A method for identifying, imaging, and characterizing bacterial spores using lanthanide-β-diketones

Abstract

The sample was treated with the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain 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.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority from Singapore Patent Application No. 10202260603W, filed December 27, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to the field of imaging and detection. In particular, non-toxic lanthanide-beta-dikenate probes and related methods for imaging and characterizing bacterial spores in situ are disclosed. [Background technology]

[0003] Bacteria belonging to the genera Bacillus and Clostridium are ubiquitous in nature and possess the ability to initiate endospore formation upon sensing unfavorable environmental conditions. Such spores can persist for many years, with the longest known survival estimated at 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 infectious diseases, particularly foodborne illnesses (e.g., Bacillus cereus, Clostridium perfringens, C. botulinum) and hospital-acquired infections (e.g., Clostridiodes difficile). It is hypothesized that certain bacterial spores, without further manipulation, could be used as biological weapons or could find their way into space, thereby jeopardizing life-detection experiments and unnoticed altering extraterrestrial ecosystems.

[0004] Aside from posing a threat as an infectious agent, spore detection is a concern for space exploration, as contamination of alien planets with life of Earth origin could jeopardize life-detection experiments and unnoticedly alter terrestrial ecosystems. Similar spore contamination concerns exist in the food, pharmaceutical manufacturing, and medical device industries, where sterility is paramount. In all of these research fields, there is a long-felt and unmet need for rapid detection and triage of suspicious spore material.

[0005] Spore detection methods can be classified as direct or indirect. The earliest attempts at bacterial spore detection relied primarily on ultraviolet (UV) absorbance to reveal the presence of dipicolinic acid (DPA), a ubiquitous constituent of bacterial spores. In contrast, more recent methods have shifted toward detecting DPA using fluorescence spectroscopy. The current state of the art involves the coupling of DPA released from spores with terbium(III) cations (Tb 3+ This signal, which is diffusible and not localized to the spore, can be visualized by immobilizing the spores on a diffusion-retarding substrate such as agarose and by detecting the fluorescent complex formed between the DPA / Tb 3+ A fluorescence microscope capable of accommodating the low excitation wavelengths (approximately 270 nm) of the complex is required. Due to these limitations, DPA detection is most practical as an indirect indicator of germinating spores at the population level.

[0006] The detection limit for such indirect detection is 10 5 CFU / ml, but when dodecylamine and heating at 60°C are used to maximize DPA release, this is 10 3 Only viable spores with intact DPA reservoirs contribute to the fluorescent signal, so pre-germinated spores that have released DPA are not detected. 3+Fluorescence does not reveal whether negative samples are devoid of spores or filled with germinated spores. This may lead to an underestimation of the pathogenic threat, especially in the second case, since germinated spores can easily transform into infectious vegetative forms. Colorimetric reagents for DPA detection have also been developed, such as erbium-pyrocatechol violet and betanin, but these have poor detection limits (approximately 10 6 CFU / ml) and are subject to similar constraints.

[0007] Bacterial endospores have a rigid morphology that prevents many dyes from penetrating the spore structure. For this reason, previous techniques required permeabilizing the spore structure to facilitate staining. For example, the Schaeffer-Fulton assay requires permeabilization by heat fixation and steam treatment to allow malachite green to penetrate the spore. Among fluorescent stains, some stain only dormant forms (ThT) or germinated forms (SYTO-16). The lack of staining for either dormant or germinated forms makes visualization and counting of unstained spores difficult or impossible. The remaining fluorescent agents stain both dormant and germinated forms equally and visually indistinguishably (DAPI, AO), making it impossible to distinguish between dormant and germinated spores. Finally, a problem specific to malachite green and ThT is that their associated protocols are inherently toxic to germinating spores, making it impossible to use these methods for live imaging of germination events.

[0008] 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 the real-time detection of bacterial spores and their germination state. There is also a need for live imaging of bacterial spores and their germination state. Summary of the Invention

[0009] The present disclosure provides compositions, methods, and kits for fluorescently labeling bacterial spores in situ and instantly without further treatment using lanthanide-β-diketones. Given their low toxicity to spores and their ability to distinguish between dormant and germinating forms, this method also enables live imaging of single spores during germination.

[0010] In one aspect, the present invention provides a method for live imaging of one or more bacterial spores in a sample, comprising: n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer; and detecting a fluorescent signal from the one or more stained bacterial spores in the sample using a fluorescent microscope.

[0011] Another aspect of the present invention is a method for distinguishing the germination state of one or more bacterial spores in situ, comprising: subjecting a sample containing one or more bacterial spores to a spore mixture of a spore mixture of a compound of the formula Ln[BD] n The method includes contacting one or more bacterial spores in a sample with a probe having the formula: (wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer) to stain the bacterial spores; live-imaging the fluorescent signal of the sample using a fluorescent microscope; and distinguishing between dormant bacterial spores and germinating or germinated bacterial spores in the sample. In some embodiments, dormant bacterial spores have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a central cavity, and germinating or germinated bacterial spores have a central core filled with fluorescence, where Ln is a member of the lanthanide series, and BD is a β-diketone.

[0012] In another embodiment, the present invention provides a method for real-time live imaging of bacterial spore germination, comprising: injecting one or more bacterial spores into one or more germinants and a compound of formula Ln[BD] nwherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample to form a mixture; depositing 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 fluorescent signals from the one or more bacterial spores on the slide over time using time-lapse fluorescence microscopy.

[0013] The method for live imaging 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 comprise using a USB-powered microscope, a digital microscope, a confocal microscope, a Raman microscope or a bright-field microscope to perform signal detection.Therefore, the advantageous features of the method of the present invention include: (i) it is instantaneous; (ii) it does not involve special processing; (iii) it does not destroy spores and allows live imaging of germinating spores; (iv) it stains spores and distinguishes between dormant spores and germinated spores; and (v) it can also use a low-cost USB-powered microscope (although it cannot visualize the details of spores), and it can provide at least preliminary on-site results of the sample using low-cost visualization.

[0014] In some embodiments, real-time imaging of one or more bacterial spore germination includes three phase contrast microscopy phases, including 1) a phase bright phase, and 2) a phase gray phase of spore germination that correlates with the strongest fluorescent signal localized to the region outside the spore's nucleus, and 3) a phase dark phase of spore germination that correlates with the strongest fluorescent signal localized to the nucleus region of the spore, wherein the dark phase indicates bacterial spore germination.

[0015] In some embodiments, any one of the above methods can be used to monitor spore bioburden in food or pharmaceutical samples, in food pathogen detection, environmental monitoring, quality assurance, or microbiology research, to monitor spore bioburden in the environment, or to monitor spore bioburden as a quality assurance step in an industrial process. In some embodiments, the industrial process is in the production of self-healing concrete.

[0016] 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 creating a video of the sample to observe changes in germination over time.

[0017] The present invention also provides a method for producing a compound of formula Ln[BD] in a first container. n The kit includes a germinant in a second container, and a germinant having the formula: (wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer). In some embodiments, the germinant (or germination-supporting compound) is L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some embodiments, the kit includes multiple germinants, each provided in a separate container.

[0018] The present invention also provides compounds of formula Ln[BD] nwherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, and a germinant, wherein the germinant (or germination-supporting compound) is L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some embodiments, L-valine or D-valine may be used as a germinant for C. novyi-NT species.

[0019] Other features and characteristics of the subject matter of the present disclosure, as well as the method of operation, function of the combination of associated elements and parts of construction, and economy of manufacture, will become more apparent from consideration of the following description and appended claims, all of which form a part hereof.

[0020] In one aspect, the present invention provides an apparatus for live imaging of one or more bacterial spores in a sample, wherein the one or more bacterial spores are labeled with a compound of the formula Ln[BD] n In some embodiments, for staining one or more bacterial spores in a sample, Ln can be a member of the lanthanide series, BD is a β-diketone, and n is an integer. In some embodiments, the means for live imaging is a camera, a microscope, an image capture device, a video capture device, or a combination thereof. A camera, a microscope, an image capture device, a video capture device, or a combination thereof is understood by those skilled in the art as having a structure for performing imaging.

[0021] In some embodiments, the device may include a means for in situ distinguishing the germination state of one or more bacterial spores in a sample. In some embodiments, the sample has a spore of the formula Ln[BD] nIn some embodiments, Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer. In some embodiments, the device can be configured to acquire a live imaging fluorescent signal of the sample and distinguish between dormant bacterial spores and germinating or germinated bacterial spores in the sample.

[0022] In some embodiments, dormant bacterial spores may have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a central cavity. In certain embodiments, germinating or germinated bacterial spores have a central core filled with fluorescence.

[0023] In some embodiments, the device comprises one or more germinants and a compound of formula Ln[BD] n The method may include means for real-time live imaging of bacterial spore germination by contacting the spore with a germinant probe solution comprising a probe having the formula:

[0024] In some embodiments, Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer for staining one or more bacterial spores in a sample to form a mixture, hi some embodiments, the device can include a slide configured to hold the mixture.

