Patent Publication Number: US-2022228202-A1

Title: Positive pressure driven flow for multiplexed fluorescence in situ hybridization imaging system

Description:
TECHNICAL FIELD 
     This specification relates to a driving flow through a flow cell in a fluorescence in-situ hybridization imaging system. 
     BACKGROUND 
     It is of great interest to the biotech community and pharmaceutical industry to develop methods for visualizing and quantifying multiple biological analytes—e.g., DNA, RNA, and protein—within a biological sample—e.g., tissue resection, biopsy, cells grown in culture. Scientists use such methods to diagnose/monitor disease, validate biomarkers, and investigate treatment. To date, exemplary methods include multiplex imaging of antibodies or oligonucleotides (e.g., RNA or DNA) labeled with a functional domain to a biological sample. 
     Multiplexed fluorescence in-situ hybridization (mFISH) imaging is a powerful technique to determine gene expression in spatial transcriptomics. In brief, a sample is exposed to multiple oligonucleotide probes that target RNA of interest. These probes have different labeling schemes that will allow one to distinguish different RNA species when the complementary, fluorescent labeled probes are introduced to the sample. Then the sequential rounds of fluorescence images are acquired with exposure to excitation light of different wavelengths. 
     Exposing the sample generally includes flowing one or more solutions through a flow cell containing a sample. Traditional methods include the use of downstream rotary peristaltic pumps to generate negative pressure and draw reagents into and through the flow cell. 
     SUMMARY 
     In one aspect, a fluorescent in-situ hybridization imaging system, including a flow cell to contain a sample to be exposed to fluorescent probes in a reagent; a plurality of reagent reservoirs, each reagent reservoir including a container to hold a liquid reagent; a valve system to control flow from one of a plurality of reagent reservoirs to the flow cell; a pressure source coupled to each of the plurality of reagent reservoirs to apply a positive pressure to liquid reagent in the container and urge the liquid reagent to flow toward the flow cell; and a fluorescence microscope including a variable frequency excitation light source and a camera positioned to receive fluorescently emitted light from the sample. 
     In some embodiments, each reagent reservoir of the plurality of reagent reservoirs further includes a sealing lid, including an inlet configured to receive pressurized gas from the pressure source above an upper surface of the liquid reagent, and an outlet configured to permit flow of the liquid reagent out of the reagent reservoir from below the upper surface of the liquid reagent. The inlet is further configured to detachably connect to the sealing lid, and the outlet is further configured to detachably connect to the sealing lid. The pressure source includes a gas pump for delivering pressurized gas to and a pressure regulator for regulating the pressurized gas. The valve system includes a plurality of valves, each valve of the plurality of valves corresponding to one reagent reservoir of the plurality of reagent reservoirs. 
     The system further includes a first solenoid valve (e.g., 3/2 valve) in fluid communication between the valve system and the flow cell. The system further includes a second solenoid valve in fluid communication with the flow cell. The system further includes a controller to control the valve system, configured to synchronize movement of the valve system such that only one liquid reagent flows to the flow cell at a time. The system further includes a manifold, the manifold including a plurality of outlet ports, each outlet port being in fluid communication with one reagent reservoir. The liquid reagent includes oligonucleotide probes. The liquid reagent includes a buffer. The liquid reagent includes a purge fluid, an imaging buffer, or a bleach buffer. 
     In another aspect, a method for using positive pressure in a fluorescent in-situ hybridization imaging system, the method including applying a positive pressure to a plurality of liquid reagents in a plurality of reagent reservoirs; selecting a first liquid reagent from the plurality of liquid reagents; supplying the first liquid reagent as driven by the positive pressure to a flow cell containing a sample; and obtaining a first fluorescence microscope image of the sample. 
