacterial spore and is not limited to the exemplary bacterial spore types used in the experiments. These experiments serve as proof of concept to support the use of the methods of the present invention for the detection of bacterial spores in general. Non-limiting examples of such bacterial spores include, for example, Bacillus subtilis (B. subtilis), which is a well-characterized model of bacterial spore biology; Bacillus thuringiensis (B. thuringiensis), an insect pathogen that expresses a pore-forming toxin with insecticidal applications and is also a close relative of B. anthracis, sometimes used as a surrogate in research studies; Bacillus cereus (B. cereus), a common foodborne pathogen that releases toxins that can cause food poisoning and related illnesses; C. novyi-NT, a gas gangrene bacterium derived from Clostridium novyi type A by removing its inherent phages, making it genetically equivalent to Clostridium botulinum type C. C); C. difficile, a nosocomial agent transmitted in hospital settings; and C. septicum, a livestock pathogen known to be associated with infections in patients with existing (often occult) hematological and gastrointestinal malignancies. These proof-of-concept bacterial spore types were selected and confirmed to be effective in a variety of endospore-forming bacteria.

[0047] The germination process of bacterial spores is generally divided into three consecutive phases, termed phase bright, phase gray, and phase dark. These terms describe the brightness of the spores as observed under a phase-contrast microscope. Both the darkening and the release of the spore biomarker DPA are hallmarks of bacterial spore germination.

[0048] In addition, bacterial spores contain sieve-like layers with different permeabilities that selectively restrict the entry of small and large molecules. Among these sieves, the cortex layer is considered to be the final physical barrier of the bacterial spore's inner membrane. During normal germination, this cortex barrier is hydrolyzed by enzymes.

[0049] The present disclosure provides compositions, methods, and kits for transient in situ fluorescent labeling of bacterial spores using lanthanide-β-diketones without further processing. Given their low toxicity to spores and the ability to distinguish between dormant and germinating forms, this method also allows for live imaging of individual germinating spores. Thus, in some aspects, the present invention provides a method that does not require an incubation period.

[0050] In one aspect, the present invention is a method for live imaging of one or more bacterial spores in a sample, comprising: contacting the sample with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain the one or more bacterial spores in the sample; and detecting a fluorescent signal from the one or more stained bacterial spores in the sample using a fluorescence microscope.

[0051] Another aspect of the present invention is a method for in situ distinguishing the germination state of one or more bacterial spores, comprising: contacting a sample containing one or more bacterial spores with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain the one or more bacterial spores in the sample; live imaging the sample using a fluorescence microscope for a fluorescent signal; and distinguishing between dormant bacterial spores and germinating or germinated bacterial spores in the sample. In some embodiments, the dormant bacterial spores have a fluorescent signal characterized by a circular fluorescent profile with a hollow center, while the germinating or germinated bacterial spores have a central core that is filled with fluorescence, wherein Ln is a member of the lanthanide series and BD is a β-diketone.

[0052] In other embodiments, the present invention is a method for real-time in vivo imaging of bacterial spore germination, comprising: contacting one or more bacterial spores with a germination probe solution comprising one or more germinants and a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample to form a mixture; placing the mixture on a slide; visualizing the morphology of the one or more bacterial spores on the slide over time using time-lapse phase contrast microscopy; and imaging the fluorescent signals of the one or more bacterial spores on the slide over time using time-lapse fluorescence microscopy.

[0053] In some embodiments, Raman microscopy can distinguish between dormant spores exhibiting a bimodal distribution and germinating spores exhibiting a unimodal distribution. In some aspects, the method further comprises detecting a peak in the bimodal distribution that includes an inner membrane region and a valley between peaks corresponding to the spore core region, and detecting a unimodal distribution that includes the spore core. In some aspects, the Raman distribution of Ln levels for the Ln[BD]n probe exhibits no statistically significant shift throughout the germination process. In some embodiments, when a single peak corresponding to the Ln[BD]n probe is present in the spore core region, dipicolinic acid (DPA) is absent.

[0054] In some embodiments, signal detection is performed using a confocal microscope, wherein an optical plane section image through a dormant spore shows a hollow region corresponding to the spore core region without any fluorescent signal, and wherein an optical plane section image through a germinating spore shows a fluorescent signal in the center of the spore corresponding to the spore core region.

[0055] The method for live imaging of one or more bacterial spores in a sample and the method for distinguishing the germination status of one or more bacterial spores in situ may further include performing signal detection using a USB-powered microscope, a digital microscope, a confocal microscope, a Raman microscope, or a bright-field microscope.

[0056] In some aspects, the real-time live imaging of one or more bacterial spore germination comprises three phase contrast microscopy stages, including: 1) a bright phase of spore germination, 2) a gray phase of spore germination, associated with a most intense fluorescent signal localized in an outer region of the spore core, and 3) a dark phase of spore germination, associated with a most intense fluorescent signal localized in an area of the spore core, wherein the dark phase is indicative of bacterial spore germination.

[0057] In some embodiments, the germination agent (or compound that supports germination) used is one or more of L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some embodiments, Ln in the Ln[BD]n probe is Europium, Samarium, Dysprosium, or Terbium. In some embodiments, BD in the Ln[BD]n probe is at least one member selected from the following group: thenoyltrifluoroacetone (TTA), 1-(2-naphthoyl)-3,3,3-trifluoroacetone (NTFA), 4,4,4-trifluoro-1-phenyl-1,3-butanedione (BTFA), acetylacetone (AA), 1,1,1-trifluoroacetylacetone (TriFAA), and 1,1,5,5-tetrafluoroacetylacetone (TetraFAA). In some embodiments, n in the Ln[BD]n probe is 1 to 5, or 2 to 4, or 3.

[0058] In some embodiments, the probe is at least one member selected from the following group: Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, Tb[TTA]3, Eu[BTFA]3, Eu[NTFA]3, Eu[AA]3, Eu[TriFAA]3 and Eu[TetraFAA]3. In some embodiments, the probe is Europium(III)thenoyltrifluoroacetone (Europium(III)thenoyltrifluoroacetone, Eu[TTA]3.

[0059] In some aspects, the ratio of Ln to BD in the Ln[BD]n probe is in the range of 9: 1 to 1: 9. In some aspects, the concentration of the probe is in the range of 0.7 mM to 10 mM, or in the range of 1 mM to 5 mM, or in the range of 1.4 mM to 2.8 mM, which ranges support the selection of any concentration or range of concentrations within the ranges.

[0060] In some embodiments, the probe binds to spores from at least one Bacillus, Clostridium, or Clostridioides. In some aspects, the probe binds to spores from at least one B. subtilis, B. thuringiensis, B. cereus, C. novyi-NT type A, C. difficile, or C. septicum.

[0061] In some embodiments, treatment with the probe does not significantly affect the viability, growth, or colony-forming ability of the spores (compared to untreated spores). In some aspects, the change in colony forming units (CFU) of the bacterial spores before and after treatment is less than 0.5 log CFU.

[0062] In some aspects, staining with the probe is instantaneous or near instantaneous. In some aspects, bacterial spores are visualized immediately after the contacting step.

[0063] In some aspects, any of the above methods do not include a spore permeabilization step or a heating step. In some aspects, no fluorescent brightener is used. In some aspects, the methods of the present disclosure do not include the use of malachite green.

[0064] In some aspects, hydrolysis of the spore cortex precedes entry of the probe into the core.

[0065] In some embodiments, the fluorescence microscope is equipped with a 4',6-diamidino-2-phenylindole (DAPI) long pass filter.

[0066] In some aspects, the method further comprises adding one or more surfactants to the sample. In some aspects, the one or more surfactants may include Triton X-100 (2-[4-(2,4,4-trimethylpentane-2-yl)phenoxy]ethanol), polysorbates, such as polysorbate 20 ( 20), sodium lauryl sulfate, or a combination thereof. In some aspects, any of the above methods can further comprise contacting the sample with one or more additional probes or dyes and detecting one or more additional signals. In some aspects, one or more of the above methods further comprise co-staining bacterial spores with Nile Red, DAPI, fluorescein isothiocyanate (FITC)-dextran, or a combination thereof.

