A general-purpose multi-detection system for microplates using confocal imaging.

Abstract

An apparatus for optically analyzing a sample can include an imaging subsystem that images the sample, and one or more analytical subsystems that analyze the sample, including a confocal imaging subsystem, a temperature control subsystem that controls the temperature of an atmosphere within the apparatus, a gas control subsystem that controls the composition of the atmosphere within the apparatus, and a control module that controls the various subsystems of the apparatus.

Description

【Technical Field】 【0001】 Devices and methods consistent with embodiments of the present disclosure provide a microplate-based detection system that offers multiple detection modes, including the detection of fluorescence, chemiluminescence, and absorbance of samples disposed within microwells, as well as imaging of the contents of microplate wells at the cellular and sub-cellular levels using wide-field and confocal microscopy. 【0002】 [Cross-reference to Related Applications] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 120,605, filed Dec. 2, 2020, the entire contents of which are incorporated herein by reference. 【Background Art】 【0003】 To evaluate sample specimens placed within containers of various shapes and sizes, various different analytical instruments can be employed in a laboratory. Conventionally, the use of a microplate format system has become widespread because it is suitable for testing many samples on a single-matrix receptacle. 【0004】 FIG. 1 is a diagram showing a conventional 96-well plate 1 including 96 circular wells 2 arranged in a matrix. FIG. 2 is a diagram showing a conventional 384-well plate 3 including 384 square wells 4. Depending on the configuration, there may be even higher-density wells, such as wells including 1536 wells while the overall dimensional sizes (height, width, etc.) of the microplate arranged in a matrix are the same. 【0005】 Microplate-based detection methods have progressed from simple absorbance to fluorescence and then to chemiluminescence. Other applications of microplate-based systems include fluid injectors and incubators for processing and analyzing microplates at controlled temperatures, which is necessary for kinetic assays where temperatures close to human body temperature are maintained for extended periods. Similarly, atmosphere control systems that regulate the gas environment surrounding microplates have been developed to enable long-term studies of living cells placed within microplates. 【0006】 Single-function, specialized instruments have evolved into multi-detection instruments that combine several detection modalities within a single instrument. Early multi-detection instruments were filter-based, with several commonly used analytical wavelengths for selection. However, next-generation multi-detection instruments have further incorporated monochromators to allow researchers to select wavelength bands from a wide spectrum that can be provided in a single instrument, typically within the 200nm–1000nm wavelength range. Thus, for example, the early monochromator-based instrument described in Patent Document 1 combined a single-grid monochromator with a bandpass filter. However, while advantageous in terms of flexible selection of arbitrary wavelengths, the purity of the selected spectral band in such units did not match that available from research-level spectroscopic measurements. 【0007】 Subsequent monochromator-based multi-detection instruments, as described in Patent Document 2, for example, have been based on double monochromators where the purity of light was sufficient to reach detection limits comparable to those of filter-based instruments. In these, two grids in series can be deployed in an excitation double monochromator, and two grids in series can be deployed in an emission double monochromator. In addition to wavelength selection, depending on the assay being analyzed, these instruments also allow spectral scanning of the sample for both absorbance and fluorescence, thus greatly increasing the usefulness of a single instrument used in modern laboratories. 【0008】 Because the market was divided into two categories—high-sensitivity filter-based instruments useful for the high-throughput screening (HTS) market and monochromator-based instruments primarily used in research—combination instruments were developed that provided both filter-based and monochromator-based readings within a single instrument, as described, for example, in Patent Documents 3 and 4. 【0009】 Hybrid filter-based and monochromator-based detection instruments addressed the dichotomy between filters and monochromators, but such instruments were primarily used in homogeneous assays where the number of samples processed per hour was critical. The research area in which systems of this design were deployed is known as high-throughput screening (HTS), for rapidly processing large volumes of samples in microplate wells using a small number of parameters recorded per sample. The speed is optimized to perform the analysis as quickly as possible to find wells exhibiting some unique characteristics, and then focus on "hit" wells to ensure further investigation. Such subsequent investigations are typically performed by a separate line of more detailed analytical instruments. 