[0025] In some embodiments, the device can include a time-lapse phase microscope configured to visualize bacterial spores in the mixture and image the fluorescent signal from the bacterial spores on the slide. In some embodiments, the real-time imaging of one or more bacterial spore germination includes three phase-contrast microscope phases, including: 1) a bright phase; 2) a gray phase of spore germination, which correlates with the strongest fluorescent signal localized in the region outside the spore nucleus; and 3) a dark phase of spore germination, which correlates with the strongest fluorescent signal localized in the nucleus region of the spore, and the dark phase indicates bacterial spore germination. [Brief explanation of the drawings]

[0026] [Figure 1]Figure 1A shows fluorescence images of germinated C. novii-NT spores stained with various ratios of Eu and TTA. Figure 1B shows fluorescence images of germinated C. novii-NT spores stained with various concentrations of Eu[TTA]3. Figure 1C shows germinated C. novii-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 shows wide-field phase-contrast images, and the bottom row shows fluorescence images. [Figure 2] Figure 2A shows dormant (left) and germinated (right) spores of Clostridium novi-NT, Clostridium septicum, Bacillus subtilis, Clostridioides difficile, and Bacillus thuringiensis stained with 1.4 mM Eu[TTA]3. The top image shows a wide-field phase-contrast image, and the bottom image shows a fluorescence image. Figure 2B shows DPA (1017 cm-1) and europium (1531 cm-1) Raman peak mapping across the longest spore axis for dormant and germinated C. novi-NT spores. Average Eu Raman peak intensity profiles (top) of spores stained with Eu[TTA]3 (left) and EuCl3 (right), and average DPA Raman peak intensity profiles (bottom) of spores stained with Eu[TTA]3 (left) and EuCl3 (right), respectively (N=6 for each sample). [Figure 3] Figure 3A shows pseudocolor confocal images of dormant spores (left) and germinated spores (right) stained with Eu[TTA]3 (1.4 mM): C. novi-NT (row 1), C. septicum (row 2), C. difficile (row 3), B. subtilis (row 4), and B. thuringiensis (row 5). Figures 3B-3D show pseudocolor confocal images of 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. [Figure 4]Figure 4 shows dormant and germinated C. novii NT spores treated with 1.4 mM of each lanthanide-TTA complex. Images represent wide-field phase-contrast (rows 1 and 3) and fluorescence (rows 2 and 4) images of dormant (top) and germinated (bottom) C. novii NT spores stained with 1.4 mM Eu[TTA] (column 1), Sm[TTA] (column 2), Dy[TTA] (column 3), and Tb[TTA] (column 4). [Figure 5] Figure 5 shows dormant and germinated C. novii-NT spores stained with 1.4 mM different Eu β-diketone complexes. Images represent wide-field phase-contrast (rows 1 and 3) and fluorescence (rows 2 and 4) images of dormant (top) and germinated (bottom) C. novii-NT spores stained with 1.4 mM Eu[BTFA]3 (column 1), Eu[NTFA]3 (column 2), Eu[AA]3 (column 3), Eu[triFAA]3 (column 4), and Eu[tetraFAA]3 (column 5). [Figure 6] Figure 6A shows ungerminated C. novi-NT and B. 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. Figures 6B and 6C show time-lapse images of Eu[TTA]3-stained C. novi-NT spores germinating with L-cysteine, hypoxanthine, and oxylase, and Eu[TTA]3-stained B. subtilis P1A1 spores germinating with L-alanine, respectively. Each time panel consists of corresponding phase-contrast (top) and fluorescence (bottom) images. The inset below shows a magnified fluorescence image of a germinating spore along with a 3D surface plot projection of the fluorescence of a single germinating spore using the Interactive 3D Surface plot plugin in ImageJ. [Figure 7]Figure 7A shows representative phase-contrast and fluorescence images of single light-, gray-, and dark-phase spores, along with their Raman spectra. Average Raman spectra of spores at three different germination stages (N = 20 for each stage). Average Raman intensity peak profiles of the DPA (1017 cm-1) and Eu (1531 cm-1) peaks for light-, gray-, and dark-phase spores (N = 20 for each stage). Figure 7B shows Eu[TTA]3 (1.4 mM) added to wild-type P1A1 and cwlD mutant spores before germination. The left panel represents wild-type spores, and the right panel represents cwlD mutant spores. The upper panel represents a phase-contrast image, and the lower panel represents a fluorescence image. Figure 7C shows dormant C. novii-NT spores treated with decoating buffer, followed by cortex lysis buffer containing lysozyme, and stained with Eu[TTA]3 (1.4 mM). Column 1 shows untreated resting spores stained with Eu[TTA]3, column 2 shows uncoated spores stained with Eu[TTA]3, column 3 shows lysozyme-treated resting spores, and column 4 shows lysozyme-treated uncoated spores. The top panel shows phase-contrast images, and the bottom panel shows fluorescence images. [Figure 8] Figure 8. (Top) RGB images of dormant and germinated Clostridium novi-NT and B. subtilis spores stained with malachite green for 15 min and counterstained with safranin. (Bottom) Phase contrast (columns 1, 3) and fluorescence (columns 2, 4) images of dormant (left) and germinated (right) Clostridium novi-NT and Bacillus subtilis spores stained with DAPI (5 μM) and acridine orange (9.3 mg / ml) for 30 min, respectively. [Figure 9] Figure 9 shows fluorescence images of resting (left) and germinated (right) spores of Clostridium novi-NT, Clostridium septicum, Clostridioides difficile, Bacillus subtilis, and Bacillus thuringiensis stained with 1.4 mM Eu[TTA]3. [Figure 10]Figure 10 shows a 2.5% (w / v) milk sample spiked with dormant C. novii-NT spores, an aliquot of which was stained with Eu[TTA]3 (1.4 mM). The images show phase-contrast (top) and fluorescence (bottom) images of C. novii-NT spores in the milk matrix stained with (left) and without (right) Eu[TTA]3. [Figure 11] Figure 11 shows 3D maximum intensity projection images of Z-stack confocal images of dormant (left) and germinated (right) C. novi-NT (top) and C. septicum (bottom) spores from Figure 3 . [Figure 12] Figure 12 shows a tabular comparison of the performance of various spore stains. [Figure 13] Figure 13A shows Z-average intensity projection images of Z-stack confocal images of germinating cultures of C. novi-NT (left) and B. subtilis (right) in BHI-FBS oxylase medium and LB medium, respectively, both stained with Eu[TTA]3 (green). Figure 13B shows a single Z-plane confocal image of an early sporulating overnight culture of B. cereus in 2xSG sporulation medium stained with Eu[TTA]3 (green). Scale bar: 2 μm. Figure 13C shows phase contrast (top) and fluorescence (bottom) images of B. cereus spores in a 2.5% (v / v) milk matrix stained with (left) and without (right) Eu[TTA]3. Scale bar: 2 μm. DETAILED DESCRIPTION OF THE INVENTION

[0027] While aspects of the presently disclosed subject matter may be embodied in a variety of forms, the following description is intended to disclose some of these forms merely as illustrative examples of the subject matter encompassed by the present disclosure, and therefore, the presently disclosed subject matter is not intended to be limited to the forms or embodiments so described.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein, but other suitable methods and materials known in the art can also be used.Materials, methods and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0029] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0030] Concentrations, amounts, and other numerical data may be expressed or presented in range format herein. It should be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​expressly recited as range limits, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. By way of example, a numerical range of "about 0.01 to 2.0" should be interpreted not only to include the explicitly recited value of about 0.01 to about 2.0, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 0.5, 0.7, and 1.5, as well as subranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5. Furthermore, such interpretation should apply regardless of the breadth or character of the range described. Furthermore, it should be noted that, unless otherwise specified, all percentages are by weight.

[0031] In understanding the scope of the present disclosure, the terms "including" or "comprising" and their derivatives, as used herein, are intended to be open-ended terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other, unrecited features, elements, components, groups, integers, and / or steps. The same also applies to words of similar meaning, such as the terms "including," "having," and their derivatives. The term "consisting of" and its derivatives, as used herein, is intended to be closed terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but excluding the presence of other, unrecited features, elements, components, groups, integers, and / or steps. The term "consisting essentially of," as used herein, is intended to specify the presence of stated features, elements, components, groups, integers, and / or steps, as well as those that do not materially affect the basic and novel characteristics of the features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transitional terms (i.e., "comprising," "consisting of," or "consisting essentially of") provides direct support for substitution of any other transitional term not specifically used. For example, amending the term "comprising" to "consisting essentially of" or "consisting of" finds direct support due to this definition for any element disclosed throughout this disclosure. Based on this definition, any element disclosed or incorporated by reference herein may be included in or excluded from the claimed invention.

[0032] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Accordingly, the individual members of such lists should not be construed as de facto equivalents of any other members of the same list solely based on their presentation in a common group, unless indicated to the contrary.