[0067] In some embodiments, any of the above methods can be used for food pathogen detection, environmental monitoring, quality assurance, or microbiological research. They can be used to monitor spore bioburden in food or pharmaceutical samples, to monitor spore bioburden in the environment, or to monitor spore bioburden as a quality assurance step in an industrial process. In some aspects, the industrial process is the production of self-healing concrete.

[0068] In some embodiments, the sample comprises a plurality of bacterial spores, and the method further comprises quantifying the plurality of bacterial spores in the sample. In some embodiments, the imaging and / or monitoring comprises generating a video of the sample to observe changes in germination over time.

[0069] The present invention also includes a kit comprising a germination probe having the formula Ln[BD]n in a first container, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, and a germination agent in a second container. In some aspects, the germination agent is L-cysteine, hypoxanthine, an oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some aspects, the kit comprises a plurality of germination agents, wherein each germination agent is provided in a separate container.

[0070] The present invention also includes a composition comprising a germinative probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, n is an integer, and a germinant, wherein the germinant is L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine.

[0071] The disclosed invention will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the scope of the disclosed invention.

[0072] Example

[0073] The following examples are intended to illustrate the present disclosure and are not intended to limit the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples and the remainder of the specification, figures, and claims form part of the present disclosure.

[0074] Example 1

[0075] Spore formation of C. novyi-NT. Spores of C. novyi-NT were generated according to the method reported by Dang et al. in 2001. Briefly, the microorganisms were cultured in a Gaspak TMThe culture medium contained 5 g Na2HPO4, 30 g polypeptide peptone, 0.5 g L-cysteine, 10 g maltose, 50 g dry cooked meat particles (Difco) and 10% v / v fetal bovine serum (FBS) per liter in an anaerobic jar at 37°C and pH 7.4. After 3 weeks of culture in this medium, the spores settled in the cooked meat particle layer. The spores were further purified from contaminating vegetative forms by a discontinuous Percoll gradient. The spore quality was confirmed by phase contrast microscopy and contained >99% bright phase spores. The spore concentration was counted using a Neubauer counting chamber with a special depth (Petroff Marenfield) and adjusted to approximately 5x10 9 The order of CFU / ml.

[0076] Bacillus sporulation. For Bacillus subtilis, Bacillus cereus and Bacillus thuringiensis spores, sporulation was performed based on the protocol of Nicholson and Setlow (1990). Bacillus strains were inoculated on Luria Bertani (LB) agar plates at 37°C overnight. The next day, a single colony was inoculated into 2xSG medium (30 ml, pH 7) and grown on a MaxQ8000 orbital shaker (200 rpm, 37°C) until its OD600 value reached 0.3-0.5. This culture (25 ml) was then inoculated into a 2 L Erlenmeyer flask containing 2xSG medium (225 ml, pH 7) and covered with a breathable membrane (Breathe ) covered to allow adequate ventilation. The flasks were then placed in a Gerhardt orbital shaker (130 rpm, 37°C) and cultured for 44 hours (all mutants) and 92 hours (wild strain). The harvested spores were washed with 1X PBS and then subjected to isopycnic centrifugation using a self-forming Percoll gradient (70% for mutants and 90% for wild strain) in a Beckmann-Coulter ultracentrifuge (JS13.1 rotor, 15000 rcf, 30 min, 4°C). Most of the bright phase spores were obtained as the bottom fraction (wild strain) or as the top fraction (mutant strain), with the exception of Bacillus cereus, whose spores were obtained as the top fraction. These fractions were washed repeatedly with 1X phosphate buffered saline (PBS) and stored at 4°C until use. The spore quality was confirmed by phase contrast microscopy and contained >99% bright phase spores. With the exception of Bacillus cereus spores, the spores were adjusted to 10 10 The spore concentration of all strains except those with CFU / ml was counted using a Neubauer counting chamber with a special depth (Petroff Marenfield) and adjusted to approximately 10 9 The order of CFU / ml.

[0077] Sporulation of C. septicum. C. septicum spores were purified according to the protocol of Dang et al., 2001, with some modifications. All steps were performed in a Plas Labs anaerobic chamber. Briefly, an overnight culture of C. septicum was diluted 50-fold into 100 ml of BHI-S medium containing 0.05% L-cysteine and grown to an OD600 of 1.5-3. The entire culture was then added to 900 ml of sporulation medium (0.05% L-cysteine, 3% bacto peptide peptone, 5% dehydrated cooked meat medium, and 10% fetal bovine serum) and incubated at 37°C for five days. Spores were purified from vegetative cells by centrifugation at 15,000 rcf for 30 minutes using an 80% discontinuous Percoll gradient in a Beckman Avanti J-20XP high-performance centrifuge. The resulting spores were washed twice, resuspended in water, and stored at 4°C until use. The spore quality was checked by phase contrast microscopy and was found to contain >99% bright phase spores. The spore concentration of all strains was counted using a Neubauer counting chamber with a special depth (Petroff Marenfield) and adjusted to the order of 10^9^ CFU / ml.

[0078] C. difficile spore formation. C. difficile spore formation was performed based on the protocol of Edwards et al. 2016. Briefly, a stock concentration of C. difficile bacteria was streaked onto 0.1% taurocholate-BHI-S agar plates (BHI 37 gm / l, yeast extract 5 gm / l, agar 15 gm / l, L-cysteine 1 gm / l) and cultured overnight at 37°C in an anaerobic chamber. The next morning, a colony was picked from the overnight culture plate and inoculated into 5 ml of 0.1% taurocholate-BHI-S liquid medium. After 6 hours, 250 μl of the culture was plated onto ten 0.1% taurocholate-BHI-S agar plates. After 6 days, spores were harvested by rinsing the plates with 1X PBS. The cells were then pelleted by centrifugation (3900 rcf, 15 min) and resuspended in 1X PBS (5 ml). Spores were then harvested by isopycnic centrifugation through Percoll (90%) at 15,000 rcf for 30 min at 4°C. The bottom fractions were collected, resuspended in 2 ml of 1X PBS, and stored at 4°C until use. Spore quality was confirmed by phase contrast microscopy and contained >99% bright phase spores. The spore concentration of all strains was counted using a Neubauer counting chamber with a special depth (Petroff Marenfield) and adjusted to approximately 10 9 The order of CFU / ml.

[0079] Example 2

[0080] Preparation of Europium thenoyl trifluoroacetone and other lanthanide complexes.

[0081] Lanthanide TTA complex stock solutions were prepared by mixing equal volumes (200 μl) of stock solutions of the corresponding lanthanide chloride (3 mM) and TTA (9 mM) in 3% ethanol. The mixed solution was vortexed at maximum speed for 30 minutes to aid complexation. A Eu:TTA ratio of 1:3 was used. The claimed concentration range was found to be within the optimal concentration range that provided sufficient signal while minimizing the amount of organic solvent required to dissolve TTA for live cell imaging. For the other europium β-diketone complexes, all were prepared in 3% ethanol, except NTFA which was prepared in 50% ethanol.

[0082] For Eu:TTA stoichiometry determination, stock solutions of EuCl3 (300 mM) and TTA (9 mM) in 3% ethanol were prepared. EuCl3 was then diluted in 3% ethanol to obtain solutions with Eu:TTA molar ratios of 9:1, 3:1, 1:1, 1:3, and 1:9. These Eu solutions (200 μl) were mixed with TTA (9 mM) in equal volumes and vortexed for 30 minutes to form EuTTA complexes with different stoichiometric ratios. Figure 1A Shown (imaged using fluorescence microscopy), germinated C. novyi-NT spores were stained using complexes with Eu:TTA ratios ranging from 9:1 to 1:9 (fixed TTA molar concentration).

[0083] For the determination of the optimal concentration of Eu[TTA]3, stock solutions of EuCl3 (6mM, 3mM, 2mM, 1.5mM and 1mM) and TTA (18mM, 9mM, 6mM, 4.5mM and 3mM) in 3% ethanol were prepared respectively. Equal volumes (200μl) of these corresponding Eu and TTA solutions were mixed and vortexed for 30 minutes to form EuTTA complexes with different concentrations (stoichiometric 1:3Eu:TTA). The concentration was limited by the solubility of TTA in the solvent (3% ethanol in water). Figure 1B As shown (imaged using fluorescence microscopy), germinated C. novyi-NT spores were stained with Eu[TTA]3 at concentrations of 2.8 mM, 1.4 mM, 0.9 mM, 0.7 mM, and 0.5 mM.

