Screening of fluorescent microorganisms using a microfabricated device

Abstract

A method for identifying fluorescent cells in a sample using a microfabricated chip having a top surface comprising a plurality of microwells. At least one cell of the sample is loaded into at least one microwell of the plurality of microwells. The microfabricated chip is incubated to allow the at least one cell in the at least one microwell to grow into a cell population. The presence of a fluorescent cell of interest in the sample is determined by analyzing an image of the microfabricated chip to detect fluorescence exhibited by the at least one microwell.

Description

Background Technology

[0001] Bacteria are ubiquitous, small, single-celled organisms, and the human body contains millions of them. While most bacteria are harmless to humans, some can cause serious health problems. Due to the widespread use of antibiotics, multidrug-resistant pathogens are gradually becoming a serious global problem. Identifying bacterial strains or species within the human microbiome may be crucial for treating bacterial infections and developing antibiotics.

[0002] Various techniques have been employed for bacterial detection, such as polymerase chain reaction (PCR), Gram staining, immunological techniques, and Raman spectroscopy. However, these methods are typically time-consuming, expensive, and complex, and may lead to false positive results.

[0003] Fluorescent dyes are small organic probes used to distinguish bacteria. Some bacteria are capable of naturally producing fluorescence. While various methods can be used to detect fluorescent bacteria, fluorescence-activated cell sorting (FACS) is typically employed to separate fluorescent bacteria from non-fluorescent bacteria for large-scale culture. FACS instruments use a laser as an excitation source and a set of wavelength detectors to detect emission, thereby identifying individual fluorescent bacterial cells. These cells are then sorted using electromagnets. Summary of the Invention

[0004] In one aspect, a method for identifying fluorescent cells in a sample is provided. The method utilizes a microfabrication chip having a top surface comprising a plurality of micropores. The method includes: loading at least one cell from the sample into at least one of the plurality of micropores; culturing the microfabrication chip to grow at least one cell in the at least one micropore into a cell population; and determining the presence of fluorescent cells of interest in the sample by analyzing images of the microfabrication chip to detect fluorescence exhibited by the at least one micropore.

[0005] In some embodiments, loading includes loading a plurality of cells of a sample into a plurality of microwells such that at least some of the microwells each contain one and only one cell, while the percentage of microwells containing more than one cell is zero or statistically insignificant.

[0006] In some embodiments, at least one of the microwells is further loaded with a metabolic indicator that indicates cellular metabolic activity. This indicator may emit fluorescence, and its fluorescence state may indicate cellular metabolic activity, such as cell growth and proliferation. In some such embodiments, detection includes simultaneously detecting the fluorescence of the metabolic indicator and the fluorescence of the cell population on a separate fluorescence detection channel.

[0007] In some embodiments, if fluorescence of a cell population is detected, at least one cell of the cell population in at least one microwell can be transferred to a target location.

[0008] In some embodiments, a membrane may be applied to a microfabrication device to retain at least one cell loaded in at least one micropore.

[0009] On the other hand, a method for identifying fluorescent cells in a sample is provided. The method utilizes a microfabricated chip having a top surface comprising a plurality of microwells, and the method includes: (a) loading at least one cell of the sample and a fluorescent metabolic indicator into at least one of the plurality of microwells, the fluorescence state of the metabolic indicator indicating the presence or absence of cellular metabolic activity (such as growth and proliferation); (b) culturing the microfabricated chip to grow at least one cell in the at least one microwell into a cell population; and (c) determining the presence of fluorescent cells of interest in the sample by analyzing images of the microfabricated chip to detect fluorescence exhibited by the at least one microwell. In some embodiments, detecting fluorescence exhibited by the at least one microwell includes simultaneously detecting the fluorescence of the metabolic indicator and the fluorescence of the cell population on a separate fluorescence detection channel. Attached Figure Description

[0010] Those skilled in the art will understand that the accompanying drawings are primarily for illustrative purposes and are not intended to limit the scope of the subject matter of the invention described herein. The drawings are not necessarily drawn to scale; in some instances, various aspects of the subject matter disclosed herein may be highlighted or enlarged in the drawings to facilitate understanding of different features. In the drawings, similar reference numerals generally refer to similar features (e.g., elements that are functionally similar and / or structurally similar).

[0011] Figure 1 This is a perspective view of a microfabrication device or chip shown according to some embodiments.

[0012] Figures 2A to 2C These are top, side, and end views of a microfabrication device or chip size, as illustrated in some embodiments.