     In some embodiments, the first liquid reagent includes oligonucleotide probes having a first nucleotide sequence. The method further includes selecting a second liquid reagent from the plurality of liquid reagents; and supplying the second liquid reagent as driven by the positive pressure to a flow cell containing a sample. The second liquid reagent includes a purge fluid or a photobleaching buffer. The method further includes selecting a third liquid reagent from the plurality of liquid reagents; supplying the third liquid reagent as driven by the positive pressure to a flow cell containing a sample; and obtaining a second fluorescence microscope image of the sample. The third reagent includes oligonucleotide probes having a third nucleotide sequence. The second liquid reagent includes a purge fluid, a washing bugger, an imaging buffer, or a photobleaching buffer. Applying the positive pressure includes applying gas pressure to the plurality of liquid reagents. Advantages of implementations can include, but are not limited to, one or more of the following. 
     Using positive pressure fluid displacement systems in conjunction with a flow cell for fluorescence in-situ hybridization imaging systems can prevent a number of standing issues. Positive pressure displacement systems distribute gas pressure equally to downstream components. Gas lines downstream of the pressure regulator can be of flexible length and design, allowing for additional regulating manifolds and the capability of multiple flow cells for imaging samples. 
     Constant pressure applied to headspace within reagent tubes allows for constant flow rates downstream in the liquid circuitry. As the liquid reagent travels to the flow cell, constant pressure reduces turbulent flow as the reagent is introduced to the sample. The reduced turbulence increases the likelihood of the sample maintaining position within the flow cell, and reduces the need for the imaging system or technician to reacquire a new position or z-height of the sample, thereby yielding higher image acquisition rates and reducing system reagent use by reducing the number of failed FOV images. 
     Positive pressure displacement of liquids also produces smoother flow at low flow rates. This is particularly useful for sensitive or small samples that could be driven from their spatial positions by higher flow rates through the flow cells. Because the positive pressure equally distributes across the available headspaces in near-real time, multiple reagents can be delivered to the sample simultaneously, rather than sequentially. 
     The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram of an apparatus for multiplexed fluorescence in-situ hybridization imaging using positive pressure displacement. 
         FIG. 2  is a schematic diagram of a positive pressure displacement system including a flow cell. 
         FIG. 3  is a schematic cross-sectional diagram of a positive pressure displacement system displacing reagents from a tube. 
         FIG. 4  is a schematic diagram of a positive pressure displacement system including two flow cells. 
         FIG. 5  is a flow chart of a method of mFISH imaging. 
     
    
    
     Like reference numbers and designations in the various drawings indicate like elements. 
     DETAILED DESCRIPTION 
     Multiplex fluorescent in-situ hybridization imaging (mFISH) systems generally include a peristaltic pump downstream of the imaging flow cell to produce negative pressures, thereby drawing liquids from the flow cell and upstream components and driving waste into a disposal container. However, negative pressure flow cells tend to generate turbulent flow of liquids, e.g., reagents, buffs, or washes, through the flow cell. This can cause turbulent motion of the sample within the flow cell, changing the position and elevation of the sample while being imaged. 
     In particular, peristaltic pumps can generate an uneven flow rate, termed pulsation. This pulsation causes turbulent flow through sensitive components, such as flow cells. Pulsation through a fluidic circuit can be affected by line length and flow rate. 
     The pulsation of peristaltic flow can also cause imaging issues for a sensitive imaging system capable of changes in focal plane and FOV lateral resolution measured in microns. The uneven flow through the flow cell can cause sensitive or small samples to be moved from their previously imaged position, resulting in reacquiring images at different focal planes, making registration between images taken at different times more difficult, or loss of imaging data. 
     The use of negative pressure downstream of a flow cell also requires that fluid tube lengths be highly uniform to maintain the pressure from the peristaltic pump, through the flow cell, the selector, reagent manifold, and individual reagent tubes. Negative pressure systems can also induce cavitation, e.g., bubble formation, in solutions with large volumes of dissolved gases in fluids being transported through the liquid circuitry. Entrained bubbles in the fluid circuitry also lead to imaging problems if the bubbles travel through the flow cell in between rounds of imaging. 