[0084] The exposure time was set to 10 milliseconds (ms) for fluorescence mode and 100 ms for phase contrast mode. The display histogram range was adjusted for each experimental image to ensure better visibility. A Zeiss Observer7 microscope equipped with a DAPI long-pass filter was used at 1600× ( Figure 1A 、 Figure 1B) and 1000 times ( Figure 1C ) magnification. Scale bar is 2 μm.

[0085] Spores stained with Eu or TTA, respectively, did not emit light, indicating that both Eu and TTA are required for spore fluorescence. Figure 1C Shown are germinating C. novyi-NT spores stained with 1.4 mM Eu[TTA]3 (left), 1.4 mM EuCl3 (center), and 4.2 mM TTA (right). The top row represents widefield phase contrast images, and the bottom row represents fluorescence images.

[0086] Example 3

[0087] Eu[TTA]3 assay for dormant and germinated spores Clostridium novyi-NT spores were germinated using a mixture of L-cysteine (100 mM), hypoxanthine (0.1 mM), and oxygenase (1:50). Clostridium septicum spores were germinated in sodium glycocholate (9.3 mM) and oxygenase (1:50). Clostridium difficile spores were germinated in sodium taurocholate (8.5 mM), glycine (45 mM), and oxygenase (1:50). Bacillus subtilis spores were germinated in L-alanine (20 mM) after heat activation at 70°C for 1 hour, while Bacillus thuringiensis spores were first treated with D-cycloserine (9.7 mM) at 70°C for 30 minutes to inactivate the conversion of the germinant L-alanine to the non-germinant D-alanine and then germinated with L-alanine (93.5 mM).

[0088] All endpoint germination assays were performed at 37°C for 30 minutes (except for cwlD spores, which germinated for 15 hours), after which the spores were centrifuged at 3900 rcf for 5 minutes and resuspended in 1X PBS (Bacillus spores) or 1X PBS + oxygenase (Clostridium spores). 50 μl of Ln-BD complex was added to 3.5 μl of spores, and an aliquot (10 μl) of this sample was used for real-time imaging.

[0089] Microscopy was performed on an inverted widefield microscope (Zeiss Axiovert 200M equipped with a Photometrics Coolsnap HQ2 camera, using 1000x magnification for all bacteria except C. difficile; Zeiss Observer 7 equipped with a Hamamatsu ORCA flash 4.0 V2 camera, using 1600x magnification for C. difficile). Imaging was performed using a 100X Plan Apochromat Ph3 objective (NA 1.4, oil immersion lens) and a DAPI longpass filter (excitation filter -365 / 12BP; dichroic mirror 395; emission filter 397LP).

[0090] Figure 2AIt was shown that when the Ln-BD complex was Eu[TTA]3, it stained both dormant and germinated spores of all tested bacteria, with dormant spores exhibiting an annular fluorescence with a central hollow, while upon germination the central hollow was filled with bright luminescence. Figure 4 It was shown that when the Ln-BD complex was Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, or Tb[TTA]3, it also stained dormant C. novyi-NT spores and caused their cores to fluoresce after germination. Moreover, in all spores tested, germination resulted in brighter fluorescence within the core. Figure 5 This indicates that when the Ln-BD complex is Eu[BTFA]3, Eu[NTFA]3, Eu[AA]3, Eu[TriFAA]3, or Eu[TetraFAA]3, the aromatic BD used in the Ln-BD complex stains dormant and germinated C. novyi-NT spores more brightly than Ln-BD complexes using aliphatic BDs. These results demonstrate that various Ln[BD]n complexes can be used to stain a variety of bacterial spores. The results also suggest that aliphatic BDs are also useful for obtaining spore fluorescence.

[0091] A germination dye solution for live cell imaging of C. novyi-NT spores was prepared by mixing appropriate amounts of the germination agent L-cysteine (1 M, 53.5 μl), hypoxanthine (0.001 M, 53.5 μl), oxygenase (10 μl) to induce hypoxic conditions to promote germination, and Eu[TTA]3 (0.0015 M, 383 μl). To an aliquot (50 μl) of this germination solution, a solution of C. novyi-NT spores (5 x 10 9 CFU / ml, 3.5μl) to a final concentration of 100mM, 0.1mM, and 1.1mM for L-cysteine, hypoxanthine, and Eu[TTA]3, respectively. 10μl of this solution was dropped onto a Superfrost Plus microscope slide, covered with a glass coverslip (#1.5), and sealed on all four sides with nail polish. For Bacillus subtilis spores, a germination dye solution was prepared by mixing appropriate amounts of L-alanine (100mM, 107μl) and Eu[TTA]3 (0.0015M, 393μl). Heat-treated spores were adhered to a regular glass slide at 4°C for 1 hour, after which the germination dye solution (50μl) was added to the adhered spores, which were immediately sealed with a coverslip and imaged. The final concentrations of L-alanine and Eu[TTA]3 were 20mM and 1.1mM, respectively.

[0092] For individual live cell imaging, the cage incubator connected to the microscope was set to 37°C and germination was tracked at this temperature. Images were captured using Metamorph Series 7.7 (M7.7) software. Imaging parameters were as follows: Binning 1, exposure time -100ms (phase contrast mode and fluorescence images), unless otherwise stated. All images of different types of bacterial spores were acquired under the same incident light intensity settings. All images were cropped and processed using ImageJ Version 1.53n. All time-lapse images were first corrected for XY drift over time using the Stackreg plugin in ImageJ before cropping.

[0093] like Figure 3A For confocal images of Eu[TTA]3 alone, slides were imaged using a Leica SP8 super-resolution microscope equipped with a 100X HCX PL APO objective (NA1.4, oil immersion lens), a 405 nm diode laser, a detection wavelength of 580-700 nm, and a 0.5 AU pinhole. Images were acquired with Leica Application Suite X 1.8 (LASX v1.8) software and deconvolved using Lightning mode. The same parameters were used for all bacteria, except for Bacillus spores, where an optimization factor of 3 was used instead of 1.

[0094] All confocal images were cropped and processed using ImageJ Version 1.53n. Videos were generated using 3D maximum projections of Z-stack images acquired with a confocal microscope and processed using ImageJ.

[0095] like Figure 3A and Figure 11 As shown, optical plane section images through dormant spores stained with Eu[TTA]3 of all tested bacteria revealed a hollow region lacking fluorescent signal that became fluorescent upon germination.

[0096] Example 4

[0097] Raman imaging of Clostridium knowlesi-NT spore germination. In order to better illustrate the effects of the present invention, Raman microscopy was used as a tool to map the distribution of Eu. The germination dye solution for Raman live cell imaging of Clostridium knowlesi-NT spores was prepared by mixing appropriate amounts of germination agent L-cysteine (1M, 160.5μl), hypoxanthine (0.001M, 53.5μl), oxygenase (10μl) and Eu[TTA]3 (0.0015M, 276μl). Compared to other experiments, a higher concentration of germination agent was used to promote rapid germination to offset the possible photodamage to the spores caused by the laser over time. To an aliquot (50μl) of the germination solution was added a solution of Clostridium knowlesi-NT spores (5x10 9 CFU / ml, 3.5 μl) was added to a quartz microscope slide (Photonik, Singapore) to obtain a final concentration of 300 mM, 0.1 mM, and 0.77 mM of L-cysteine, hypoxanthine, and Eu[TTA] 3 or EuCl 3. 10 μl of this solution was added dropwise to a quartz microscope slide (Photonik, Singapore), covered with a quartz cover slip (#1.5), and all four edges were sealed with nail polish. Colony germination studies were performed by placing the slides on a heating stage (Linkam Controller DC95) set to 37°C, while X-mapping studies were performed at room temperature without a heating stage.