【0010】 Typically, most assays were primarily biochemistry-based, and HTS techniques were the preferred method for drug discovery research and analysis. As the cost of drug discovery increased, the need for more biologically relevant models drove the growth of two-dimensional (2D) cell-based assays using a single monolayer of cells. Researchers recognized the limitations of homogeneous assays and wanted to study the behavior of individual cells. Therefore, they redistributed cells from conventional experimental vessels such as petri dishes to microplates. As a result, they were able to dramatically increase the number of parameters for analysis. 【0011】 First, the wells of the microplate were imaged using a conventional microscope, and later imaged using a dedicated imaging device. Due to the enormous amount of information available even from a single image, imaging of cells in a microplate can be known as high-content screening (HCS), as described, for example, in Patent Document 5. 【0012】 HTS non-imaging instrumentation and HCS imaging instrumentation are sold in parallel on the market. Systems that leverage the capabilities of both types have been developed, allowing researchers to access non-imaging analysis modalities such as fluorescence, absorbance, and chemiluminescence, as well as microwell imaging at the cellular level, within the same instrument, as described, for example, in Patent Documents 6 and 7. These instruments perform wide-field microscopy in which the entire field of view of each microscope objective lens is illuminated and imaged. This type of analysis is suitable for studying monolayers of cells. 【0013】 Figure 3 shows cells seeded in the wells of a microplate. Cells 42 seeded at the bottom of well 41 of the microplate grow, for example, in an environment where the atmosphere and humidity are controlled. There, the growing cells 42 proliferate and spread to cover the bottom of well 41. The depth of the cell layer can be limited to single cells, and clear, highly informative images of the cells can be obtained through wide-field imaging and microscopy. 【0014】 Recently, researchers have recognized that while adherent cells in microwells, appearing as two-dimensional (2D) layers, are very useful, they have limitations in accurately representing biological tissues. This is because cells in biological tissues eventually grow three-dimensionally. Therefore, three-dimensional (3D) cell culture will be the next step in cell research. 【0015】 3D cell culture is an artificially created environment in which biological cells are induced to grow and interact with their surroundings in three dimensions. This can closely mimic the actual growth of cells in vivo. 【0016】 Three-dimensional cell cultures are called spheroids. Spheroids can also be grown in the wells of microplates. Pharmaceutical studies of cells in spheroids grown in microwells aim to more accurately reproduce in vivo cell behavior. For example, for the purpose of drug toxicity screening, testing the gene expression of in vitro cells grown in three dimensions is more useful than testing in two dimensions because gene expression in 3D spheroids more closely resembles gene expression in vivo. Furthermore, 3D cell cultures have higher stability and a longer lifespan than cells in 2D cultures, making them more suitable for long-term studies and for demonstrating the long-term effects of drugs. 【0017】 Figure 4 is a schematic diagram showing 3D cells in a microwell. As shown in Figure 4, the 3D clusters of individual cells 62 forming the spheroid 63 are located at the bottom of the microwell 61. For example, 3D assays may include spheroids, tumoroids, organoids, Matrigel, Drosophila, zebrafish, and 3D-printed biomaterial scaffolds for cell growth. In each of these cases, wide-field fluorescence imaging has limitations as an imaging detection method due to illuminating the entire sample, and as the sample thickness increases, it introduces heavy background "noise" that reduces the ability to obtain sufficiently high-resolution images. [Prior art documents] [Patent Documents] 【0018】 [Patent Document 1] U.S. Patent No. 6,313,471 [Patent Document 2] U.S. Patent No. 6,654,119 [Patent Document 3] U.S. Patent No. 7,782,454 [Patent Document 4] U.S. Patent No. 8,218,141 [Patent Document 5] U.S. Patent No. 5,989,835 [Patent Document 6] U.S. Patent No. 9,557,217 [Patent Document 7] U.S. Patent No. 10,072,982 [Overview of the Initiative] [Problems that the invention aims to solve] 【0019】 Therefore, further methods for screening 3D spheroids would be desirable. 