     In contrast, a positive pressure can provide more precise control over pressure at each gas and liquid junction in an mFISH imaging system. Because the gas pressure can be evenly distributed from the gas manifold, the flow rate from each reagent tube can be more finely controlled without pressure fluctuations that peristaltic pumps introduce. The flexibility of positive gas pressure distribution also allows for simplified addition of further flow cells and necessary components. 
     Changes in gas pressure can occur at shorter time scales than liquid pressure, and the headspace in reagent tubes can provide a buffer, reducing pressure fluctuations in the downstream liquid components. This reduces turbulence in the flow cell and reduces the chance a sample can be moved from the imaging position. 
     Referring to  FIG. 1 , a multiplexed fluorescent in-situ hybridization (mFISH) imaging system  100  includes a flow cell  102  to hold a sample  10 , a fluorescence microscope  120  to obtain images of the sample  10 , and a control system  140  to control operation of the various components of the mFISH imaging system  100 . The control system  140  can include a computer  142 , e.g., having a memory, processor, etc., that executes control software. 
     The fluorescence microscope  120  includes an excitation light source  122  that can generate excitation light  130  of multiple different wavelengths. In particular, the excitation light source  122  can generate narrow-bandwidth light beams having different wavelengths at different times. For example, the excitation light source  122  can be provided by a multi-wavelength continuous wave laser system, e.g., multiple laser modules  122   a  that can be independently activated to generate laser beams of different wavelengths. Output from the laser modules  122   a  can be multiplexed into a common light beam path. 
     The fluorescence microscope  120  includes a microscope body  124  that includes the various optical components to direct the excitation light from the light source  122  to the flow cell  102 . For example, excitation light from the light source  122  can be coupled into a multimode fiber, refocused and expanded by a set of lenses, then directed into the sample by a core imaging component, such as a high numerical aperture (NA) objective lens  136 . When the excitation channel needs to be switched, one of the multiple laser modules  122   a  can be deactivated and another laser module  122   a  can be activated, with synchronization among the devices accomplished by one or more microcontrollers  144 ,  146 . 
     The objective lens  136 , or the entire microscope body  124 , can be installed on a vertically movable mount coupled to a Z-drive actuator. Adjustment of the Z-position, e.g., by a microcontroller  146  controlling the Z-drive actuator, can enable fine tuning of focal position. Alternatively, or in addition, the flow cell  102  (or a stage  118  supporting the sample in the flow cell  102 ) could be vertically movable by a Z-drive actuator  118   b , e.g., an axial piezo stage. Such a piezo stage can permit precise and swift multi-plane image acquisition. 
     The sample  10  to be imaged is positioned in the flow cell  102 . The flow cell  102  can be a chamber with cross-sectional area (parallel to the object or image plane of the microscope) with an area of about 2 cm by 2 cm. The sample  10  can be supported on a stage  118  within the flow cell, and the stage (or the entire flow cell) can be laterally movable, e.g., by a pair of linear actuators  118   a  to permit XY motion. This permits acquisition of images of the sample  10  in different laterally offset fields of view (FOVs). Alternatively, the microscope body  124  could be carried on a laterally movable stage. 
     A positive pressure reagent delivery system  104  connects to an entrance to the flow cell  102 . Broadly, the reagent delivery system  104  includes a pressure management system, directing pressurized gas to the headspace of a number of reservoirs  112 , at least some of which are reagent reservoirs. The pressurized gas drives liquid, e.g., a liquid reagent, e.g., from reservoir  112   a , to a valve system  114  and further to the flow cell  102 . The positive pressure reagent delivery system  104  and included components are explained in more detail with regard to  FIG. 2 . 
       FIG. 2  shows an example positive pressure reagent delivery system  104  including gas flow subsystem  202  to create and distribute the gas pressure to the headspace of the reservoirs  112 , and liquid flow subsystem  204  for supplying selected liquids to the flow cell  102  and directing spent solutions to the chemical waste management system  119 . 