[0098] Simultaneous phase contrast, fluorescence and Raman imaging were performed using a Nikon Eclipse Ci microscope equipped with a PhotoFluor LM 75 fluorescence unit, a 532 nm laser and a uRaman spectrometer (Einst Technologies, Singapore). Phase contrast and fluorescence images were captured using a CoolSnap HQ2 monochrome camera. The objective lens used was a 100X Nikon Plan Ph3DL objective lens (NA1.25, oil immersion lens), and fluorescence imaging used a DAPI filter (excitation filter -350 / 5BP; emission filter -412LP). Images were captured using Metamorph software, and Raman spectra were acquired using uSoft software (Einst Technologies, Singapore). Raman spectra were processed using uRaman process software (Einst Technologies, Singapore), and baseline correction was performed for all samples using the same parameters using the Savitzky-Golay method. Only the fluorescence images were further deconvoluted using the CMLE method of Hyugens Professional v16.10 software.

[0099] Figure 2BThe distribution of Eu along the longest axis of C. knowlesi-NT spores treated with Eu[TTA]3 or uncomplexed Eu (in the form of EuCl3) is shown. C. knowlesi-NT spores were used in this study because of their larger size relative to the other bacteria used in this study, allowing for high-resolution mapping. Raman microscopy not only provided fluorescence-independent evidence for the localization of Eu in the spores, but also allowed for the simultaneous detection of the spore biomarker dipicolinic acid (DPA). Figure 2B As shown in the bottom row, DPA (1017 cm -1 ) is concentrated in the core during dormancy, while germination results in spore DPA concentrations no higher than ambient, indicating its release from the core. This DPA distribution pattern is true regardless of whether the spores are treated with Eu[TTA]3 or EuCl3.

[0100] In contrast to the DPA distribution characteristics, Eu[TTA]3 and EuCl3 produced completely different results in dormant and germinated spores, e.g. Figure 2B As shown in the top row, spores treated with Eu[TTA]3 exhibit a bimodal distribution, with a peak coinciding with the inner membrane region and a valley located within the core region. After germination, the Eu signal spatially redistributes to form a single peak within the core, confirming the bright core fluorescence observed after germination. Furthermore, there is no net increase in Eu within spores after germination, but the spore core fluorescence appears brighter to the human eye than that of dormant spores.

[0101] To investigate the relationship between DPA and Eu[TTA]3 fluorescence distribution in bacterial spores, Clostridium novosporium-NT spores were imaged in all three phases (bright, gray, and dark), with 20 spores analyzed in each phase. Each spore was first classified by phase contrast, then imaged under fluorescence, and subsequently subjected to Raman spectroscopy analysis as described above. The Raman laser was aimed at the center of the spore to generate a general Raman distribution map of the spore. The data for a typical spore is shown in Figure 2. Figure 7A As shown. The bright phase spores showed a characteristic DPA peak (Raman shift 1017cm -1 ) and annular fluorescence. In gray phase spores, the DPA peak disappears, indicating DPA release, while the annular pattern is still observed. Dark phase spores lack the DPA peak as expected, but now exhibit spore core fluorescence. These results indicate that DPA release precedes the appearance of Eu[TTA]3 fluorescence in the core. In addition, Figure 7A The Raman distribution of Eu levels showed no statistically significant shift between the various stages, indicating that the amount of Eu in the spores remained largely stable throughout the germination process.

[0102] Example 5

[0103] Co-staining assay. Germinated C. novyi-NT spores were incubated with Eu[TTA]3 (1.4 mM) and Nile Red (0.2 μg / ml) or DAPI (2.5 μM, 30 min) or FITC-dextran 3k-5k (0.45 mg / ml). Aliquots of these samples were used for imaging. Slides were imaged using a Leica SP8 super-resolution microscope equipped with a 100X HCX PL APO objective (NA1.4, oil immersion lens). Images were acquired using Leica Application Suite X 1.8 (LASX v1.8) software and deconvolution was performed using Lightning mode.

[0104] Figures 3B-3D This indicates that the Ln[BD]n complex can be used in combination with other dyes to examine other elements of spore morphology. Figure 3B As shown, co-staining with Nile Red labels the endospore and exospore membranes of the spore, which are lipid-rich regions of the spore. Nile Red staining of the inner membrane strongly overlaps with Eu[TTA]3 fluorescence, although Eu[TTA]3 fluorescence extends beyond the inner membrane borders, filling the spore core outlined by the Nile Red-stained inner membrane. Figure 3C As shown, co-staining with the DNA intercalator DAPI revealed a comma-shaped crystalline nuclear DNA. This comma structure is nested within the spore core defined by Eu[TTA]3 fluorescence. Figure 3D As shown, co-staining with FITC-dextran (molecular weight 3-5 kD) revealed that the spore exine membrane remained intact as a physical barrier and was therefore impermeable to FITC-dextran. In contrast, Eu[TTA]3, due to its smaller molecular size and lipophilic properties, was able to penetrate the exine membrane and stain the inner membrane as well as the core.

[0105] Example 6

[0106] Viability Assay. C. novyi-NT spores were germinated as previously described in Example 3, and B. subtilis was germinated in 93.5 mM L-alanine. Eu[TTA]3 (1.5 mM, 142.8 μl) was added to a 10 μl aliquot of germinated spores and incubated at room temperature for 15 minutes to a final concentration of 1.4 mM. Spores were then plated onto BHI (10%) FBS agar plates (C. novyi-NT) or onto LB agar plates under aerobic conditions (B. subtilis) in a Plas Labs anaerobic chamber. Colonies were counted the following day.

[0107] Figure 6A It was shown that treatment with Eu[TTA]3 did not result in any statistically significant loss of viability, with both C. novyi-NT and B. subtilis retaining their growth and colony-forming abilities.

[0108] Since Eu[TTA]3 does not affect the viability of spores, spore germination was followed in real time. Spores were placed on a glass slide and incubated at 37°C with Clostridium novyi-NT ( Figure 6B ) and Bacillus subtilis spores ( Figure 6C ) with an appropriate germination agent. Time-lapse phase contrast and fluorescence microscopy were then performed according to the conditions outlined in Example 3 to capture initial spore germination. Spores initially appeared in the bright phase and exhibited an annular fluorescence. This fluorescence pattern remained as the spores transitioned to the gray phase. Only upon entering the dark phase did the annular core become fluorescent. This process occurred within 10-20 minutes, resulting in the core fluorescing brighter than the surrounding annular core.

[0109] In the case of C. noursei-NT, the initial fluorescence of the core was observed as a pattern of punctate fluorescent spots that gradually grew larger and eventually covered the entire spore core, e.g. Figure 6B Bacillus subtilis follows the same sequence of events, with a hollow spore core that then rapidly fills with fluorescence as it enters a dark phase, as shown in Figure 6C shown.

[0110] Example 7

[0111] Cortex Degradation Studies. To investigate the role of the bacterial spore cortex layer in potentially promoting or hindering the penetration of lanthanide-β-diketones into the spore core, the following studies were performed. For C. novosporii-NT cortex removal, dormant spores were treated in decoating buffer (0.09 M NaOH / 0.9% SDS / 0.09 M DTT / 0.09 M NaCl) at 37°C for 1 hour. After washing the spores six times with sterile water to remove the decoating buffer, the spores were then incubated in cortex lysis buffer (1X Halt TM / 50mM Tris pH 7.4 / 2mg / ml lysozyme / 40μg / ml MgCl2) at 37℃ for 30 minutes. Spores were washed with water again and resuspended in Tris-Halt TM The cells were then placed in 1X PBS (MgCl₂ buffer, a cortex lysis buffer without the lysozyme component) for further analysis. An aliquot of spores was taken at each stage for imaging. Spores of the cwlD mutant Bacillus subtilis, lacking the muramic acid-δ-lactam necessary for cortex hydrolysis, were germinated and stained with Eu[TTA]₃ as described in Example 3.

[0112] Microscopy was performed on an inverted widefield microscope. For C. novosporii NT spores, imaging conditions were a Leica DMI6000 equipped with a Hamamatsu ORCA flash 4.0LT camera. Images were taken at 1000x magnification using a DAPI longpass filter. Exposure time was 50 ms in both modes, except for fluorescence images of dormant and decoated spores, which were 100 ms. For Bacillus subtilis spores, imaging conditions were a Zeiss Axiovert 200M equipped with a Photometrics Coolsnap HQ2 camera, 1000x magnification using a DAPI longpass filter, and 100 ms exposure time in both modes.