[0013] Figure 3A and Figure 3B These are exploded views and top views of a microfabrication device or chip, as illustrated in some embodiments.

[0014] Figure 4 This is a scatter plot illustrating, based on some embodiments, the differentiation between presumed GFP (low-red / high-blue-cloud, blue dot (triangle symbol) E. coli and presumed non-GFP (low-red / low-blue-cloud, green dot (square symbol) E. coli).

[0015] Figure 5This is a box-and-whisker diagram showing the fold variability of fluorescence difference between GFP-expressing and GFP-non-GFP-expressing transfected 96-well plates according to some embodiments. Detailed Implementation

[0016] One object of the present invention is to provide a method for identifying fluorescent microorganisms using a high-density cell culture and screening platform.

[0017] In some embodiments, the high-density cell culture platform may be a microfabrication device (or “microfabrication chip”). As used herein, a microfabrication device or chip may define a high-density array of micropores (or experimental units). For example, a microfabricated chip including "high-density" microvias may include approximately 150 microvias per square centimeter to approximately 160,000 or more microvias per square centimeter (e.g., at least 150 microvias per square centimeter, at least 250 microvias per square centimeter, at least 400 microvias per square centimeter, at least 500 microvias per square centimeter, at least 750 microvias per square centimeter, at least 1,000 microvias per square centimeter, at least 2,500 microvias per square centimeter, at least 5,000 microvias per square centimeter, at least 7,500 microvias per square centimeter, at least 10,000 microvias per square centimeter, at least 50,000 microvias per square centimeter, at least 100,000 microvias per square centimeter, or at least 160,000 microvias per square centimeter). The substrate of the microfabricated chip may include approximately 10,000,000 or more microvias or locations. For example, a micropore array may include at least 96 sites, at least 1,000 sites, at least 5,000 sites, at least 10,000 sites, at least 50,000 sites, at least 100,000 sites, at least 500,000 sites, at least 1,000,000 sites, at least 5,000,000 sites, or at least 10,000,000 sites. The micropore array may form a grid pattern and be divided into individual regions or segments. The size of the micropores can range from the nanometer scale (e.g., diameters from about 1 nanometer to about 100 nanometers) to the micrometer scale. For example, the diameter of each micropore may be from about 1 μm to about 800 μm, from about 25 μm to about 500 μm, or from about 30 μm to about 100 μm. The diameter of the micropores can be about 1 μm or less, about 5 μm or less, about 10 μm or less, about 25 μm or less, about 50 μm or less, about 100 μm or less, about 200 μm or less, about 300 μm or less, about 400 μm or less, about 500 μm or less, about 600 μm or less, about 700 μm or less, or about 800 μm or less. In an exemplary embodiment, the diameter of the micropores can be about 100 μm or less, or about 50 μm or less. The depth of the micropores can be from about 25 μm to about 100 μm, for example, about 1 μm, about 5 μm, about 10 μm, about 25 μm, about 50 μm, and about 100 μm. Micropores can also have greater depths, for example, about 200 μm, about 300 μm, about 400 μm, and about 500 μm. The spacing between adjacent micropores can be in the range of about 25 μm to about 500 μm, or about 30 μm to about 100 μm.

[0018] Microfabricated chips may have two main surfaces: a top surface and a bottom surface, with microvias having openings on the top surface. Each microvia may have an opening or cross-section of any shape, such as circular, hexagonal, square, or other shapes. Each microvia may include sidewalls. For microvias whose openings or cross-sections are not circular, the diameter of a microvia as described herein refers to the effective diameter of a circle with an equivalent area. For example, for a square microvia with sides of 10 μm × 10 μm, the diameter of a circle with an equivalent area (100 square μm) is 11.3 μm. Each microvia may include one or more sidewalls. The sidewalls may have straight, sloping, and / or curved cross-sectional profiles. Each microvia includes a bottom, which may be flat, circular, or other shapes. Microfabricated chips (with microvias thereon) may be manufactured from polymers (e.g., cyclic olefin polymers) via precision injection molding or through some other process (such as embossing). Other structural materials, such as silicon and glass, may also be used. The chip may have a substantially flat main surface. Figure 1 A schematic diagram of a microfabricated chip is shown, with its edges generally parallel to the direction of the rows and columns of micro-holes on the chip.