     The gas flow subsystem  202  of the reagent delivery system  104  includes a pressure source for supplying gas pressure to the downstream components.  FIG. 2  depicts the pressure source as a gas pump  106  in fluid communication with a gas supply  105 , e.g., and a pressure controller  108 . The gas supply  105  can be a source of purified gas, e.g., substantially particle free gas, such as a tank or an industrial gas supply line, or the gas supply can simply be surrounding atmosphere. The gas can be air, nitrogen, argon, or other molecular inert gas or mixture thereof. 
     The gas pump  106  is a pneumatic pump that is compatible with the imaging environment. Because the pressurized gas will be in contact with sensitive reagent solutions, a pump that generates relatively little contamination, such as an oil-free compressor pump, can be used as the gas pump  106 . The gas pump  106  intakes gas from the gas supply  105 , compresses the gas, and supplies gas (e.g., air) compressed to above local atmospheric pressure. For example, the gas supplied to the gas circuitry can be 1 psi above local atmospheric pressure or more (e.g., 1 psi, 1.5 psi, 2 psi, 2.5 psi or more). The pressure controller  108  intakes the compressed gas and outputs the gas at a regulated pressure to the manifold  110 . Examples of pressure controller  108  include a pressure reducing regulator (e.g., reduces the input pressure of the gas to a desired value at its output) or a back-pressure regulator (e.g., maintains the set pressure at its inlet side by opening to allow flow when the input pressure exceeds the set value). In some implementations, the pressure source can be a container (e.g., a tank) of compressed gas. 
     In some implementations, the compressed gas can be filtered by a filter  109 , for example, to remove particulates, aerosolized liquids, or biological agents to prevent contamination of the liquid reagents in the reservoirs  112 . The filtration can occur before being compressed, between the gas pump  106  and the pressure controller  108 , or following the pressure controller  108 . 
     The pressure controller  108  outputs the compressed gas at a regulated pressure to a gas manifold  110 . The gas manifold  110  includes an inlet port to accept the pressurized gas, and fluidly connects the inlet port to one or more outlet ports, e.g., multiple outlet ports, thereby equally distributing the compressed gas to each outlet port. For example, if pressure controller  108  supplies compressed gas at 1 psi to the input of manifold  110 , each output will supply compressed gas at 1 psi to connected components. 
     Each outlet port of the manifold  110  is in fluid connection with the headspace of one corresponding reservoir  112  through a gas line  111 . In general, the gas manifold  110  includes at least as many outlet ports as there are reservoirs  112 . In some implementations, gas manifold  110  can have 5 or more outlet ports (e.g., 8 or more, 10 or more, 12 or more, 15 or more, or 20 or more). 
       FIG. 3  shows an example reservoir  112   a  and the corresponding connections to the gas flow subsystem  202  and liquid flow subsystem  204 . The reservoir  112   a  includes a lid  302 , a vessel  304 , a first conduit  306  (which provides the gas line  111 ), and a second conduit  308  (which provides a liquid line  113 ). The reagent vessel  304  is a receptacle for containing an amount of liquid  310 , e.g., a reagent. The remaining volume of the vessel  304  above the surface of the liquid  310  is termed “headspace”  312 . The first conduit  306  has an opening positioned to be in the headspace  312  during operation of the reagent delivery system  104 , i.e., when the reservoir is partially filled with liquid. In contrast, the second conduit  308  has an opening positioned to be below the surface of liquid  310  during operation. Although the implementation in  FIG. 3  illustrates the first and second conduits  306 ,  308  extending through the lid  302 , the conduits could instead open directly into sidewalls of the vessel  304 . 
     The first conduit  306  can be provided by a material capable of maintaining the regulated gas pressure from the manifold  110 , e.g., flexible or rigid plastic tubing, glass or metal piping, etc. Flexible materials such as plastic, rubber, or silicone provide possible advantages in reducing pressure fluctuations in the gas being distributed. The second conduit  308  can be composed of similar materials to the first conduit  306 . Examples of second conduits  308  include high pressure liquid chromatography tubing composed of ethylene tetrafluoroethylene (ETFE) or poly ether ether ketone (PEEK). The first and second conduits  306 ,  308  can have equal cross-sectional area, or have different cross-sectional areas. 