【0020】 Cell-based assays, especially live cell assays, are becoming more common in the field of life science research. Microplates are increasingly used as containers for investigating the cell growth process by qualitative and quantitative means. In many cases, work with cells is performed by researchers using multiple dedicated instruments. 【0021】 Fluorescence readings using an instrument with a light beam diameter large enough to obtain representative measurements of the total well fluorescence, or a beam size large enough to perform area scanning and mapping of the signal across the well, can be achieved using a dedicated conventional fluorescence reader or a multi-detection reader. Most instruments offer plate incubation, fluid injection, and the option of gas control (CO2 and / or O2 (i.e., CO2 or O2, or both)) similar to a tissue culture incubator. 【0022】 Much more information can be obtained from cells using wide-field imaging modalities than simply the fluorescence signal level of the wells. Laboratory microscopes with brightfield and phase contrast for unstained cells, and fluorescence imaging for stained cells, are commonly used. Some instruments allow control of the incubation chamber and environment. Confocal microscopy is used as a third measurement option for clearer imaging or sectioning of 3D cell clusters such as spheroids. 【0023】 Typically, these sets of equipment are supplied from various vendors, and the user may be forced to physically move the microplate from device to device as needed, track the entire sample analysis process, and manipulate data from several units to obtain complete analysis results. Without robotics, it may be nearly impossible to properly conduct long-term complex experiments. This further increases both the analysis cost and complexity. By combining non-imaging analysis modalities (fluorescence, absorbance, and chemiluminescence), wide-field fluorescence imaging at the cell level, confocal fluorescence imaging, environmental control, and reagent injection into a single device, a complete analysis solution is provided, and the researcher is freed from the tedious handling of microplates, tracking of microplates, and data transmission. 【Means for Solving the Problems】 【0024】 The embodiments described herein overcome the above-mentioned drawbacks and other drawbacks not described above. Also, the embodiments do not necessarily need to overcome the above-mentioned drawbacks, and an exemplary embodiment may not overcome any of the above-mentioned problems. 【0025】 According to one aspect of an exemplary embodiment, a device for analyzing one or more samples is provided, the device including a support for a receptacle for holding the sample, an imaging subsystem for imaging the sample, and an analysis subsystem for analyzing the sample. 【0026】 According to one embodiment of an exemplary model, a sample analysis method is provided, which involves selecting at least one subsystem from a plurality of subsystems of a sample analysis device for examining one or more samples, wherein the plurality of subsystems include an imaging subsystem for imaging one or more samples and an analysis subsystem for analyzing one or more samples, and controlling the selected at least one subsystem to perform an examination on one or more samples, wherein the examination includes imaging operations of the imaging subsystem for imaging one or more samples and analysis operations of the analysis subsystem for analyzing one or more samples. 【0027】 According to one embodiment of an exemplary model, a non-temporary computer-readable medium is provided on which a program is embodied that causes a computer to perform a sample inspection method when executed by a computer, the method comprising selecting at least one subsystem from a plurality of subsystems of a sample analysis device for inspecting one or more samples, wherein the plurality of subsystems include an imaging subsystem for imaging one or more samples and an analysis subsystem for analyzing one or more samples, and controlling the selected at least one subsystem to perform an inspection on one or more samples, wherein the inspection includes imaging operations of the imaging subsystem for imaging one or more samples and analysis operations of the analysis subsystem for analyzing one or more samples. 【0028】 According to one embodiment of an exemplary design, a device for analyzing a sample is provided. The device may comprise a receptacle support configured to support a microplate having a microplate well configured to hold a sample; an objective lens configured to image the sample; a laser point scanning confocal system configured to image the sample through the objective lens; and a spinning disk and / or wide-field (i.e., spinning disk and / or wide-field) imaging system configured to image the sample through the objective lens, wherein at least a portion of both the laser point scanning confocal system and the spinning disk and / or wide-field imaging system is movably provided such that the laser point scanning confocal system and the spinning disk and / or wide-field imaging system are configured to be selectively aligned with the objective lens for imaging the sample. 