[0019] High-density microwells on microfabricated chips can be used to accommodate samples comprising at least one biological entity (e.g., at least one cell). The term "biological entity" may include, but is not limited to, organisms, cells, cellular components, cell products, and viruses. The term "species" may be used to describe taxonomic units, including but not limited to operational taxonomic units (OTUs), genotypes, germ types, phenotypes, ecotypes, histories, behaviors or interactions, products, variants, and evolutionarily significant units. High-density microwells on microfabricated chips can be used for a variety of experiments, such as the growth, culture, or screening of various species of bacteria and other microorganisms (or microorganisms) such as aerobic, anaerobic, and / or facultative aerobic microorganisms. Microwells can be used for experiments involving eukaryotic cells such as mammalian cells. Furthermore, microwells can be used for various genomics or proteomics experiments and may contain cell products or components, or other chemical or biological substances or entities, such as cell surfaces (e.g., cell membranes or cell walls), metabolites, vitamins, hormones, neurotransmitters, antibodies, amino acids, enzymes, proteins, carbohydrates, ATP, lipids, nucleosides, nucleotides, nucleic acids (e.g., DNA or RNA), chemicals, such as dyes, enzyme substrates, etc.

[0020] In some embodiments, high-density cell culture platforms can be droplet-based; for example, dispersed droplet swarms can be used to hold cells, culture media, and other components for cell culture, replacing the array of pores used as experimental units on a microfabricated chip. Droplet generation methods (especially when used in conjunction with on-chip cell sorter-type instruments) can be used to grow and screen microorganisms in complex environmental samples. Droplets can be generated at hundreds of hertz, meaning millions of droplets can be generated in hours. Simple chip-based devices can be used to generate droplets, and droplets can be designed to contain single cells. Systems for generating droplets containing cell suspensions can contain one cell or a small number of cells. Droplets can be emulsions, dual emulsions, hydrogels, bubbles, and composite particles, etc. For example, aqueous droplets can be suspended in immiscible liquids, keeping them separate from each other and preventing contact with or contamination of any surfaces. Droplet volumes can range from 10 fl to 1 µL, and highly monodisperse droplets can be fabricated with diameters ranging from a few nanometers to 500 µm. Droplet-based microfluidic systems can be used to generate, manipulate, and / or culture small droplets. Cell survival and proliferation can be simulated similarly to control experiments in bulk solution. Fluorescent screening of droplets can be performed on a chip at a rate of, for example, 500 drops per second. Droplets can be merged to form new droplets or reagents can be added to droplets. Droplets can be passed through microchannels arranged in a single row and detected by spectroscopic methods, such as detecting the fluorescence emitted by the droplets using a fluorescence detector. Droplets meeting specific criteria (e.g., emitting fluorescence at a specific wavelength) can be selected by diverting the fluid to branch channels that can converge or collect the droplets. Fluid diversion or switching can be achieved using valves, pumps, the application of external electric fields, etc.

[0021] In various embodiments, the cell can be an archaea, bacteria, or eukaryote (e.g., a fungus). For example, the cell can be a microorganism, such as an aerobic, anaerobic, or facultative aerobic microorganism. A virus can be a bacteriophage. Other cellular components / products may include, but are not limited to, proteins, amino acids, enzymes, carbohydrates, adenosine triphosphate (ATP), lipids, nucleic acids (e.g., DNA and RNA), nucleosides, nucleotides, cell membranes / cell walls, flagella, pili, organelles, metabolites, vitamins, hormones, neurotransmitters, and antibodies.

[0022] To culture cells, nutrients are typically provided. Nutrients can be defined (e.g., culture media with defined chemical compositions or synthetic media) or undefined (e.g., basal media or composite media). Nutrients may include laboratory-prepared media and / or commercially manufactured media (e.g., mixtures of two or more compounds), or components thereof. Nutrients may include liquid nutrient media (i.e., nutrient broths), such as marine broth, lysogenic broths (e.g., Luria broth), or components thereof. Nutrients may include liquid media mixed with agar to form solid media and / or commercially manufactured agar plates, such as blood agar, or components thereof.

[0023] Nutrients may include, or be components of, selective culture media. For example, selective culture media may be used only for the growth of specific biological entities or only for the growth of biological entities with specific characteristics (e.g., antibiotic resistance or synthesis of specific metabolites). Nutrients may include, or be components of, differential culture media to distinguish one type of biological entity from another type or other types of biological entities by using biochemical characteristics in the presence of specific indicators (e.g., neutral red, phenol red, eosin Y, or methylene blue).