[0113] like Figure 7B As shown, Eu[TTA]3 failed to stain the core of germinated spores of cwlD mutant Bacillus subtilis with an intact cortex, indicating that the cortex restricts the entry of Eu[TTA]3. Figure 7C We showed that bright core fluorescence was observed when dormant C. novyi-NT spores were treated with the decoating and lysozyme steps as described above, but not with either step alone. These results suggest that Eu[TTA]3 can enter the dormant spore core after complete removal of all layers prior to the inner membrane and that cortical hydrolysis is required for Eu[TTA]3 entry into the spore core.

[0114] Example 8 (Comparative Example)

[0115] DAPI staining. Clostridium knowlesi-NT and Bacillus subtilis spores (dormant and germinated) were incubated with DAPI (5 μM), and aliquots of these samples were used for imaging, as shown in Figure 5. Figure 8 (Bottom, left column). Imaging was performed using an inverted Zeiss Axiovert 200M widefield microscope equipped with a 100X PlanApochromat Ph3 objective (NA1.4, oil immersion lens) and a DAPI longpass filter, and a Photometrics Coolsnap HQ2 monochrome camera. Images were captured using Metamorph Series 7.7 (M7.7) software. All image processing and cropping were performed using ImageJ.

[0116] Acridine orange staining. For C. novyi-NT spores, dormant or germinated spores (3.5 μl) were incubated with acridine orange solution (50 μl, 10 mg / ml) for 30 min (final concentration 9.3 mg / ml) and then imaged. Figure 8 (bottom, right column, row 1). For Bacillus subtilis 1A1 wild-type spores, heat-treated dormant and germinated spore slides were prepared as described in the previous section, followed by addition of acridine orange solution and immediate imaging, as shown in Figure 8 (Bottom, right column, row 2). Imaging was performed using an inverted Zeiss Axiovert 200M widefield microscope equipped with a 100X PlanApochromat Ph3 objective (NA1.4, oil immersion lens) and EGFP filters (excitation filter -470 / 40BP; dichroic mirror 495; emission filter 525 / 50BP), and a CoolsnapHQ2 monochrome camera. Images were captured using Metamorph Series 7.7 (M7.7) software. All image processing and cropping were performed using ImageJ.

[0117] Malachite Green Staining. Spores of Clostridium novyi-NT and Bacillus subtilis (dormant and germinated) were heat-fixed on a 50°C heating block for 5 minutes, followed by continuous addition of malachite green solution (5%) to the heat-fixed smears covered with filter paper in a 95°C water bath steam for 15 minutes. The slides were then rinsed with deionized water to remove excess malachite green stain and then counterstained with safranin for 5 minutes. Finally, the slides were rinsed with water again and blotted dry with a paper towel. Images were then taken using a Zeiss Axioplan2 upright microscope equipped with a 100X Plan ApochromatPh3 objective (1.4NA, oil immersion lens) and a Leica DFC 7000T color camera, as shown in Figure 5. Figure 8 (top). Images were captured using Leica Application Suite X 1.8 (LASX v1.8) software. Imaging parameters: exposure time - 50 ms. Images were processed using a minimum filter (2 pixel radius). All image processing and cropping were performed using ImageJ.

[0118] The Schaeffer-Fulton method uses malachite green and safranin counterstaining to distinguish dormant and germinating forms. Sample processing is long and destroys spore viability, thus precluding live imaging. DAPI stains dormant and germinating spores with high fluorescence but cannot distinguish between them. Finally, AO provides some differentiation between dormant and germinating spores but fails to clearly stain germinated spores. Both DAPI and AO require incubation times of at least 30 minutes to achieve adequate staining contrast. A comparison of the performance of various stains is available at Figure 12 .

[0119] Example 9

[0120] Dino-Lite imaging. Eu[TTA]3-stained bacterial samples for dormant and terminal germination spores were prepared as described in Example 3. Images were acquired at 55x magnification using a Dino-Lite Premier AM4113T digital microscope equipped with a DAPI excitation light source. Images were cropped and processed using ImageJ. Figure 9As shown, despite the low image resolution, spores stained with Eu[TTA]3 were observed to be significantly more fluorescent compared to the background.

[0121] Example 10

[0122] Milk matrix experiments. A 5% w / v skim milk solution was prepared by dissolving blot-grade milk powder in water. An equal volume of dormant C. novyi-NT spores was added to this solution to a final milk concentration of 2.5% (w / v) and a spore concentration of 2.5 x 10 9 CFU / ml. An aliquot (3.5 μl) of this sample was then stained with Eu[TTA]3 (1.4 mM). The samples were imaged at 1600x magnification using a Zeiss inverted Axio Observer 7 widefield microscope equipped with a DAPI longpass filter. Exposure times were 100 ms for phase contrast mode and 50 ms for fluorescence mode. Display histograms were adjusted for each acquisition mode to ensure maximum visibility.

[0123] Figure 10 This shows that spores can only be clearly observed under fluorescence in the presence of Eu[TTA]3, and the annular fluorescent shape of spores suspended in milk can be easily distinguished even in the presence of background fluorescence.

[0124] Although the subject matter of the present disclosure has been described and shown in considerable detail with reference to certain illustrative embodiments (including various combinations and subcombinations of features), other embodiments and variations and modifications thereof within the scope of the present disclosure will be readily apparent to those skilled in the art. Furthermore, the description of such embodiments, combinations, and subcombinations is not intended to convey that the claimed subject matter requires features or combinations other than those expressly recited in the claims. Accordingly, the scope of the present disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the appended claims.

[0125] Example 11 (complex mixture sample)

[0126] Spore growth staining: Dormant spores of Clostridium novyi-NT were incubated anaerobically (37°C, 7 hours) in a medium containing L-cysteine (100 mM), hypoxanthine (0.1 mM), BHI (0.62X), FBS (9.3% v / v), and oxygenase (1:50 v / v). Dormant spores of Bacillus subtilis were first heat-activated in 1X PBS (70°C, 1 hour) and then incubated aerobically in LB medium (37°C, 4 hours). The medium was then removed by centrifugation (3900 rcf, 15 minutes), followed by the addition of Eu[TTA]3 (1.4 mM). Aliquots of these samples were then used for imaging.

[0127] Spore-forming culture staining: Vegetative Bacillus cereus cells were cultured aerobically (37°C overnight to allow early sporulation) in 2xSG sporulation medium with shaking at 220 rpm (Infors HT Multitron Standard shaking incubator) and harvested the next day after 24 hours. Cells were then harvested by centrifugation (3900 rcf, 15 min). Cells were then stained with Eu[TTA]3 (1.4 mM) and imaged.

[0128] In the natural environment, it is expected that spores will mix with vegetative bacteria. Figure 13A ) or overnight cultures of sporulating vegetative bacteria ( Figure 13B ) were stained. In cultures of Clostridium novyi-NT or Bacillus subtilis, spores exhibited brighter fluorescence compared to their vegetative counterparts and were easily distinguished based on their morphology and size. A similar trend was observed in early sporulating cultures of Bacillus cereus, where the developing forespores of sporulating cells were labeled by a hollow region, highlighting the utility of Eu[TTA]3 for spore visualization, regardless of the presence of the vegetative form.

[0129] Milk matrix experiments: A 5% w / v skim milk solution was prepared by dissolving blot-grade milk powder in water. An equal volume of dormant Bacillus cereus spores was added to this solution to give a final milk concentration of 2.5% (w / v) and a spore concentration of 1.75 x 10 10 CFU / ml (Bacillus cereus). An aliquot (3.5 μl) of the sample was then stained with Eu[TTA]3 (1.4 mM) and imaged.