     An amount of liquid can be delivered into the vessel  304 , e.g., manually by an operator or automatically through another unillustrated delivery line, so that the headspace includes 5% or more of the total interior volume of the reservoir (e.g., 5% or more, 10% or more, 20% or more, or 30% or more). The example vessel  304  of  FIG. 3  is cylindrical with a hemispherical bottom (e.g., a test tube), though in some implementations, the vessel  304  can be a different shape (e.g., a bottle). Examples of the vessel  304  can include glass or polymer vessels, and the vessel  304  can have an interior volume sufficient to contain 1 mL or more (e.g., 2 mL or more, 5 mL or more, 10 mL or more, 20 mL or more, or 50 mL or more). 
     The reservoir lid  302  includes an upper surface, a side surface extending from the rim of the upper surface, and two ports on the upper surface. The reservoir lid  302  detachably secures to the opening of vessel  304  to form a gas-tight seal (e.g., a sealing lid). The ports can be cylindrical in shape and provide access from the outer surface of the seal to the interior volume of the reagent vessel  304  when the reservoir lid  302  is secured to the vessel  304 . In some implementations, the ports include gaskets to form gas-tight seals with objects traversing the port. 
     The first conduit  306  delivers pressurized gas from the manifold  110  to the headspace  312  of the reservoir  112   a , thereby increasing the gas pressure within the vessel  304 . The increased gas pressure of the headspace  312  exerts a downward force equally distributed across the upper surface of the liquid  310 . 
     At least one reservoir holds a liquid reagent. In particular, different reservoirs can hold different reagents. Each different liquid reagent includes a different set of one or more different types of oligonucleotide readout probes. Each different type of readout probe targets a different nucleotide sequence on a different encoding probe (and thus targets a different RNA sequence), and/or has a different set of one or more fluorescent materials, e.g., phosphors, that are excited by different combinations of wavelengths. 
     One or more of the reservoirs  112  can contain other liquids for delivery to the flow cell  102 , such as a purge fluid (e.g., DI water), or a buffer (e.g., a wash buffer, an imaging buffer, or a bleaching buffer), instead of a reagent. 
     The second conduit  308  extends below the upper surface of the liquid  310 , for example, near to the bottom of the vessel  304 . The increased gas pressure of the headspace  312  drives the liquid  310  into the open end of second conduit  308  and through second conduit  308  to downstream liquid flow subsystem  204 . 
     Referring again to  FIG. 2 , the pressure controller  108  maintains the gas pressure within the manifold  110  which distributes the compressed gas to the headspace  312  of each connected reservoir  112 . The liquid flow subsystem  204  of the reagent delivery system  104  includes the liquid lines  113  through which liquid  310  is driven from the reservoirs  112 , and a valve system  114  for selecting one or more liquids to deliver to the flow cell  102 . The liquid flow subsystem  204  can also include a first valve  116 , e.g., a solenoid valve, to control flow of the liquid into the flow cell  102 , and a second valve  117 , e.g., a solenoid valve, to control flow of liquid out of the flow cell  102 . 
     Liquid  310  from reservoirs  112  is driven through the second liquid lines  113 , which terminate at the valve system  114 . The valve system  114  is a selector valve, e.g., a rotary valve, including at least as many input ports as reservoirs  112  of the reagent delivery system  104 , and a single output port. Each second conduit  308  extending from a corresponding reservoir  112  is in fluid connection with a single input port of the valve system  114 . The valve system  114  connects a single input port corresponding to a single reservoir  112  to the output port, supplying a single liquid  310  to downstream components. The control system  140  controls the valve system  114  to switch between reservoirs  112  thereby selecting which liquid  310  is supplied to the solenoid valve  116 . 