[0024] Nutrients may include extracts or culture media derived from the natural environment, or components thereof. For example, nutrients may originate from an environment that is natural for a particular type of biological entity, different environments, or multiple environments. Environments may include, but are not limited to, one or more of the following: biological tissues (e.g., connective tissue, muscle, nerves, epithelium, plant epidermis, blood vessels, ground, etc.), biological fluids or other biological products (e.g., amniotic fluid, bile, blood, cerebrospinal fluid, cerumen, exudate, feces, gastric juice, interstitial fluid, intracellular fluid, lymph, milk, mucus, rumen contents, saliva, sebum, semen, sweat, urine, vaginal secretions, vomit, etc.), microbial suspensions, and air (including, for example, different gas concentrations). Supercritical carbon dioxide, soil (including minerals, organic matter, gases, liquids, organisms, etc.), sediments (e.g., agricultural, marine, etc.), living organic matter (e.g., plants, insects, other small organisms and microorganisms), dead organic matter, feed (e.g., grass, legumes, silage, crop residues, etc.), minerals, oils or oil products (e.g., animal, plant, petrochemical), water (e.g., natural freshwater, drinking water, seawater, etc.) and / or wastewater (e.g., sanitary, commercial, industrial and / or agricultural wastewater and surface runoff).

[0025] Figure 1This is a perspective view of a microfabrication apparatus or chip illustrated according to some embodiments. Chip 100 includes a substrate formed in the form of a microscope slide, having injection-molded feature structures on a top surface 102. These feature structures include four separate arrays (or microarrays) of micropores 104 and injector markings 106. The micropores 113 in each microarray (separated by gap spaces 114) are arranged in a grid pattern, and there are pore-free edges around the edges of chip 100 and between the microarrays 104.

[0026] Figures 2A to 2C These are, respectively, top, side, and end views of the dimensions of chip 100 shown according to some embodiments. Figure 2A In the middle, the top of chip 100 is approximately 25.5mm × 75.5mm. In Figure 2B In the middle, the end of chip 100 is approximately 25.5mm × 0.8mm. In Figure 2C In the middle, the side of chip 100 is approximately 75.5mm × 0.8mm.

[0027] After the sample is loaded onto the microfabrication device, the membrane can be applied to at least a portion of the microfabrication device. Figure 3A According to some embodiments, from Figure 3B The exploded view of the microfabrication device 300 shown in the top view is illustrated in the figure. The device 300 includes a chip having a pore array 302, which contains, for example, soil microorganisms or human microbiota. A membrane 304 is placed on top of the pore array 302. A gasket 306 is placed on top of the membrane 304. A cap 308 having filling holes 310 is placed on top of the gasket 306. Finally, a sealing tape 312 is applied to the cap 308.

[0028] The membrane can cover at least a portion of a microfabrication device comprising one or more experimental units or micropores. For example, after a sample is loaded onto the microfabrication device, at least one membrane can be applied to at least one micropore of a high-density micropore array. Multiple membranes can be applied to multiple portions of the microfabrication device. For example, a single membrane can be applied to a single sub-segment of a high-density micropore array.

[0029] The membrane can be attached, adhered, partially adhered, bonded, sealed, and / or partially sealed to a microfabrication device to retain at least one biological entity in at least one micropore of a high-density micropore array. For example, the membrane can be reversibly bonded to the microfabrication device using lamination. The membrane can be punctured, peeled, separated, partially separated, removed, and / or partially removed to access at least one biological entity in at least one micropore of the high-density micropore array.

[0030] A portion of a cell population from at least one experimental unit, pore, or micropore can be attached to the membrane (e.g., by adsorption). If this is the case, the cell population from at least one experimental unit, pore, or micropore can be sampled by peeling off the membrane, such that a portion of the cell population from at least one experimental unit, pore, or micropore remains attached to the membrane.

[0031] The membrane can be impermeable, semi-permeable, selectively permeable, differentially permeable, and / or partially permeable to allow at least one nutrient to diffuse into at least one micropore of a high-density micropore array. For example, the membrane may comprise natural and / or synthetic materials. The membrane may include a hydrogel layer and / or filter paper. In some embodiments, the membrane is selected to have a sufficiently small pore size to retain at least some or all of the cells within the micropores. For mammalian cells, the pore size may be only a few micrometers, yet still sufficient to retain the cells. However, in some embodiments, the pore size may be less than or equal to about 0.2 μm, such as 0.1 μm. Impermeable membranes have pore sizes close to zero. It should be understood that the membrane may have a complex structure and may or may not have a defined pore size.