[0130] Finally, the ability of Eu[TTA]3 to address the problem of spore detection in food matrices was also investigated. The problem of contamination of dairy products by spore-forming bacteria, such as Bacillus cereus and Clostridium botulinum, is well documented, for example in [Christiansson, A.; Bertilsson, J.; Svensson, B. Bacillus Cereus Spores in Raw Milk: Factors Affecting the Contamination of Milk during the Grazing Period. J. Dairy Sci. 1999, 82(2), 305-314; Gupta, TB; Brightwell, G. Farm Level Survey of Spore-Forming Bacteria on Four Dairy Farms in the Waikato Region of New Zealand. Microbiology open 2017, 6(4); Barash, JR; Hsia, JK; Arnon, SS Presence of Soil-Dwelling Clostridia in Commercial Powdered Infant Formulas. J. Pediatr. 2010, 156(3), 402-408.]{.mark}. This contamination was simulated by inoculating milk with Bacillus cereus spores and then imaging with and without Eu[TTA]3 ( Figure 13C Despite the presence of moderate background fluorescence, visible fluorescent spores with hollow cores were readily observed only in the presence of Eu[TTA]3. These results demonstrate the potential of Eu[TTA]3 for detecting spores in complex food matrices.

[0131] Technical Solution Group

[0132] Notwithstanding the appended claims, the following technical solutions also constitute part of the instant disclosure and are also examples and representative species of the present invention.

[0133] 1. A method for live imaging of one or more bacterial spores in a sample, the method comprising:

[0134] contacting the sample with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer, to stain one or more bacterial spores in the sample; and

[0135] The fluorescent signal of the one or more stained bacterial spores in the sample is detected using a fluorescence microscope.

[0136] 2. A method for in situ distinguishing the germination state of one or more bacterial spores, the method comprising:

[0137] contacting a sample comprising one or more bacterial spores with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer, to stain the one or more bacterial spores in the sample;

[0138] Performing in vivo imaging of the sample using a fluorescence microscope to detect a fluorescent signal; and

[0139] Distinguish between dormant bacterial spores and germinating or germinated bacterial spores in a sample;

[0140] wherein dormant bacterial spores have a fluorescent signal characterized by a circular fluorescent outline with a hollow center, and

[0141] The germinating or germinated bacterial spores have a central core that is fluorescent.

[0142] 3. A method for real-time in vivo imaging of bacterial spore germination, the method comprising:

[0143] contacting the one or more bacterial spores with a germination probe solution comprising one or more germinants and a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer, to stain the one or more bacterial spore-forming mixtures in the sample;

[0144] placing the mixture on a glass slide;

[0145] visualizing the morphology of the one or more bacterial spores on the slide over time using time-lapse phase contrast microscopy; and

[0146] imaging the fluorescent signal of the one or more bacterial spores on the slide over time using time-lapse fluorescence microscopy,

[0147] The real-time live imaging of one or more bacterial spore germination comprises three phase contrast microscopy stages, including: 1) a bright phase of spore germination, 2) a gray phase of spore germination, associated with a most intense fluorescent signal localized in an outer region of the spore core, and 3) a dark phase of spore germination, associated with a most intense fluorescent signal localized in an area of the spore core, wherein the dark phase indicates bacterial spore germination.

[0148] 4. The method of any one or combination of technical solutions disclosed herein, wherein signal detection is performed using a confocal microscope,

[0149] The optical plane slice image through the dormant spore shows a hollow area corresponding to the spore core region without any fluorescent signal, and

[0150] An optical slice image through a germinating spore shows a fluorescent signal in the center of the spore corresponding to the spore core region.

[0151] 5. The method of any one or combination of the technical solutions disclosed herein, wherein the germination agent is one or more of L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine or D-cycloserine.

[0152] 6. The method according to any one or combination of the technical solutions disclosed herein, wherein Ln in the Ln[BD]n probe is europium, samarium, dysprosium or terbium.

[0153] 7. A method according to any one or combination of the technical solutions disclosed herein, wherein the BD in the Ln[BD]n probe is at least one member selected from the following groups: thenoyltrifluoroacetone (TTA), 1-(2-naphthoyl)-3,3,3-trifluoroacetone (NTFA), 4,4,4-trifluoro-1-phenyl-1,3-butanedione (BTFA), acetylacetone (AA), 1,1,1-trifluoroacetylacetone (TriFAA) and 1,1,5,5-tetrafluoroacetylacetone (TetraFAA).

[0154] 8. The method according to any one or combination of the technical solutions disclosed herein, wherein n in the Ln[BD]n probe is 1 to 5.

[0155] 9. The method according to any one or combination of the technical solutions disclosed herein, wherein n in the Ln[BD]n probe is 2 to 4.

[0156] 10. The method according to any one or combination of the technical solutions disclosed herein, wherein n in the Ln[BD]n probe is 3.

[0157] 11. A method as described in any one or combination of the technical solutions disclosed herein, wherein the probe is at least one member selected from the following groups: Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, Tb[TTA]3, Eu[BTFA]3, Eu[NTFA]3, Eu[AA]3, Eu[TriFAA]3 and Eu[TetraFAA]3.

[0158] 12. The method of any one or combination of the technical solutions disclosed herein, wherein the probe binds to spores from at least one of the genus Bacillus, Clostridium, or Clostridioides.

[0159] 13. A method as described in any one or combination of the technical solutions disclosed herein, wherein the probe binds to spores from at least one Bacillus subtilis, Bacillus thuringiensis, Bacillus cereus, Clostridium novyi-NT type A, Clostridium difficile, or Clostridium septicum.

[0160] 14. The method of any one or combination of the technical solutions disclosed herein, comprising performing signal detection using a USB-powered microscope, a digital microscope, a confocal microscope, a Raman microscope, or a bright-field microscope.

[0161] 15. The method of any one or combination of the technical solutions disclosed herein, wherein treatment with the probe does not significantly affect the viability, growth or colony-forming ability of the spores (compared to untreated spores).

[0162] 16. The method of any one or combination of technical solutions disclosed herein, wherein the change in colony forming units (CFU) of the bacterial spores before and after treatment is less than 0.5 log CFU.

[0163] 17. A method as described in any one or combination of technical solutions disclosed herein, wherein staining with the probe is instantaneous or near-instantaneous.

[0164] 18. The method of any one or combination of technical solutions disclosed herein, wherein the method further comprises contacting the sample with one or more additional probes or dyes and detecting one or more additional signals.

[0165] 19. The method according to any one or combination of the technical solutions disclosed herein, wherein the ratio of Ln to BD in the Ln[BD]n probe is in the range of 9:1 to 1:9.

[0166] 20. The method according to any one or combination of technical solutions disclosed herein, wherein the concentration of the probe is in the range of 0.7 mM to 10 mM.

[0167] 21. The method according to any one or combination of technical solutions disclosed herein, wherein the concentration of the probe is in the range of 1 mM to 5 mM.

[0168] 22. The method of any one or combination of technical solutions disclosed herein, wherein the concentration of the probe is in the range of 1.4 mM to 2.8 mM.

[0169] 23. The method of any one or combination of the technical solutions disclosed herein, wherein the concentration of the probe is about 1.4 mM or about 2.8 mM.

[0170] 24. The method of any one or combination of technical solutions disclosed herein, wherein the method does not include a spore permeabilization step or a heating step.

[0171] 25. The method according to any one or combination of the technical solutions disclosed herein, wherein the spore cortex is hydrolyzed before the probe enters the core.

[0172] 26. The method of any one or combination of the technical solutions disclosed herein, for use in food pathogen detection, environmental monitoring, quality assurance, or microbiological research. In some aspects, the food is milk.

[0173] 27. The method of any one or combination of the technical solutions disclosed herein, comprising using Raman microscopy to distinguish between dormant spores showing a bimodal distribution and germinated spores showing a unimodal distribution.

[0174] 28. The method of any one or combination of the technical solutions disclosed herein, comprising detecting a peak in the bimodal distribution comprising an inner membrane region and a valley between the peaks corresponding to a spore core region, and detecting a unimodal distribution comprising a spore core.

[0175] 29. The method of any one or combination of technical solutions disclosed herein, wherein the Raman distribution of the Ln level of the Ln[BD]n probe has no statistically significant shift throughout the germination process.

[0176] 30. The method of any one or combination of the technical solutions disclosed herein, comprising detecting the absence of dipicolinate (DPA) when a single peak corresponding to the Ln[BD]n probe in the spore core region is present.

[0177] 31. A method as described in any one or combination of technical solutions disclosed herein, wherein the probe is europium (III) thenoyltrifluoroacetone (Eu[TTA]3).