[0033] The present disclosure includes detecting any bacterial spore and is not limited by the exemplary bacterial spore types used in the experiments, which serve as proof of concept to support the use of the methods of the present invention for detecting bacterial spores generally. Non-limiting examples of such bacterial spores include, for example, B. subtilis (a well-characterized model for bacterial spore biology); B. thuringiensis (which expresses pore-forming toxins for insecticidal use and is a close relative of B. anthracis, sometimes serving as a surrogate in research studies); B. cereus (a common foodborne pathogen that can release toxins that can cause food poisoning and related illnesses); C. novyi-NT (a gas gangrene bacterium derived from Clostridium novyi type A by removing a unique bacteriophage, making it genetically equivalent to Clostridium botulinum type C); C. difficile (a nosocomial agent transmitted in hospital settings); and C. septicum (a pathogen of livestock that is also known to be associated with infections in patients with existing (and often occult) hematologic and gastrointestinal malignancies). These proof-of-concept bacterial spore types were selected to confirm that the invention is effective across a diverse cross-section of endospore-forming bacteria.

[0034] The bacterial spore germination process can be essentially divided into three successive phases called phase bright, phase gray, and phase dark. These terms describe the brightness of the spores observed under a phase-contrast microscope. Phase darkening and the release of the spore biomarker DPA are both hallmarks of bacterial spore germination.

[0035] Furthermore, bacterial spores contain various permeable sieve-like layers that selectively restrict the access of small and large molecules. Among these sieves, the cortex layer is known to be the final physical barrier to the bacterial spore inner membrane. During the normal process of germination, this cortex barrier is hydrolyzed by enzymes.

[0036] The present disclosure provides compositions, methods, and kits for fluorescently labeling bacterial spores in situ and instantly without further treatment using lanthanide-β-diketonates. Given their low toxicity to spores and their ability to distinguish between dormant and germinating forms, this method also allows for live imaging of single spores during germination. Thus, in some embodiments, the present invention provides methods that do not require incubation time.

[0037] In one aspect, the present invention provides a method for live imaging of one or more bacterial spores in a sample, comprising: n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer; and detecting a fluorescent signal from the one or more stained bacterial spores in the sample using a fluorescent microscope.

[0038] Another aspect of the present invention is a method for distinguishing the germination state of one or more bacterial spores in situ, comprising: subjecting a sample containing one or more bacterial spores to a spore mixture of a spore mixture of a compound of the formula Ln[BD] nThe method includes contacting one or more bacterial spores in a sample with a probe having the formula: (wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer) to stain the bacterial spores; live-imaging the fluorescent signal of the sample using a fluorescent microscope; and distinguishing between dormant bacterial spores and germinating or germinated bacterial spores in the sample. In some embodiments, dormant bacterial spores have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a central cavity, and germinating or germinated bacterial spores have a central core filled with fluorescence, where Ln is a member of the lanthanide series, and BD is a β-diketone.

[0039] In another embodiment, the present invention provides a method for real-time live imaging of bacterial spore germination, comprising: injecting one or more bacterial spores into one or more germinants and a compound of formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample to form a mixture; depositing 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 fluorescent signals from the one or more bacterial spores on the slide over time using time-lapse fluorescence microscopy.

[0040] In some embodiments, the Raman microscope can distinguish dormant spores, which exhibit a double-peak profile, from germinated spores, which exhibit a single-peak profile. In some embodiments, the method can further include detecting a peak in the double-peak profile that includes an inner membrane region and a valley between the peaks that corresponds to the nucleus region of the spore, and detecting a single-peak profile that includes the nucleus of the spore. In some embodiments, during germination, Ln[BD] n There is no statistically significant shift in the Raman profile of the Ln level of the probe. In some embodiments, the Ln[BD] in the core region of the spore nIf there is a single peak corresponding to the probe, dipicolinic acid (DPA) is not present.

[0041] In some embodiments, signal detection is performed using a confocal microscope, where an optical plane slice image through a dormant spore shows a hollow region completely devoid of fluorescent signal corresponding to the core region of the spore, and an optical plane slice image through a germinated spore shows a fluorescent signal in the center of the spore corresponding to the core region of the spore.

[0042] The methods for live imaging of one or more bacterial spores in a sample and for differentiating the germination state 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.

[0043] In some embodiments, real-time imaging of one or more bacterial spore germination includes three phase contrast microscopy phases, including 1) a light phase, and 2) a gray phase of spore germination that correlates with the strongest fluorescent signal localized to the region outside the spore's nucleus, and 3) a dark phase of spore germination that correlates with the strongest fluorescent signal localized to the nucleus region of the spore, wherein the dark phase indicates bacterial spore germination.

[0044] In some embodiments, the germinant (or compound that supports germination) used is one or more of L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. n Ln of the probe is europium, samarium, dysprosium, or terbium. In some embodiments, Ln[BD] nThe BD of the probe is at least one selected from the group consisting of 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, Ln[BD] n The n of the probes is 1 to 5, or 2 to 4, or 3.

[0045] In some embodiments, the probe is at least one selected from the group consisting of 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 (Eu[TTA]3).

[0046] In some embodiments, Ln[BD] n The ratio of Ln to BD in the probe ranges from 9:1 to 1:9. In some embodiments, the concentration of the probe ranges from 0.7 mM to 10 mM, or from 1 mM to 5 mM, or from 1.4 mM to 2.8 mM, which ranges support the selection of any concentration or range of concentrations from within the range.

[0047] In some embodiments, the probe binds to spores from at least one of the genera Bacillus and Clostridium, or Clostridioides, hi some embodiments, the probe binds to spores from at least one of B. subtilis, B. thuringiensis, B. cereus, C. novi-NT type A, C. difficile, or C. septicum.

[0048] 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 embodiments, the change in colony-forming units (CFU) of bacterial spores before and after treatment with the probe is less than 0.5 log CFU.

[0049] In some embodiments, staining with the probe occurs instantaneously or nearly instantaneously, hi some embodiments, the bacterial spores are visualized immediately after the contacting step.

[0050] In some embodiments, any one of the above methods does not include a spore permeabilization step or a heating step. In some embodiments, no optical brightener is used. In some embodiments, the methods of the present disclosure do not include the use of malachite green.

[0051] In some embodiments, spore cortex hydrolysis occurs prior to probe entry into the nucleus.

[0052] In some embodiments, the fluorescence microscope is equipped with a 4',6-diamidino-2-phenylindole (DAPI) long-pass filter.

[0053] In some embodiments, the method further comprises adding one or more detergents to the sample. In some embodiments, the one or more detergents may include Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol), a polysorbate such as polysorbate 20 (Tween® 20), sodium dodecyl sulfate, or a combination thereof. In some embodiments, any one of the above methods may further comprise contacting the sample with one or more additional probes or dyes and detecting one or more additional signals. In some embodiments, one or more of the above methods may further comprise co-staining bacterial spores with Nile Red, DAPI, fluorescein isothiocyanate (FITC)-dextran, or a combination thereof.

[0054] In some embodiments, any one of the above methods can be used in food pathogen detection, environmental monitoring, quality assurance or microbiology 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 industrial processes.In some embodiments, the industrial process is in the production of self-repairing concrete.

[0055] 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 creating a video of the sample to observe changes in germination over time.

[0056] The present invention also provides a method for producing a compound of formula Ln[BD] in a first container. n wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, and a germinant is contained in a second container. In some embodiments, the germinant is L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. In some embodiments, the kit includes multiple germinants, each provided in a separate container.

[0057] The present invention also provides compounds of 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 germinant, wherein the germinant is L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine.

[0058] The invention of the present disclosure 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. [Example]

[0059] The following examples are intended to illustrate the present disclosure, but not to limit the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the Examples and other sections, figures, and claims herein form part of the present disclosure.

[0060] Example 1

[0061] Clostridium novi-NT spore formation. Spores of Clostridium novi-NT strain were produced according to the method reported by Dang et al. 2001. Briefly, spores were grown in a Gaspak broth at 37°C, pH 7.4, in a medium containing 5 g NaHPO, 30 g polypeptone peptone, 0.5 g L-cysteine, 10 g maltose, 50 g dried cooked meat particles (Difco), and 10% v / v FBS per liter. TM The organism was grown anaerobically in an anaerobic jar. After 3 weeks in this medium, spores had settled in the cooked meat particle layer. The spores were further purified from contaminating vegetative forms on a discontinuous Percoll gradient. Spore quality was confirmed by phase contrast microscopy and contained greater than 99% phase-clear spores. Spore concentrations were counted using a special depth Neubauer counting chamber (Petroff Marenfield) and were approximately 5 x 10 9 The concentration was adjusted to the order of CFU / ml.