[0032] In one aspect, a method is provided for identifying fluorescent cells in a sample using a microfabricated chip having a top surface comprising a plurality of microwells. The method includes: (a) loading at least one cell from the sample into at least one of the plurality of microwells; (b) culturing the microfabricated chip (at a suitable temperature, for a suitable duration, conforming to cell type and growth conditions, etc.) to allow at least one cell in the at least one microwell to grow into a cell population; and (c) determining the presence of fluorescent cells of interest in the sample by analyzing images of the microfabricated chip to detect fluorescence exhibited by the at least one microwell.

[0033] In some embodiments, loading includes loading a plurality of cells from a sample into a plurality of microwells such that at least some of the microwells each contain one cell, and the percentage of microwells containing more than one cell is zero or statistically insignificant. "Statistically significant" means that the probability of any microwell containing more than one cell is less than 0.1%, preferably less than 0.01%, and more preferably 0.001%. This ensures that the cell population grown in a single microwell belongs to a single strain or species, thereby facilitating the recovery and analysis of downstream purified isolates.

[0034] In some embodiments, at least one of the microwells is also loaded with a metabolic indicator, such as resazurite, that indicates the metabolic activity of the cells. This loading can be performed before, simultaneously with, or after cell loading. The metabolic indicator may have fluorescent properties that may be affected by the metabolic activity of the cells, such as cell growth and proliferation. For example, resazurite, a commonly used fluorescent dye, can be used. The fluorescence of resazurite decreases in the environment of living cell growth and changes from blue / purple to pink.

[0035] In some implementations, detecting fluorescence in the microwells includes detecting the fluorescence of metabolic indicators and cell populations on separate fluorescence detection channels. Detection can be performed simultaneously in different channels.

[0036] In this paper, the detection of fluorescence in a cell population indicates the presence of intrinsic fluorescence in the fluorescent cells of interest (naturally occurring fluorescent cells or transgenic cells with fluorescent properties), which naturally increases as the cells grow and multiply in the microwells. In some embodiments, if fluorescence of the cell population contained in the microwells is detected, at least one cell from at least one microwell of the cell population can be transferred to a target location, for example, a 96-well plate containing growth medium for further culture, growth, analysis, etc.

[0037] In some implementations, a membrane is applied after loading and before culturing to hold a cell in at least one micropore.

[0038] The at least one cell may include a cell, a bacterial cell, an archaea cell, or a eukaryotic cell.

[0039] In one example, the screening method described herein utilizes the applicant's Prospector system, a high-throughput isolation, culture, and screening system that uses the microfabricated chip described herein to employ an automated workflow to culture hundreds to thousands of microorganisms in parallel in individual microwells of a composite sample. The system integrates onboard optics capable of emitting excitation wavelengths and capturing fluorescence images in the red (635 nm), green (532 nm), and blue (488 nm) channels. The optics can be used to detect bacteria expressing GFP, which fluoresces when exposed to light in the blue to ultraviolet range.

[0040] Example 1

[0041] A mixed community of *E. coli* ATCC 25922 and *E. coli* ATCC 25922GFP (American Center for Type Culture Collection, Manassas, Virginia, USA) was constructed. ATCC 25922GFP was derived from ATCC 25922 and contained a multi-copy vector encoding GFPmut3. GFPmut3 is present in P... lac Expression is controlled by a promoter, the Plac promoter being a construct designed for fluorescent labeling of Gram-negative bacteria.

[0042] Liquid cultures of two *E. coli* strains were grown separately overnight in LB broth at room temperature. The cultures were then mixed 50 / 50, diluted 1:100,000 in LB broth, and infused with resazurite indicator to a final concentration of 100 µM. This diluted culture was then loaded onto 6109 wells of a microfabrication chip, resulting in an average of less than 0.4 cells per well. The dilution factor and microwell loading technique were adjusted to ensure that at least some wells were loaded with exactly one cell, and that no well was loaded with more than one cell (or that it was unlikely any well would be loaded with more than one cell). The microfabrication chip was incubated at room temperature for 24 hours. Approximately 1600 positives were detected by varying the color of the resazurite on the microfabrication device used in the following analyses, indicating that microbial growth was detected in more than a quarter of the 6109 wells of the microfabrication chip.