[0178] 32. A method as described in any one or combination of technical solutions disclosed herein, wherein the fluorescence microscope is equipped with a 4′,6-diamidino-2-phenylindole (DAPI) long-pass filter.

[0179] 33. The method of any one or combination of technical solutions disclosed herein, further comprising adding one or more surfactants to the sample.

[0180] 34. A method as described in any one or combination of technical solutions disclosed herein, wherein the one or more surfactants are 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polysorbate, sodium lauryl sulfate or a combination thereof.

[0181] 35. The method of any one or combination of the technical solutions disclosed herein, further comprising co-staining bacterial spores with Nile Red, DAPI, fluorescein isothiocyanate (FITC)-dextran, or a combination thereof.

[0182] 36. A method as described in any one or combination of the technical solutions disclosed herein, comprising monitoring spore bioburden in food or pharmaceutical samples, monitoring spore bioburden in the environment, or monitoring spore bioburden as a quality assurance step in an industrial process.

[0183] 37. A method as described in any one or combination of technical solutions disclosed herein, wherein the industrial process is the production of self-repairing concrete.

[0184] 38. The method of any one or combination of the technical solutions disclosed herein, wherein the sample comprises a plurality of bacterial spores, and the method further comprises quantifying the plurality of bacterial spores in the sample.

[0185] 39. The method of any one or combination of the technical solutions disclosed herein, comprising visualizing the stained bacterial spores immediately after the contacting step.

[0186] 40. A method as described in any one or combination of the technical solutions disclosed herein, wherein the imaging and / or monitoring includes generating a video of the sample to observe changes in germination over time.

[0187] 41. A method as described in any one or combination of technical solutions disclosed herein, wherein no fluorescent whitening agent is used.

[0188] 42. The method of any one or combination of technical solutions disclosed herein, wherein the method does not involve a permeabilization step.

[0189] 43. A kit comprising in a first container a germinative probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer, and in a second container a germinant.

[0190] 44. A kit according to any one or combination of the technical solutions disclosed herein, wherein the germination agent is L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine or D-cycloserine.

[0191] 45. A kit according to any one or combination of technical solutions disclosed herein, wherein the kit comprises a plurality of germinants, wherein each germinant is provided in a separate container.

[0192] 46. A composition comprising a germinative probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, n is an integer, and a germinant.

[0193] 47. A composition as described in any one or combination of technical solutions disclosed herein, wherein the germination agent is L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine or D-cycloserine.

[0194] 48. A live imaging apparatus for live imaging one or more bacterial spores in a sample that has been contacted with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain the one or more bacterial spores in the sample, comprising a live imaging capture device configured to detect staining of the one or more bacterial spores in the sample.

[0195] 49. A device for in situ distinguishing the germination status of one or more bacterial spores in a sample, wherein the sample comprises one or more bacterial spores stained with a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, wherein the device is configured to acquire a live image fluorescence signal of the sample using a fluorescence microscope and distinguish dormant bacterial spores from germinating or germinated bacterial spores in the sample,

[0196] Wherein dormant bacterial spores have a fluorescent signal characterized by a circular fluorescent outline with a hollow center, and wherein germinating or germinated bacterial spores have a central core that is filled with fluorescence.

[0197] 50. An apparatus for performing real-time, live imaging of germination of bacterial spores contacted with a germination probe solution, the germination probe solution comprising one or more germinants and a probe having the formula Ln[BD]n, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, to stain one or more bacterial spore-forming mixtures in a sample, wherein the apparatus comprises a slide configured to hold the mixture, wherein the apparatus comprises a time-lapse phase-contrast microscope configured to visualize the bacterial spores in the mixture and image fluorescent signals of the bacterial spores on the slide, wherein the real-time, live imaging of the germination of the one or more bacterial spores comprises three phase-contrast microscopy phases, including: 1) a bright phase of spore germination, 2) a gray phase of spore germination, which is associated with a most intense fluorescent signal localized to an outer region of the spore core, and 3) a dark phase of spore germination, which is associated with a most intense fluorescent signal localized to an area of the spore core, wherein the dark phase is indicative of bacterial spore germination.

[0198] 51. Use of the method as described in any one or combination of the technical solutions disclosed herein for monitoring spore bioburden in food or pharmaceutical samples, for monitoring spore bioburden in the environment, or for monitoring spore bioburden as a quality assurance step in an industrial process.

[0199] 52. Use of the method according to any one or combination of technical solutions disclosed herein as a quality assurance step in the production of self-repairing concrete.

[0200] 53. Use of the method according to any one or combination of the technical solutions disclosed herein for quantifying a plurality of bacterial spores in a sample.

[0201] 54. Use of the method according to any one or combination of the technical solutions disclosed herein for visualizing the stained bacterial spores immediately after the contacting step.

[0202] 55. Use of the method according to any one or combination of the technical solutions disclosed herein, for generating a video of the sample to observe germination changes over time.

[0203] 56. Use of the kit as described in any one or combination of technical solutions disclosed herein, for use in the method as described in any one or combination of technical solutions disclosed herein.

[0204] 57. Use of the composition as described in any one or combination of technical solutions disclosed herein, used in the method as described in any one or combination of technical solutions disclosed herein.

[0205] Although the subject matter of the present disclosure has been described and shown in considerable detail with reference to certain illustrative aspects (including various combinations and subcombinations of features), those skilled in the art will readily appreciate other aspects and variations and modifications thereof within the scope of the present disclosure. Furthermore, the description of such aspects, combinations, and subcombinations is not intended to convey that the claimed subject matter requires features or combinations other than those expressly recited in the claims. Accordingly, the scope of the present disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the appended claims. The section headings, materials, methods, and examples are illustrative only and not restrictive.

[0206] Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for live imaging of one or more bacterial spores in a sample, the method comprising: The sample is mixed with a mixture having the formula Ln[BD] n contacting a probe of to stain one or more bacterial spores in a sample, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer; and The fluorescent signal of the one or more stained bacterial spores in the sample is detected using a fluorescence microscope.

2. A method for in situ distinguishing the germination state of one or more bacterial spores, the method comprising: A sample containing one or more bacterial spores is mixed with a sample having the formula Ln[BD] n contacting a probe of to stain one or more bacterial spores in a sample, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer; Performing in vivo imaging of the sample using a fluorescence microscope to detect a fluorescent signal; and Distinguish between dormant bacterial spores and germinating or germinated bacterial spores in a sample; wherein dormant bacterial spores have a fluorescent signal characterized by a circular fluorescent outline with a hollow center, and Germinating or germinated bacterial spores have a central core that is fluorescent.

3. A method for real-time in vivo imaging of bacterial spore germination, the method comprising: contacting the one or more bacterial spores with a germination probe solution comprising one or more germination agents and a spore having the formula Ln[BD] to stain the one or more bacterial spores in the sample to form a mixture. n A probe wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer; placing the mixture on a glass slide; visualizing the morphology of the one or more bacterial spores on the slide over time using time-lapse phase contrast microscopy; and imaging the fluorescent signal of the one or more bacterial spores on the slide over time using time-lapse fluorescence microscopy, The real-time live imaging of one or more bacterial spore germination comprises three phase contrast microscopy stages, including: 1) a bright phase of spore germination, 2) a gray phase of spore germination, associated with a most intense fluorescent signal localized in an outer region of the spore core, and 3) a dark phase of spore germination, associated with a most intense fluorescent signal localized in an area of the spore core, wherein the dark phase indicates bacterial spore germination.

4. The method according to claim 2, wherein signal detection is performed using a confocal microscope, The optical plane slice image through the dormant spore shows a hollow area corresponding to the spore core region without any fluorescent signal, and An optical slice image through a germinating spore shows a fluorescent signal in the center of the spore corresponding to the spore core region.

5. The method according to claim 3, wherein the germination agent is one or more of L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine or D-cycloserine.

6. The method according to any one of claims 1 to 3, wherein the Ln[BD] n The Ln in the probe is europium, samarium, dysprosium or terbium.