[0062] Bacillus sporulation. For B. subtilis, B. cereus, and B. thuringiensis spores, sporulation was performed according to the protocol of Nicholson and Setlow, 1990. Bacillus strains were inoculated onto Luria Bertani (LB) agar plates overnight at 37°C. The next day, a single colony was inoculated into 2x SG medium (30 ml, pH 7) and incubated at OD on a MaxQ8000 orbital shaker (200 rpm, 37°C). 600 The culture was grown until a pH value of 0.3–0.5 was reached. This culture (25 ml) was then reinoculated into a 2 L Erlenmeyer flask containing 2x SG medium (225 ml, pH 7) and covered with a gas-permeable membrane (Breathe Easyr®) to allow sufficient aeration. The flask was then incubated in a Gerhardt orbital shaker (130 rpm, 37°C) for 44 hours for all mutant strains and 92 hours for the wild-type strain. The recovered spores were washed with 1x PBS and subsequently subjected to isopycnic centrifugation using a self-forming Percoll gradient (70% for mutants and 90% for wild-type strains) in a Beckmann-Coulter ultracentrifuge (JS 13.1 rotor, 15,000 rcf, 30 minutes, 4°C). The majority of the phase-clear spores were obtained in the bottom fraction for the wild-type strain and in the top fraction for the mutant strain, except for B. cereus, which was obtained in the top fraction. These fractions were washed repeatedly with 1x phosphate-buffered saline (PBS) and stored at 4°C until further use. The quality of the spores was confirmed by phase-contrast microscopy and contained more than 99% phase-clear spores. The spore concentrations of all strains were counted using a special depth Neubauer counting chamber (Petroff Marenfield) and counted at 10 10 Approximately 10 for each strain, except for B. cereus spores, which were adjusted to the order of CFU / ml. 9 The concentration was adjusted to the order of CFU / ml.

[0063] Clostridium septicum sporulation. C. septicum spores were purified according to the protocol by 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 the OD 600 The culture was incubated until the pH reached 1.5-3, after which the entire culture was added to 900 ml of sporulation medium (0.05% L-cysteine, 3% bactopolypeptone, 5% dehydrated cooked meat medium, and 10% fetal bovine serum) and incubated at 37°C for 5 days. Spores were purified from vegetative cells on an 80% discontinuous Percoll gradient in a Beckman Avanti J-20 XP high-performance centrifuge at 15,000 rcf for 30 minutes. The resulting spores were washed twice, resuspended in water, and stored at 4°C until further use. Spore quality was confirmed by phase-contrast microscopy and found to contain >99% phase-clear spores. Spore concentrations for all strains were counted using a special-depth Neubauer counting chamber (Petroff Marenfield), yielding approximately 10 spores for each strain. 9 The concentration was adjusted to the order of CFU / ml.

[0064] Clostridioides difficile sporulation. C. difficile sporulation was performed according to the protocol of Edwards et al., 2016. Briefly, stock concentrations of C. difficile bacteria were streaked onto 0.1% taurocholate-BHI-S agar plates (37 gm / L BHI, 5 gm / L yeast extract, 15 gm / L agar, and 1 gm / L L-cysteine) and incubated overnight at 37°C in an anaerobic chamber. The following morning, a single colony was picked from the overnight plate and inoculated into 5 ml of 0.1% taurocholate-BHI-S liquid medium. After 6 h, 250 μl of the culture was plated onto 10 0.1% taurocholate-BHI-S agar plates. After 6 days, spores were harvested by washing 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 collected by isopycnic centrifugation with Percoll (90%) at 15000 rcf for 30 min at 4°C. The bottom fraction was collected, resuspended in 2 ml of 1x PBS, and stored at 4°C until further use. The quality of the spores was confirmed by phase contrast microscopy and contained more than 99% phase-clear spores. The spore concentrations of all strains were counted using a special-depth Neubauer counting chamber (Petroff Marenfield), and approximately 10% of each strain were counted. 9 The concentration was adjusted to the order of CFU / ml.

[0065] Example 2

[0066] Preparation of europium thenoyltrifluoroacetone and other lanthanide complexes. Lanthanide TTA complex stock solutions were prepared by mixing equal volumes (200 μl) of stock solutions of the respective lanthanide chlorides (3 mM) and TTA (9 mM) in 3% ethanol. The mixture was vortexed at maximum speed for 30 minutes to promote complex formation. A 1:3 Eu:TTA ratio was used. The claimed concentration range was found to be within the optimal range that could provide adequate signal while minimizing the amount of organic solvent required for TTA solubilization for live cell imaging. All other europium β-diketone complexes were prepared in 3% ethanol, except for NTFA, which was prepared in 50% ethanol.

[0067] For Eu:TTA stoichiometry determination, a stock solution of EuCl3 (300 mM) and TTA (9 mM) in 3% ethanol was prepared. EuCl3 was then diluted in 3% ethanol to obtain Eu:TTA molar ratios of 9:1, 3:1, 1:1, 1:3, and 1:9. Equal volumes (200 μl) of these Eu solutions and TTA (9 mM) were mixed and vortexed for 30 min to form EuTTA complexes with various stoichiometric ratios. Germinated C. novii-NT spores were stained with Eu:TTA complexes in ratios ranging from 9:1 to 1:9 with a fixed TTA molar concentration, as shown in Figure 1A, imaged using a fluorescence microscope.

[0068] To determine the optimal concentration of Eu[TTA]3, stock solutions of EuCl3 (6 mM, 3 mM, 2 mM, 1.5 mM, and 1 mM) and TTA (18 mM, 9 mM, 6 mM, 4.5 mM, and 3 mM) were prepared in 3% ethanol. Equal volumes (200 μl) of these Eu and TTA solutions were mixed and vortexed for 30 min to form EuTTA complexes with various concentrations at a 1:3 Eu:TTA stoichiometry. The concentration was limited by the solubility of TTA in the solvent (3% ethanol in water). Germinated C. novii 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, as shown in Figure 1B, imaged using a fluorescence microscope.

[0069] Exposure times were set at 10 milliseconds (ms) for fluorescence mode and 100 ms for phase contrast mode. The display histogram range was adjusted for images from each experiment to ensure better visibility. Images were acquired with a Zeiss Observer 7 microscope at 1600x (Figure 1A, Figure 1B) and 1000x (Figure 1C) magnification using a long-pass DAPI filter. Scale bars are 2 µm.

[0070] Spores stained separately with either Eu or TTA did not fluoresce, indicating that both Eu and TTA are required for spore fluorescence. Figure 1C shows germinated C. novii-NT spores stained with 1.4 mM Eu[TTA] (left), 1.4 mM EuCl (center), and 4.2 mM TTA (right). The top row represents wide-field phase-contrast images, and the bottom row represents fluorescence images.

[0071] Example 3

[0072] Eu[TTA] assay of dormant and germinated spores. C. novi-NT spores were germinated using a cocktail mixture of L-cysteine ​​(100 mM), hypoxanthine (0.1 mM), and Oxyrase (1:50). C. septicum spores were germinated in sodium glycocholate (9.3 mM) and Oxyrase (1:50). C. difficile spores were germinated in sodium taurocholate (8.5 mM), glycine (45 mM), and Oxyrase (1:50). B. subtilis spores were germinated in L-alanine (20 mM) after heat activation at 70°C for 1 h, whereas B. thuringiensis were first treated with D-cycloserine (9.7 mM) at 70°C for 30 min to inactivate the conversion of germinative L-alanine to nongerminative D-alanine and then germinated in L-alanine (93.5 mM).

[0073] All endpoint germination assays were performed at 37°C for 30 min (except for cwlD spores, which were germinated for 15 h). After centrifugation at 3900 rcf for 5 min, spores were resuspended in 1x PBS (for Bacillus spores) or 1x PBS + Oxyrase (for Clostridium spores). To 3.5 μl of spores, 50 μl of Ln-BD complex was added, and a 10 μl aliquot of this sample was used for immediate imaging.

[0074] Microscopy was performed with an inverted wide-field microscope (Zeiss Axiovert 200M with a Photometrics Coolsnap HQ2 camera, 1000x magnification for all bacteria except C. difficile, and Zeiss Observer 7 with a Hamamatsu ORCA Flash 4.0 V2 camera, 1600x magnification for C. difficile). Imaging was performed with a 100X Plan Apochromate Ph3 objective (NA 1.4, oil objective) and DAPI long-pass filters (excitation filter 365 / 12 BP; dichroic 395; emission filter 397 LP).

[0075] Figure 2A shows that when the Ln-BD complex was Eu[TTA]3, it stained both dormant and germinated spores of all bacteria tested. The dormant spores fluoresced as donut-shaped spores with a central cavity, which filled with bright fluorescence after germination. Figure 4 shows that when the Ln-BD complex was Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, or Tb[TTA]3, it similarly stained dormant C. novii spores, turning their nuclei fluorescent after germination. Furthermore, germination resulted in brighter fluorescence in the nuclei of all spores tested. Furthermore, Figure 5 shows that when the Ln-BD complex was 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 stained dormant and germinated C. novii-NT spores more brightly than the Ln-BD complex using an aliphatic BD. These results suggest that various Ln[BD] nWe demonstrate that the conjugate can be used to stain various bacterial spores. The results also demonstrate that aliphatic BDs are useful for obtaining spore fluorescence.