[0043] After 24 hours of incubation at room temperature, the microfabricated chip was imaged under each fluorescence channel, and scatter plots comparing blue and red fluorescence were generated. Figure 4 Each point in the scatter plot represents a microwell on the microfabricated chip. Because the resplenium detected in the red channel has been metabolized into halogenated fluorescein, which fluoresces in the green channel, the red fluorescence of all microwells containing microbial growth is weak. Only the microwells containing GFP E. coli show higher blue fluorescence. (In the scatter plot...) Figure 4 Two distinct microbial growth clouds are clearly visible on the image: one is a low-red / high-blue cloud indicating presumed GFP E. coli, and the other is a low-red / low-blue cloud indicating presumed wild-type E. coli. The high-red / low-blue cloud (dots) indicates micropores where no microbial growth is observed.

[0044] Selected microwells were transferred to a 96-well plate containing -LB to verify that the blue and green positive clouds detected by the Prospector's optics indeed represented GFP-expressing *E. coli* and wild-type *E. coli*, respectively. A total of 48 microwells indicating the growth of GFP-expressing microorganisms were randomly selected from the clouds of low red / high blue fluorescence wells. Figure 4(blue dots), and randomly selected a total of 44 microwells from the cloud of low red / low blue fluorescence wells to indicate wild-type microbial growth (i.e., no GFP expression). Figure 4 (Green dot). Four wells of the 96-well plate were retained as negative controls containing only LB.

[0045] After incubation at 30°C for 24 hours, the 96-well transfer plate was measured at 526 nm using a fluorescent plate reader with excitation at 488 nm. The fluorescence difference between wild-type *E. coli* and GFP-expressing *E. coli* was compared relative to the average of the four negative control wells (Table 1). The results showed 100% accuracy in selecting GFP and non-GFP wells for transfer, and the expected target was retrieved each time.

[0046]

[0047] A box-and-whisker diagram of all wells containing GFP E. coli as shown in Table 1 ( Figure 5 The wider fold change range shown in the fluorograms is likely due to the natural variability of GFP expression. These measurements demonstrate that Prospector's imaging system can clearly distinguish wild-type bacteria from GFP-labeled bacteria on microfabricated chips and successfully transfer these isolates to 96-well plates. These results indicate that the disclosed method can screen for GFP-expressing bacteria in live cultures.

[0048] Those skilled in the art will understand that various changes and / or modifications can be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention generally described. Therefore, all aspects of the embodiments of the invention are to be regarded as illustrative rather than restrictive.

Claims

1. A method for identifying fluorescent cells using a microfabricated chip having a top surface comprising a plurality of micropores, the method comprising: (a) Loading at least one cell of a sample into at least one of the plurality of microwells, the microwell being loaded with a metabolic indicator that indicates the metabolic activity of the cell, the metabolic indicator emitting fluorescence, and the fluorescence state of the metabolic indicator indicating the metabolic activity of the cell; (b) Cultivating the microfabricated chip to grow the at least one cell in the at least one microwell into a cell population; and (c) Determining the presence of fluorescent cells of interest in the sample by analyzing images of the microfabricated chip to detect fluorescence exhibited by at least one micropore, the detection including simultaneous detection of the fluorescence of the metabolic indicator and the fluorescence of the cell population on different fluorescence detection channels.

2. The method according to claim 1, wherein, The loading includes loading a plurality of cells of the sample into a plurality of microwells such that at least some of the microwells each contain one and only one cell, while the percentage of microwells containing more than one cell is zero or statistically insignificant.

3. The method according to claim 1, further comprising: If fluorescence of the cell population is detected, at least one cell of the cell population in the at least one microwell is transferred to the target location.

4. The method of claim 1, further comprising applying a membrane to retain the at least one cell in the at least one micropore.

5. The method according to claim 1, wherein, The at least one cell includes at least one of cells, bacterial cells, archaea cells, and eukaryotic cells.

6. A method for identifying fluorescent cells in a sample using a microfabricated chip having a top surface comprising a plurality of micropores, the method comprising: (a) Loading at least one cell of the sample and a fluorescent metabolic indicator into at least one of the plurality of microwells, wherein the fluorescence state of the metabolic indicator indicates the presence or absence of cellular metabolic activity; (b) Cultivating the microfabricated chip to grow the at least one cell in the at least one micropore into a cell population; (c) Determine the presence of fluorescent cells of interest in the sample by analyzing images of the microfabricated chip to detect fluorescence displayed by at least one microwell, wherein detecting fluorescence displayed by at least one microwell includes simultaneously detecting the fluorescence of the metabolic indicator and the fluorescence of the cell population on different fluorescence detection channels.

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