     The first valve  116  can be a selector valve with one inlet port and two outlet ports, e.g., a 3/2 valve. The first valve  116  operates to fluidly connect the inlet port with a single outlet port. The control system  140  controls which outlet port is connected. When the inlet port is in fluid connection with a first outlet port, there is no fluid connection to the second outlet port, and vice versa. One outlet is in fluid connection with the flow cell  102  and the other outlet port is in fluid connection with the chemical waste management system  119 . When the first valve  116  connects the valve system  114  to the flow cell  102 , the selected liquid  310  can flow to the flow cell  102 . Any volume of liquid  310  remaining in the first valve  116  can be purged when the flow cell  102  is connected to the chemical waste management system  119 . This additionally can prevent backflow from flow cell  102  entering the first valve  116 . 
     The flow cell  102  receives the liquid  310  supplied from the first valve  116 . The flow cell  102  also has an outlet in fluid connection with a second valve  117 , e.g., a 2/2 valve, also controlled by the control system  140  to control flow of liquid, e.g., the reagent or purge fluid, through the flow cell  102 . When the valves  116 ,  117  are operated by the control system  140  in unison, liquid flows from first valve  116 , through the flow cell  102 , and through the solenoid valve  117 . In this manner, a selected liquid  310  is supplied to the flow cell  102  and used solution is displaced and directed to the chemical waste management system  119 . In contrast, both valves  116 ,  117  can be closed to seal the flow cell  102 . 
     Referring now to  FIG. 4 , in some implementations, the reagent delivery system  104  can provide liquid  310  to multiple flow cells  402 . The example system of  FIG. 4  shows gas circuitry  400  including an gas pump  406  in fluid connection with a pressure controller  408  fluidly connected to two manifold  410   a ,  410   b . In some implementations, the pressure controller  408  delivers equal gas pressure to manifolds  410   a ,  410   b.    
     The manifolds  410   a ,  410   b  connect to a number of reservoirs  412 , with manifold  410   a  connecting to the headspace  312  of reservoirs  412   a - c  and manifold  410   b  connecting to the headspace  312  of reservoirs  412   d - f.    
     Valve system  414   a  receives liquid  310   a - c  from reservoirs  412   a - c , whereas valve system  414   b  receives liquid  310   d - f  from reservoirs  412   d - f . Each valve system  414   a ,  414   b  is controlled by control system  140  to deliver one liquid  310  to respective first solenoid valves  416   a ,  416   b . As described above, the first solenoid valves  416   a ,  416   b  direct, upon control by the control system  140 , the selected liquid  310  to the respective flow cell  402   a ,  402   b  or to the chemical waste management system  419 . Assuming liquid is directed to a flow cell, liquid that is displaced from the flow cells  402   a ,  402   b  is driven to the second solenoid valves  417   a ,  417   b , and on to the chemical waste management system  419 . 
     Returning to  FIG. 1 , in operation, the control system  140  causes the light source  122  to emit the excitation light  130 , which causes emission of light from fluorophores in the sample  10 , e.g., of fluorophores in the probes that are bound to RNA in the sample and that are excited by the wavelength of the excitation light. The emitted light  132 , as well as back propagating excitation light, e.g., excitation light scattered from the sample, stage, etc., are collected by an objective lens  136  of the microscope body  124 . 
     The collected light can be filtered by a multi-band dichroic mirror  138  in the microscope body  124  to separate the emitted fluorescent light from the back propagating illumination light, and the emitted light is passed to a camera  134 . The multi-band dichroic mirror  138  can include a pass band for each emission wavelength expected from the probes under the variety of excitation wavelengths. Use of a single multi-band dichroic mirror (as compared to multiple dichroic mirrors or a movable dichroic mirror) can provide improved system stability. 
     When triggered by a signal, e.g., from a microcontroller, image data from the camera can be captured, e.g., sent to an image processing system  150 . Thus, the camera  134  can collect a sequence of images from the sample. The sequence of images can provide the visualization and can be analyzed to quantify a biological analyte, e.g., the expression of a gene. 