[0076] A germination dye solution for live cell imaging of C. novii-NT spores was prepared by mixing appropriate amounts of the germinants L-cysteine ​​(1 M, 53.5 μl) and hypoxanthine (0.001 M, 53.5 μl), oxylase (10 μl) to induce hypoxic conditions for germination, and Eu[TTA] (0.0015 M, 383 μl). An aliquot (50 μl) of this germination solution was added to the C. novii-NT spore solution (5 × 10 9 A solution of 100 μl of L-alanine (100 mM, 107 μl) and Eu[TTA]3 (0.0015 M, 393 μl) was added to a Superfrost Plus microscope slide, covered with a #1.5 cover slip, and sealed on all four sides with nail polish. For B. subtilis spores, a germinative dye solution was prepared by mixing the appropriate amounts of L-alanine (100 mM, 107 μl) and Eu[TTA]3 (0.0015 M, 393 μl). Heat-treated spores were allowed to adhere to a regular slide for 1 hour at 4°C, after which 50 μl of the germinative dye solution was added to the adhered spores, which were immediately sealed with a cover slip, and imaged. The final concentrations of L-alanine and Eu[TTA]3 were 20 mM and 1.1 mM, respectively.

[0077] For live cell imaging alone, the cage incubator attached 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 100 ms (phase contrast mode and fluorescence images) unless otherwise noted. 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 for ImageJ before cropping.

[0078] As shown in Figure 3A, a confocal image of Eu[TTA]3 alone, slides were imaged using a Leica SP8 super-resolution microscope equipped with a 100X HCX PL APO objective (NA 1.4, oil objective), a 405 nm diode laser, detection at 580-700 nm, and a 0.5 AU pinhole. Images were acquired using Leica Application Suite X 1.8 (LASX v1.8) software and deconvolved using Lightning mode with the same parameters for all bacteria except for Bacillus spores, where an optimization factor of 3 was used instead of 1.

[0079] All confocal images were cropped and processed using ImageJ Version 1.53n. Videos were generated using the 3D maximum projection function of Z-stack images acquired using a confocal microscope and processed using ImageJ.

[0080] As shown in Figures 3A and 11, optical plane slice images through Eu[TTA]3-stained dormant spores of all bacteria tested showed hollow spaces lacking the fluorescent signal that would become fluorescent upon germination.

[0081] Example 4

[0082] Raman imaging of Clostridium novi-NT spore germination. To better elucidate the effects of this invention, Raman microscopy was used as a tool to map the distribution of Eu. A germinant dye solution for Raman live-cell imaging of C. novi-NT spores was prepared by mixing appropriate amounts of germinants L-cysteine ​​(1 M, 160.5 μl), hypoxanthine (0.001 M, 53.5 μl), oxylase (10 μl), and Eu[TTA]3 (0.0015 M, 276 μl). A higher concentration of germinant was used compared to other experiments to promote rapid germination and counteract potential photodamage to spores caused by the laser over time. An aliquot (50 μl) of this germinant solution was added to the C. novi-NT spore solution (5 × 10 9 CFU / ml, 3.5 μl) was added to the slides to give final concentrations of 300 mM, 0.1 mM, and 0.77 mM L-cysteine, hypoxanthine, Eu[TTA], or EuCl, respectively. 10 μl of this solution was added to a quartz microscope glass slide (Photonik, Singapore), covered with a quartz cover slip (#1.5), and sealed on all four sides with nail polish. Population germination assays were performed by placing the slides on a heating stage (Linkam Controller DC95) set at 37°C, whereas X-mapping assays were performed at room temperature without a heating stage.

[0083] Simultaneous phase-contrast, fluorescence, and Raman imaging was achieved 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. A 100X Nikon Plan Ph3 DL objective (NA 1.25, oil objective) and a DAPI filter (excitation filter 350 / 5 BP; emission filter 412 LP) were used for fluorescence imaging. 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 baselines were corrected for all samples using the Savitzky-Golay method with the same parameters. Only the fluorescent images were further processed for deconvolution by the CMLE method using Hyugens Professional v16.10 software.

[0084] Figure 2B shows the distribution of Eu along the longest axis of C. novi-NT spores treated with either Eu[TTA] or uncomplexed Eu (in the form of EuCl). C. novi-NT spores were used in this study because of their large size compared to the other bacteria used in this study, allowing for high-resolution mapping. Raman microscopy not only provides fluorescence-independent evidence for Eu localization in spores, but also allows for the simultaneous detection of the spore biomarker dipicolinic acid (DPA). As shown in the bottom panel of Figure 2B, DPA (1017 cm) -1 ) was observed to be concentrated in the nucleus during dormancy, and germination resulted in spore DPA concentrations that were not higher than the ambient level, indicating release from the nucleus. This DPA distribution profile held true whether spores were treated with Eu[TTA]3 or EuCl3.

[0085] In contrast to the DPA profile, Eu[TTA]3 and EuCl3 produced very different results in dormant and germinated spores, as shown in the upper panel of Figure 2B. Spores treated with Eu[TTA]3 exhibited a double-peak profile, with peaks corresponding to valleys within the inner membrane and nuclear regions. Upon germination, the Eu signal spatially redistributed to form a single peak within the nucleus, confirming the bright nuclear fluorescence observed after germination. Furthermore, although there was no net increase in Eu within spores after germination, spore nuclear fluorescence appeared brighter to the eye compared to dormant spores.

[0086] To investigate the relationship between DPA and Eu[TTA]3 fluorescence distribution in bacterial spores, C. novii-NT spores from all three phase stages (light, gray, and dark) were imaged, with 20 spores analyzed per phase stage. Each spore was first phase-classified by phase contrast, then imaged under fluorescence, and subsequently analyzed by Raman spectroscopy as described above. The Raman laser was aimed at the center of the spore to generate a typical Raman profile of the spore. Data for a typical spore are shown in Figure 7A. Phase-bright spores exhibit a Raman shift of 1017 cm, similar to donut fluorescence. -1 The characteristic DPA peak was absent in the gray-phase spores, suggesting DPA emission, although the donut pattern was still observed. Dark-phase spores lacked the DPA peak, as expected, but exhibited spore nucleus fluorescence. These results indicate that DPA emission precedes Eu[TTA]3 fluorescence in the nucleus. Figure 7A also shows that there is no statistically significant shift in the Raman profile of Eu levels between the phases, suggesting that the amount of Eu in the spores generally remains stable throughout germination.

[0087] Example 5

[0088] Co-staining assay. Germinated C. novii spores were incubated with Eu[TTA]3 (1.4 mM) and Nile Red (0.2 μg / ml), DAPI (2.5 μM, 30 min), or FITC-dextran 3k-5k (0.45 mg / ml). Aliquots from these samples were used for imaging. Slides were imaged using a Leica SP8 super-resolution microscope equipped with a 100X HCX PL APO objective (NA 1.4, oil objective). Images were acquired using Leica Application Suite X 1.8 (LASX v1.8) software and deconvolved using Lightning mode.

[0089] Figures 3B to 3D show Ln[BD] n We demonstrate that the complex can be used in combination with other dyes to investigate further elements of spore morphology. Co-staining with Nile Red labeled the inner and outer membranes of the spore, which are lipid-rich regions within the spore, as shown in Figure 3B. Nile Red staining of the inner membrane strongly overlapped with Eu[TTA]3 fluorescence, but Eu[TTA]3 fluorescence extended beyond the inner membrane boundary and filled the spore nucleus outlined by the Nile Red-stained inner membrane. Co-staining with the DNA intercalator DAPI revealed a comma-shaped, crystalline nucleoid of DNA, as shown in Figure 3C. This comma structure was squarely nested within the spore nucleus as defined by Eu[TTA]3 fluorescence. Co-staining with FITC-dextran (molecular weight 3-5 kD) revealed that the spore outer membrane remained intact as a physical barrier and was therefore impermeable to FITC-dextran, as shown in Figure 3D. In contrast, Eu[TTA]3, with its smaller molecular size and lipophilic characteristics, is able to penetrate the outer membrane and stain the inner membrane as well as the nucleus.

[0090] Example 6

[0091] Viability Assay. C. novi-NT spores were germinated as described above in Example 3, and B. subtilis was germinated in 93.5 mM L-alanine. To a 10 μl aliquot of germinated spores, Eu[TTA]3 (1.5 mM, 142.8 μl) was added and incubated at room temperature for 15 minutes, resulting in a final concentration of 1.4 mM. Spores were then plated on BHI (10%) FBS agar plates in a Plas Labs anaerobic chamber for C. novi-NT or aerobically on LB agar plates for B. subtilis. Colonies were counted the following day.