7. The method according to any one of claims 1 to 3, wherein the Ln[BD] n The BD in the probe is at least one selected from the following group: thenoyltrifluoroacetone (TTA), 1-(2-naphthoyl)-3,3,3-trifluoroacetone (NTFA), 4,4,4-trifluoro-1-phenyl-1,3-butanedione (BTFA), acetylacetone (AA), 1,1,1-trifluoroacetylacetone (TriFAA) and 1,1,5,5-tetrafluoroacetylacetone (TetraFAA).

8. The method according to any one of claims 1 to 3, wherein the Ln[BD] n n in the probe is 1 to 5.

9. The method according to any one of claims 1 to 3, wherein the Ln[BD] n n is 2 to 4 in the probe.

10. The method according to any one of claims 1 to 3, wherein the Ln[BD] n The n in the probe is 3.

11. The method according to any one of claims 1 to 3, wherein the probe is at least one selected from the following group: Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, Tb[TTA]3, Eu[BTFA]3, Eu[NTFA]3, Eu[AA]3, Eu[TriFAA]3 and Eu[TetraFAA]3.

12. The method of any one of claims 1-3, wherein the probe binds to spores from at least one of the genus Bacillus, Clostridium, or Clostridioides.

13. The method of claim 12, wherein the probe binds to spores from at least one of B. subtilis, B. thuringiensis, B. cereus, C. novyi-NT type A, C. difficile, or C. septicum.

14. The method of any one of claims 1-2, comprising performing signal detection using a USB-powered microscope, a digital microscope, a confocal microscope, a Raman microscope, or a bright-field microscope.

15. The method of any one of claims 1-3, wherein treatment with the probe does not significantly affect the viability, growth, or colony-forming ability of the spores compared to untreated spores.

16. The method according to any one of claims 1 to 3, wherein the change in colony forming units (CFU) of bacterial spores before and after treatment with the probe is less than 0.5 log CFU.

17. The method of any one of claims 1-3, wherein staining with the probe is instantaneous or near-instantaneous.

18. The method of any one of claims 1-3, wherein the method further comprises contacting the sample with one or more additional probes or dyes and detecting one or more additional signals.

19. The method according to any one of claims 1 to 3, wherein the Ln[BD] n The ratio of Ln to BD in the probe ranged from 9:1 to 1:

9.

20. The method of any one of claims 1-3, wherein the concentration of the probe is in the range of 0.7 mM to 10 mM.

21. The method of any one of claims 1-3, wherein the concentration of the probe is in the range of 1 mM to 5 mM.

22. The method of any one of claims 1-3, wherein the concentration of the probe is in the range of 1.4 mM to 2.8 mM.

23. The method of any one of claims 1-3, wherein the probe is at a concentration of about 1.4 mM or about 2.8 mM.

24. The method of any one of claims 1-3, wherein the method does not comprise a spore permeabilization step or a heating step.

25. The method of any one of claims 1-3, wherein the spore cortex is hydrolyzed prior to the probe entering the core.

26. The method of any one of claims 1-3, for use in food pathogen detection, environmental monitoring, quality assurance, or microbiological research.

27. The method of claim 2, comprising using Raman microscopy to distinguish between dormant spores that exhibit a bimodal distribution and germinated spores that exhibit a unimodal distribution.

28. The method of claim 27, comprising detecting a peak in the bimodal distribution comprising an inner membrane region and a valley between the peaks corresponding to a spore core region, and detecting a unimodal distribution comprising a spore core.

29. The method of claim 27, wherein during the entire germination process, Ln[BD] n There was no statistically significant shift in the Raman distribution of the probe's Ln level.

30. The method of claim 27, comprising detecting the presence of Ln[BD] in the region corresponding to the spore core. n When a single peak of the probe is present, dipicolinic acid (DPA) is not present.

31. The method of any one of claims 1-30, wherein the probe is europium(III) thenoyltrifluoroacetone (Eu[TTA]3).

32. The method of any one of claims 1-31, wherein the fluorescence microscope is equipped with a 4',6-diamidino-2-phenylindole (DAPI) long pass filter.

33. The method of any one of claims 1-32, further comprising adding one or more surfactants to the sample.

34. The method of claim 33, wherein the one or more surfactants is 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polysorbate, sodium lauryl sulfate, or a combination thereof.

35. The method of any one of claims 1-34, further comprising co-staining the bacterial spores with Nile red, DAPI, fluorescein isothiocyanate (FITC)-dextran, or a combination thereof.

36. The method of any one of claims 1 to 35, comprising monitoring spore bioburden in a food or pharmaceutical sample, monitoring spore bioburden in the environment, or monitoring spore bioburden as a quality assurance step in an industrial process.

37. The method of claim 36, wherein the industrial process is the production of self-repairing concrete.

38. The method of any one of claims 1-37, wherein the sample comprises a plurality of bacterial spores, and the method further comprises quantifying the plurality of bacterial spores in the sample.

39. The method of any one of claims 1-38, comprising visualizing the stained bacterial spores immediately after the contacting step.

40. The method of any one of claims 1-39, wherein the imaging and / or monitoring comprises generating a video of the sample to observe changes in germination over time.

41. The method of any one of claims 1 to 39, wherein no fluorescent whitening agent is used.

42. The method of any one of claims 1-39, wherein the method does not involve a permeabilization step.

43. A kit comprising in a first container a compound having the formula Ln[BD] n A germination probe wherein Ln is a member of the lanthanide series, BD is a beta-diketone, n is an integer, and a germinant in a second container.

44. The kit of claim 43, wherein the germination agent is L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine.

45. The kit of claim 43 or 44, wherein the kit comprises a plurality of germinants, wherein each germinant is provided in a separate container.

46. A composition comprising a compound having the formula Ln[BD] n A germination probe and a germination agent, wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer.

47. The composition of claim 46, wherein the germination agent is L-cysteine, hypoxanthine, oxygenase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine.

48. A live imaging apparatus for live imaging of one or more bacterial spores in a sample, the sample having been treated with a microorganism having the formula Ln[BD] n to stain one or more bacterial spores in a sample, wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer, the apparatus comprising a live image capture device configured to detect staining of the one or more bacterial spores in the sample.

49. A device for in situ distinguishing the germination state of one or more bacterial spores in a sample, wherein the sample comprises one or more bacterial spores having the formula Ln[BD] n Bacterial spores stained with a probe comprising: ... Wherein dormant bacterial spores have a fluorescent signal characterized by a circular fluorescent outline with a hollow center, and wherein germinating or germinated bacterial spores have a central core that is filled with fluorescence.

50. An apparatus for real-time in vivo imaging of germination of bacterial spores that have been contacted with a germination probe solution to stain one or more bacterial spores in a sample to form a mixture, the germination probe solution comprising one or more germinants and a molecule having the formula Ln[BD] n wherein Ln is a member of the lanthanide series, BD is a beta-diketone, and n is an integer, wherein the apparatus comprises a slide configured to hold the mixture, wherein the apparatus comprises a time-lapse phase-contrast microscope configured to visualize bacterial spores in the mixture and to image fluorescent signals of the bacterial spores on the slide, wherein the real-time live imaging of the germination of one or more bacterial spores comprises three phase-contrast microscope stages, including: 1) the light phase of spore germination, 2) the gray phase of spore germination, which is associated with the most intense fluorescence signal localized in the outer region of the spore core, and 3) the dark phase of spore germination, which is associated with the most intense fluorescence signal localized in the spore core region, wherein the dark phase is indicative of bacterial spore germination.

51. Use of the method according to any one of claims 1 to 42 for monitoring spore bioburden in food or pharmaceutical samples, for monitoring spore bioburden in the environment, or as a quality assurance step in an industrial process.

52. Use of the method according to any one of claims 1 to 42 as a quality assurance step in the production of self-healing concrete.

53. Use of the method according to any one of claims 1 to 42 for quantifying a plurality of bacterial spores in a sample.

54. Use of the method according to any one of claims 1 to 42 for visualizing the stained bacterial spores immediately after the contacting step.

55. Use of the method of any one of claims 1-42 to generate a video of the sample to observe changes in germination over time.

56. Use of a kit according to any one of claims 43-45, for use in a method according to any one of claims 1-42.

57. Use of a composition according to any one of claims 46-47, for use in a method according to any one of claims 1-42.

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