     To provide context, the conventional mFISH traditional round of mFISH imaging and genetic identification relies on a series of nested steps including hybridization, imaging, and photobleaching.  FIG. 5  demonstrates the workflow for a round of mFISH. Prior to mFISH imaging, encoding probes are added to a biological sample containing sequences to be targeted, e.g., by flowing the reagent from a selected reagent reservoir to the flow cell. The target nucleotide sequences are bound with a library of encoding probes, each encoding probe containing an encoding sequence that binds to a specific targeting sequence, and a hybridization region at each end of the encoding sequence. The hybridization regions are designed to bind the targeting sequences present in the set of readout probes, but not to the sequences of the sample. 
     The first round of hybridization of the readout probes ( 502 ) begins with the valve system  114  supplying a reagent containing readout probes to the flow cell  102  containing the sample  10 . As described above, each readout probe includes a fluorophore coupled to an oligonucleotide targeting sequence designed to bind to one of the hybridization regions of the encoding probes. In practice, there can be multiple groups of readout probes, with readout probes within a group having the same oligonucleotide targeting sequence and the same fluorophore, but readout probes of different groups having oligonucleotide targeting sequences and different fluorophores that emit at different wavelengths. The total number of groups of readout probes can be equal to or less than the number of color channels the system is capable of imaging. For example, a control system  140  with a light source  122  with four laser modules can excite a set of four unique fluorophores in a sequence of four rounds of excitation and imaging. 
     The system performs an incubation step allowing the set of readout probes to penetrate the sample and hybridize with the encoding probes. The system then supplies one or more buffers from one or more selected reservoirs to the flow cell  102  via the valve system  114  to prepare the sample for imaging. The buffers can include a wash buffer which can include reagents to displace unbound and excessive components which may interfere with the assay, such as an astringent reagent (e.g., formamide). The buffers can further include a hybridization buffer to control stringency and eliminate residual fluorescent material or autofluorescence of the sample. The buffers can further include an imaging buffer to prepare the sample and probes for imaging, such as performing oxygen scavenging (e.g., glucose oxidase). 
     The system then performs an imaging step ( 504 ). The light source  122  consecutively excites the fluorophores of the set of readout probes localized within a selected FOV while the filter wheel  128  allows for the collection of the emitted fluorescence to form a fluorescent image. In some implementations, the imaging system may be configured, e.g., with a color camera, to image multiple fluorophores of different emission wavelengths simultaneously. This captures the lateral and vertical position of the readout probes hybridized in the preceding step. 
     The fluorophore of the hybridized readout probes are then photobleached ( 506 ). This begins by the valve system  114  supplying a volume of bleaching buffer to the flow cell  102  to displace and purge the imaging buffer. The photobleaching includes bathing the sample  10  in the flow cell  102  with high intensity light to photochemically render the readout probes hybridized within the sample fluorophores permanently unable to fluoresce. The light source is chosen to correspond to the fluorophores used in combination with the readout probes. 
     The process can then be repeated ( 507 ) with additional rounds of hybridization, buffer washes, imaging, and photobleaching. For example, an mFISH experiment can include between 4 and 20 rounds of hybridization and mFISH imaging with unique readout probes used in each round. Each round of hybridization can use a reagent from a different reservoir. 
     While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any invention or on the scope of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially be claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination. 
     Similarly, while operations are depicted in the drawings and recited in the claims in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In certain circumstances, multitasking and parallel processing may be advantageous. Moreover, the separation of various system modules and components in the embodiments described above should not be understood as requiring such separation in all embodiments, and it should be understood that the described program components and systems can generally be integrated together in a single software product or packaged into multiple software products. 
     Particular embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims. For example, the actions recited in the claims can be performed in a different order and still achieve desirable results. As one example, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In some cases, multitasking and parallel processing may be advantageous.