[0092] Figure 6A shows that treatment with Eu[TTA]3 did not result in a statistically significant decrease in viability, and both C. novi-NT and B. subtilis retained their growth and colony-forming abilities.

[0093] Because Eu[TTA]3 did not affect spore viability, spore germination was tracked in real time. Spores were mounted on slides and incubated at 37°C with germinants appropriate for C. novi-NT (Figure 6B) and B. subtilis spores (Figure 6C). Time-lapse phase-contrast and fluorescence microscopy were then performed to capture early spore germination, following the conditions outlined in Example 3. Spores began in the light phase with a donut-shaped fluorescence pattern. This fluorescence pattern did not change as the spores entered the gray phase. The hollow donut became fluorescent only when they entered the dark phase. This process occurred within 10–20 min, resulting in the nucleus being brighter than the fluorescence of the donut surrounding it.

[0094] In the case of C. novi-NT, the initial fluorescence in the nucleus was observed as a pattern of punctate fluorescent spots that eventually increased in size to cover the entire spore nucleus, as shown in Figure 6 B. B. subtilis followed the same sequence of events, having a hollow spore nucleus that then rapidly filled with fluorescence as it entered phase darkening, as shown in Figure 6 C.

[0095] Example 7

[0096] Cortex Decomposition Test. To investigate the possibility that the cortex layer of bacterial spores may facilitate or hinder the penetration of lanthanide-β-diketones into the spore nucleus, the following test was performed. For cortex removal of C. novi-NT, dormant spores were treated with 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 lysed in cortex lysis buffer (1x Halt) at 37°C for 30 minutes. TM The spores were washed again with water and stored in Tris-Halt for further analysis. TM The spores were resuspended in 1× PBS or 1× MgCl2 buffer (which was the cortex lysis buffer without the lysozyme component). An aliquot of spores from each stage was set aside for imaging. cwlD mutant B. subtilis spores lacking the muramic acid-δ-lactam essential for cortex hydrolysis were germinated and stained with Eu[TTA]3 as described in Example 3.

[0097] Microscopy was performed with an inverted widefield microscope. For C. novi-NT spores, imaging conditions were a Leica DMI6000 with a Hamamatsu ORCA flash 4.0 LT camera. Imaging was performed at 1000x magnification using a long-pass DAPI filter. Exposure times were 50 ms for both modes, except for fluorescent images of dormant and uncoated spores, which were 100 ms. For B. subtilis spores, conditions were a Zeiss Axiovert 200M with a photometric Coolsnap HQ2 camera and a long-pass DAPI filter at 1000x magnification and 100 ms exposure times for both modes.

[0098] As shown in Figure 7B, Eu[TTA]3 did not stain the nuclei of germinated cwlD mutant B. subtilis spores with an intact cortex, indicating that the cortex restricted access to Eu[TTA]3. In contrast, Figure 7C shows that bright nuclear fluorescence was observed when dormant C. novi-NT spores were treated with both the uncoating and lysozyme steps described above, but not when treated with either step alone. These results indicate that complete removal of all layers before the inner membrane allowed Eu[TTA]3 to access the dormant spore nucleus and that cortex hydrolysis was required for Eu[TTA]3 to access the spore nucleus.

[0099] Example 8 (Comparative Example)

[0100] DAPI staining. Both C. novi-NT and B. subtilis spores (dormant and germinated) were incubated with DAPI (5 μM), and aliquots from these samples were used for imaging, as shown in Figure 8 (bottom, left column). Imaging was performed using a widefield inverted Zeiss Axiovert 200M microscope equipped with a 100X Plan Apochromat Ph3 objective (NA 1.4, oil objective) and a long-pass DAPI filter fitted with a Photometrics Coolsnap HQ2 monochrome camera. Images were captured using Metamorph Series 7.7 (M7.7) software. All image processing and cropping was performed using ImageJ.

[0101] Acridine orange staining. For C. novi-NT spores, dormant or germinated spores (3.5 μl) were incubated with acridine orange solution (50 μl, 10 mg / ml) for 30 minutes (final concentration 9.3 mg / ml) and then imaged as shown in Figure 8 (bottom, right column, row 1). For B. subtilis 1A1 wild-type spores, heat-treated dormant and germinated spore slides were prepared as described in the previous section, followed by the addition of acridine orange solution, and the slides were immediately imaged as shown in Figure 8 (bottom, right column, row 2). Imaging was performed using a wide-field inverted Zeiss Axiovert 200M microscope equipped with a 100X Plan Apochromat Ph3 objective (NA 1.4, oil objective) and EGFP filters (excitation filter - 470 / 40 BP; dichroic 495; emission filter 525 / 50 BP) attached to a Coolsnap HQ2 monochrome camera. Images were captured using Metamorph Series 7.7 (M7.7) software. All image processing and cropping was performed using ImageJ.

[0102] Malachite green staining. Both C. novi-NT and B. subtilis spores (dormant and germinated) were heat-fixed on a heating block at 50°C for 5 minutes. Malachite green solution (5%) was then added continuously to the heat-fixed smears covered with filter paper and placed in a water bath steamer at 95°C 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 again with water and blotted with paper towels. They were then imaged using a Zeiss Axioplan 2 upright microscope equipped with a 100X Plan Apochromate Ph3 objective (1.4 NA, oil objective) and a Leica DFC 7000T color camera, as shown in 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 with a 2-pixel radius. All image processing and cropping was performed using ImageJ.

[0103] The Schaeffer-Fulton method uses malachite green and safranin counterstains to distinguish between dormant and germinated forms. Sample processing is lengthy and destroys spore viability, thus preventing live imaging. DAPI stained both dormant and germinated spores with high fluorescence but did not distinguish between them. Finally, AO provided some differentiation between dormant and germinated spores but did not visibly stain germinated spores. Both DAPI and AO required an incubation time of at least 30 minutes to achieve sufficient contrast in the staining. A comparison of the performance of various stains can be seen in Figure 12.

[0104] Example 9

[0105] Dino-Lite Imaging. Eu[TTA]3-stained bacterial samples were prepared for dormant and terminally germinated spores as in Example 3. Images were acquired using a Dino-Lite Premier Digital Microscope AM4113T with DAPI excitation at 55x magnification. Images were cropped and processed using ImageJ. Figure 9 shows that despite the low image resolution, Eu[TTA]3-stained spores were observed to be significantly more fluorescent than the background.

[0106] Example 10

[0107] Milk matrix experiments. A 5% w / v skim milk solution was prepared by dissolving blotting-grade milk powder in water. An equal volume of dormant C. novi-NT spores was added to this solution to give a final milk concentration of 2.5% (w / v) and 2.5 × 10 spores. 9 The CFU / ml was calculated. An aliquot (3.5 μl) of this sample was then stained with Eu[TTA]3 (1.4 mM). Samples were imaged using a Zeiss inverted Axio Observer 7 widefield microscope equipped with a long-pass DAPI filter at 1600x magnification. Exposure times were 100 ms for phase contrast mode and 50 ms for fluorescence mode. Display histograms were adjusted differently for each acquisition mode to ensure maximum visibility.

[0108] Figure 10 shows that spores were clearly visualized under fluorescence only in the presence of Eu[TTA]3, and the donut-shaped fluorescent shape of the spores suspended in milk was easy to discern even in the presence of background fluorescence.

[0109] While the subject matter of the present disclosure has been described and illustrated in considerable detail with reference to specific exemplary embodiments, including various combinations and subcombinations of features, those skilled in the art will readily appreciate other embodiments, as well as variations and modifications thereof, that are encompassed within the scope of the present disclosure. Moreover, the description of such embodiments, combinations, and subcombinations is not intended to convey that the claimed subject matter requires any features or combinations of features 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 following appended claims.

[0110] Example 11 (complex mixture sample)

[0111] Spore growth staining: C. novi-NT dormant spores were anaerobically incubated (37°C, 7 h) in medium containing L-cysteine ​​(100 mM), hypoxanthine (0.1 mM), BHI (0.62X), FBS (9.3% v / v), and oxylase (1:50 v / v). B. subtilis dormant spores were first heat-activated in 1X PBS (70°C, 1 h) and then aerobically incubated (37°C, 4 h). The medium was then removed by centrifugation (3900 rcf, 15 min), followed by the addition of Eu[TTA]3 (1.4 mM). Aliquots of these samples were then imaged.

[0112] Sporulation culture staining: Vegetative B. cereus cells were grown aerobically in 2x SG sporulation medium (Informers HT Multitron Standard Shaking Incubator) with shaking at 220 rpm (37°C overnight to allow for early sporulation) and harvested the next day after 24 hours by centrifugation (3900 rcf, 15 min). Cells were then stained with Eu[TTA]3 (1.4 mM) and imaged.

[0113] In natural environments, a mixture of spores and vegetative bacteria is expected. Taking this into account, staining was performed on samples derived from either spore growth (Figure 13A) or overnight cultures of sporulating vegetative bacteria (Figure 13B). In cultures of C. novi-NT or B. subtilis, spores exhibited brighter fluorescence compared to their vegetative counterparts and were easily distinguishable based on their morphology and size. A similar trend was observed in early sporulation cultures of B. cereus, where developing prespores of sporulating cells were marked by hollow regions, highlighting the usefulness of Eu[TTA]3 in visualizing spores regardless of the presence of vegetative forms.

[0114] Milk matrix experiments: A 5% w / v skim milk solution was prepared by dissolving blotting-grade milk powder in water. An equal volume of dormant B. cereus spores was added to this solution to give a final milk concentration of 2.5% (w / v) and 1.75 × 10 spores. 10 CFU / ml (B. cereus). An aliquot (3.5 μl) of this sample was then stained with Eu[TTA]3 (1.4 mM) and imaged.

[0115] Finally, the ability of Eu[TTA]3 to address spore detection in food matrices was also tested. The problem of dairy product contamination by spore-forming bacteria such as B. cereus and C. botulinum has been well reported, 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, T.B.; Brightwell, G. Farm Level Survey of Spore-Forming Bacteria on Four Dairy Farms in the Waikato Region of New Zealand. Microbiologyopen 2017, 6 (4); Barash, J.R.; Hsia, J.K.; Arnon, S.S. Presence of Soil-Dwelling Clostridia in Commercial Powdered Infant Formulas. J. Pediatr. 2010, 156 (3), 402-408. Such contamination was simulated by inoculating milk with B. cereus spores and subsequently imaging with or without Eu[TTA]3 (Figure 13C). Despite moderate background fluorescence, visible fluorescent spores with hollow cores were easily visualized only in the presence of Eu[TTA]3. These results demonstrate the potential of Eu[TTA]3 for spore detection in complex food matrices.

[0116] Numbered Aspects Notwithstanding the appended claims, the following numbered embodiments also form part of this disclosure and are exemplary and representative of the present invention: 1. A method for live imaging of one or more bacterial spores in a sample, comprising: The sample was treated with the formula Ln[BD] nwherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample; and Detecting a fluorescent signal from one or more stained bacterial spores in a sample using a fluorescence microscope. A method comprising: 2. A method for differentiating the germination state of one or more bacterial spores in situ, the method comprising: A sample containing one or more bacterial spores is analyzed by the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample; Performing signal detection by live imaging of the fluorescent signal of the sample using a fluorescence microscope; and To distinguish between dormant and germinating bacterial spores in a sample Including, Dormant bacterial spores have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a central cavity, and Germinating or germinated bacterial spores have a central core filled with fluorescence, method. 3. A method for real-time live imaging of bacterial spore germination, the method comprising: One or more bacterial spores are combined with one or more germinants and a mixture of the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample to form a mixture; placing the mixture on a slide; Visualizing the morphology of one or more bacterial spores on a slide over time using time-lapse phase-contrast microscopy; and Imaging the fluorescent signal from one or more bacterial spores on a slide over time using time-lapse fluorescence microscopy Including, The method includes real-time imaging of one or more bacterial spore germination, including three phase contrast microscopy phases, including 1) a light phase, and 2) a gray phase of spore germination that correlates with the strongest fluorescent signal localized to the region outside the spore's nucleus, and 3) a dark phase of spore germination that correlates with the strongest fluorescent signal localized to the nucleus region of the spore, wherein the dark phase indicates bacterial spore germination. 4. Signal detection is performed using a confocal microscope. an optical plane slice image through the dormant spore shows a hollow region completely devoid of fluorescent signal corresponding to the core region of the spore; and The method of any one or combination of numbered aspects disclosed herein, wherein an optical plane slice image through a germinated spore shows a fluorescent signal in the center of the spore, corresponding to the core region of the spore. 5. The method of any one or combination of numbered aspects disclosed herein, wherein the germinant is one or more of L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. 6. Ln[BD] n The method according to any one or combination of numbered aspects disclosed herein, wherein Ln of the probe is europium, samarium, dysprosium, or terbium. 7. Ln[BD] n The method of any one or combination of numbered aspects disclosed herein, wherein the BD of the probe is at least one selected from the group consisting of 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. Ln[BD] n The method according to any one or combination of numbered aspects disclosed herein, wherein n of the probes is 1 to 5. 9. Ln[BD] nThe method according to any one or combination of numbered aspects disclosed herein, wherein n of the probes is 2 to 4. 10. Ln[BD] n The method according to any one or combination of numbered aspects disclosed herein, wherein n of the probes is 3. 11. The method of any one or combination of numbered aspects disclosed herein, wherein the probe is at least one selected from the group consisting of 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 or combination of numbered aspects disclosed herein, wherein the probe binds to spores from at least one of the genera Bacillus, Clostridium, or Clostridioides. 13. The method of any one or combination of numbered aspects disclosed herein, wherein the probe binds to spores from at least one of B. subtilis, B. thuringiensis, B. cereus, C. novi-NT type A, C. difficile, or C. septicum. 14. The method of any one or combination of numbered aspects 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. 15. The method of any one or combination of numbered aspects 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. 16. The method according to any one or combination of numbered aspects disclosed herein, 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 according to any one or combination of numbered aspects disclosed herein, wherein staining with the probe occurs instantaneously or near instantaneously. 18. The method of any one or combination of numbered aspects 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. 19. Ln[BD] n The method according to any one or combination of numbered aspects disclosed herein, wherein the ratio of Ln to BD in the probe ranges from 9:1 to 1:9. 20. The method according to any one or combination of numbered aspects disclosed herein, wherein the concentration of the probe ranges from 0.7 mM to 10 mM. 21. The method according to any one or combination of numbered aspects disclosed herein, wherein the concentration of the probe is in the range of 1 mM to 5 mM. 22. The method according to any one or combination of numbered embodiments disclosed herein, wherein the concentration of the probe is in the range of 1.4 mM to 2.8 mM. 23. The method according to any one or combination of numbered aspects disclosed herein, wherein the concentration of the probe is about 1.4 mM or about 2.8 mM. 24. The method according to any one or combination of numbered aspects disclosed herein, wherein the method does not include a spore permeabilization step or a heating step. 25. The method according to any one or combination of numbered embodiments disclosed herein, wherein spore cortex hydrolysis occurs prior to probe entry into the nucleus. 26. The method of any one or combination of numbered aspects disclosed herein for use in food pathogen detection, environmental monitoring, quality assurance, or microbiology research. In some embodiments, the food is milk. 27. The method of any one or combination of numbered aspects disclosed herein, comprising using a Raman microscope to distinguish dormant spores that exhibit a double-peak profile from germinated spores that exhibit a single-peak profile. 28. The method of any one or combination of numbered aspects disclosed herein, comprising detecting a peak in a double-peak profile comprising an inner membrane region and a valley between the peaks corresponding to a spore nucleus region, and detecting a single-peak profile comprising the spore nucleus. 29. Through germination, Ln[BD] n The method according to any one or combination of numbered aspects disclosed herein, wherein there is no statistically significant shift in the Raman profile of the Ln level of the probe. 30. Ln[BD] in the nuclear region of the spore n The method according to any one or combination of numbered aspects disclosed herein, comprising detecting the absence of dipicolinic acid (DPA) if a single peak corresponding to the probe is present. 31. The method of any one or combination of numbered aspects disclosed herein, wherein the probe is europium(III) thenoyltrifluoroacetone (Eu[TTA]3). 32. The method of any one or combination of numbered aspects disclosed herein, wherein the fluorescence microscope is equipped with a 4',6-diamidino-2-phenylindole (DAPI) long-pass filter. 33. The method of any one or combination of numbered aspects disclosed herein, further comprising adding one or more surfactants to the sample. 34. The method of any one or combination of numbered aspects disclosed herein, wherein the one or more surfactants is 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polysorbate, sodium dodecyl sulfate, or a combination thereof. 35. The method of any one or combination of numbered aspects disclosed herein, further comprising co-staining bacterial spores with Nile red, DAPI, fluorescein isothiocyanate (FITC)-dextran, or combinations thereof. 36. The method of any one or combination of numbered aspects disclosed herein, 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 any one or combination of numbered aspects disclosed herein, wherein the industrial process is in the production of self-repairing concrete. 38. The method of any one or combination of numbered aspects 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. 39. The method of any one or combination of numbered aspects disclosed herein, comprising visualizing the stained bacterial spores immediately after the contacting step. 40. The method of any one or combination of numbered aspects disclosed herein, wherein imaging and / or monitoring comprises creating a video of the sample to observe changes in germination over time. 41. The method of any one or combination of the numbered aspects disclosed herein, wherein the method does not use an optical brightener. 42. The method according to any one or combination of numbered aspects disclosed herein, wherein the method does not include a permeabilization step. 43. In a first container, add a solution of formula Ln[BD] n wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer, and in a second container, a germinant. 44. The kit of any one or combination of the numbered embodiments disclosed herein, wherein the germinant is L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. 45. The kit of any one or combination of the numbered embodiments disclosed herein, wherein the kit comprises a plurality of germinants, each germinant provided in a separate container. 46. ​​Expression Ln[BD] n wherein Ln is a member of the lanthanide series, BD is a β-diketone, and n is an integer; and a germinant. 47. The composition of any one or combination of the numbered aspects disclosed herein, wherein the germinant is L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine. 48. A live imaging apparatus for use in live imaging one or more bacterial spores in a sample, wherein the one or more bacterial spores are immersed in a solution of a compound of formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample, the apparatus comprising a live imaging capture device configured to detect the staining of the one or more bacterial spores in the sample. 49. An apparatus for in situ differentiating the germination state of one or more bacterial spores in a sample, the sample comprising a spore having a germination state of the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, and the apparatus is configured to acquire live imaging fluorescent signals of the sample using a fluorescence microscope and distinguish between dormant bacterial spores and germinating or germinated bacterial spores in the sample; Dormant bacterial spores have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a hollow cavity in the middle, and germinating or germinated bacterial spores have a central core filled with fluorescence. 50. One or more germinants and the formula Ln[BD] nwherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample to form a mixture, the apparatus comprising: a slide configured to hold the mixture; and a time-lapse phase microscope configured to visualize the bacterial spores in the mixture and image fluorescent signals from the bacterial spores on the slide, wherein the real-time imaging of the germination of the one or more bacterial spores comprises three phase-contrast phases, including 1) a bright phase, and 2) a gray phase of spore germination that correlates with the strongest fluorescent signal localized to a region outside the nucleus of the spore, and 3) a dark phase of spore germination that correlates with the strongest fluorescent signal localized to the nuclear region of the spore, the dark phase indicating bacterial spore germination. 51. Use of the method according to any one or combination of the numbered aspects 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. 52. Use of the method according to any one or combination of the numbered aspects disclosed herein for a quality assurance process in the production of self-repairing concrete. 53. Use of the method according to any one or combination of the numbered aspects disclosed herein for quantifying a plurality of bacterial spores in a sample. 54. Use of the method according to any one or combination of the numbered aspects disclosed herein to visualize stained bacterial spores immediately after the contacting step. 55. Use of the method according to any one or combination of the numbered aspects disclosed herein to create a video of a sample to observe changes in germination over time. 56. Use of a kit according to any one or combination of the numbered aspects disclosed herein in a method according to any one or combination of the numbered aspects disclosed herein. 57. Use of a composition according to any one or combination of the numbered aspects disclosed herein in a method according to any one or combination of the numbered aspects disclosed herein.

[0117] While the subject matter of the present disclosure has been described and illustrated in considerable detail with reference to specific exemplary embodiments, including various combinations and subcombinations of features, those skilled in the art will readily recognize other embodiments, as well as variations and modifications thereof, that are encompassed within the scope of the present disclosure. Moreover, the description of such embodiments, combinations, and subcombinations is not intended to convey that the claimed subject matter requires any features or combinations of features 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 following appended claims. Section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

[0118] Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for live imaging of one or more bacterial spores in a sample, comprising: The sample was treated with the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample; and Detecting a fluorescent signal from one or more stained bacterial spores in a sample using a fluorescence microscope. A method comprising:

2. 1. A method for differentiating the germination state of one or more bacterial spores in situ, the method comprising: A sample containing one or more bacterial spores is analyzed by the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample; Performing signal detection by live imaging of the fluorescent signal of the sample using a fluorescence microscope; and To distinguish between dormant and germinating bacterial spores in a sample Including, Dormant bacterial spores have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a central cavity, and Germinating or germinated bacterial spores have a central core filled with fluorescence, method.

3. 1. A method for real-time live imaging of bacterial spore germination, the method comprising: One or more bacterial spores are combined with one or more germinants and a mixture of the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample to form a mixture; placing the mixture on a slide; visualizing the morphology of one or more bacterial spores on the slide over time using time-lapse phase contrast microscopy; and Imaging the fluorescent signal from one or more bacterial spores on a slide over time using time-lapse fluorescence microscopy Including, The method includes real-time imaging of one or more bacterial spore germination, including three phase contrast microscopy phases, including 1) a light phase, and 2) a gray phase of spore germination that correlates with the strongest fluorescent signal localized to the region outside the spore's nucleus, and 3) a dark phase of spore germination that correlates with the strongest fluorescent signal localized to the nucleus region of the spore, wherein the dark phase indicates bacterial spore germination.

4. Signal detection was performed using a confocal microscope. an optical plane slice image through the dormant spore shows a hollow region completely devoid of fluorescent signal corresponding to the core region of the spore; and An optical plane slice image through a germinated spore shows a fluorescent signal in the center of the spore, corresponding to the nucleus region of the spore. The method of claim 2.

5. 4. The method of claim 3, wherein the germinant is one or more of L-cysteine, hypoxanthine, oxylase, sodium glycocholate, taurocholate, glycine, L-alanine, L-valine, D-valine, or D-cycloserine.

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

7. Ln[BD] n The method according to any one of claims 1 to 3, wherein the BD of the probe is at least one selected from the group consisting of 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. Ln[BD] n The method according to any one of claims 1 to 3, wherein n in the probe is 1 to 5.

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

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

11. The probe is 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 The method according to any one of claims 1 to 3, wherein the method is at least one selected from the group consisting of:

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

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

14. 3. The method of any one of claims 1 to 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. 4. The method of any one of claims 1 to 3, wherein treatment with the probe does not significantly affect the viability, growth or colonization ability of the spores compared to untreated spores.

16. 4. The method of 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 to 3, wherein staining with the probe occurs instantaneously or nearly instantaneously.

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

9.

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

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

22. The method according to any one of claims 1 to 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 to 3, wherein the concentration of the probe is about 1.4 mM or about 2.8 mM.

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

25. 4. The method of any one of claims 1 to 3, wherein spore cortex hydrolysis occurs before entry of the probe into the nucleus.

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

27. 3. The method of claim 2, comprising using a Raman microscope to distinguish dormant spores, which exhibit a double-peak profile, from germinated spores, which exhibit a single-peak profile.

28. 28. The method of claim 27, comprising detecting a peak in a double-peak profile comprising an inner membrane region and a valley between the peaks corresponding to a spore nucleus region, and detecting a single-peak profile comprising the spore nucleus.

29. Through germination, Ln[BD] n 28. The method of claim 27, wherein there is no statistically significant shift in the Raman profile of the Ln level of the probe.

30. Ln[BD] in the nuclear region of the spore n 28. The method of claim 27, comprising detecting the absence of dipicolinic acid (DPA) if a single peak corresponding to the probe is present.

31. The probe was europium(III) thenoyltrifluoroacetone (Eu[TTA] 3 31. The method according to any one of claims 1 to 30, wherein

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

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

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

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

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

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

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

41. 40. The method of any one of claims 1 to 39, wherein no optical brightener is used.

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

43. In a first container, a solution of 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 in a second container, the kit includes a germinant.

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

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

46. 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 germinant.

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

48. 1. A live imaging apparatus for use in live imaging one or more bacterial spores in a sample, wherein the one or more bacterial spores are quantified by a method comprising the steps of: n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in the sample, wherein the apparatus comprises a live imaging capture device configured to detect the staining of the one or more bacterial spores in the sample.

49. 1. An apparatus for in situ differentiating the germination state of one or more bacterial spores in a sample, the sample comprising: a spore having a germination state of one or more bacterial spores of the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, and the apparatus is configured to obtain live imaging fluorescent signals of the sample using a fluorescence microscope and distinguish between dormant bacterial spores and germinating or germinated bacterial spores in the sample; Dormant bacterial spores have a fluorescent signal characterized by a doughnut-shaped fluorescent profile with a hollow cavity in the middle, and germinating or germinated bacterial spores have a central core filled with fluorescence.

50. One or more germinants and the formula Ln[BD] n wherein Ln is a member of the lanthanide series of elements, BD is a β-diketone, and n is an integer, to stain one or more bacterial spores in a sample to form a mixture, the apparatus comprising: a slide configured to hold the mixture; and a time-lapse phase microscope configured to visualize the bacterial spores in the mixture and image fluorescent signals from the bacterial spores on the slide, wherein the real-time imaging of the germination of the one or more bacterial spores comprises three phase-contrast phases, including 1) a light phase, and 2) a gray phase of spore germination that correlates with the strongest fluorescent signal localized to a region outside the nucleus of the spore, and 3) a dark phase of spore germination that correlates with the strongest fluorescent signal localized to the nuclear region of the spore, the dark phase being indicative of bacterial spore germination.

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

52. Use of the method according to any one of claims 1 to 42 for a quality assurance process in the production of self-repairing concrete.

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

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

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

56. The method according to any one of claims 1 to 42, using the kit according to any one of claims 43 to 45.

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

Images