【0029】 The above and other embodiments will become clearer by describing the exemplary embodiments in detail with reference to the attached drawings. [Brief explanation of the drawing] 【0030】 [Figure 1] This is a diagram showing a conventional 96-well plate. [Figure 2] This figure shows a conventional 384-well plate. [Figure 3] This figure shows cells seeded in the wells of a microplate. [Figure 4] This is a schematic diagram showing 3D cells within a microwell. [Figure 5A] This is a comparison diagram of spheroids. [Figure 5B] This is a comparison diagram of spheroids. [Figure 6] Block diagram of a multi-detection system according to one embodiment. [Figure 7] Block diagram of a multi-detection system according to one embodiment. [Figure 8]Block diagram of a multi-detection system according to one embodiment. [Figure 9] Block diagram of a multi-detection system according to one embodiment. [Figure 10] This figure shows a spinning disc according to one embodiment. [Figure 11] This figure shows a confocal disk imaging module according to one embodiment. [Figure 12] This diagram shows a disk replacement mechanism and a disk focusing mechanism according to one embodiment. [Figure 13] This is a diagram of a non-imaging analysis subsystem according to one embodiment. [Figure 14] This figure shows an injection subsystem according to one embodiment. [Figure 15] This figure shows a multi-detection system according to one embodiment. [Figure 16A] This is a perspective view showing an environmental control subsystem according to one embodiment. [Figure 16B] This is a rear view showing the environmental control subsystem according to the above embodiment. [Figure 16C] This is a front view showing the environmental control subsystem according to the above embodiment. [Figure 17] This is a functional block diagram showing the control of modalities in a device according to one embodiment. [Figure 18] This is a flowchart of the control method for a multi-detection system according to one embodiment. [Figure 19A] This is the first figure showing an immersion objective lens according to one embodiment. [Figure 19B] This is a second figure showing the immersion objective lens according to the above embodiment. [Figure 20] This figure shows a fluid pump system according to one embodiment. [Figure 21] This diagram shows an objective lens connecting portion according to one embodiment. [Figure 22A] This is a perspective view showing an immersion objective lens according to the first embodiment. [Figure 22B]This is a top view showing an immersion objective lens according to the first embodiment. [Figure 22C] This is a first cross-sectional view along line AA in Figure 22B, showing an immersion objective lens according to the first embodiment with a liquid valve installed. [Figure 22D] This is a second cross-sectional view along line AA in Figure 22B, showing an immersion objective lens according to the first embodiment, with a microplate provided on top. [Figure 23A] This is a top view showing an immersion objective lens according to a second embodiment. [Figure 23B] This is a first cross-sectional view along line BB in Figure 23A, showing an immersion objective lens according to a second embodiment with a liquid valve installed. [Figure 23C] This is a second cross-sectional view along line BB in Figure 23A, showing an immersion objective lens according to a second embodiment, with a microplate provided on top. [Figure 24A] This is a top view showing an immersion objective lens according to a third embodiment. [Figure 24B] This is a first cross-sectional view along line CC in Figure 24A, showing an immersion objective lens according to a third embodiment with a liquid valve installed. [Figure 24C] This is a second cross-sectional view along line CC in Figure 24A, showing an immersion objective lens according to a third embodiment, with a microplate provided on top. [Figure 25A] This is a top view showing an immersion objective lens according to the fourth embodiment. [Figure 25B] This is a first cross-sectional view along line DD in Figure 25A, showing an immersion objective lens according to a fourth embodiment with a liquid valve installed. [Figure 25C] This is a second cross-sectional view along line DD in Figure 25A, showing an immersion objective lens according to a fourth embodiment, with a microplate provided on top. [Figure 26A] This is a first figure of a multi-detection system in a laser point scanning confocal modality according to one embodiment. [Figure 26B]This is a second figure of a multi-detection system in a wide-field or spinning disk confocal modality according to the same embodiment. [Figure 27] This is a diagram illustrating an example of a user interface according to one embodiment. [Modes for carrying out the invention] 【0031】 The imaging modality known as confocal imaging can be very well suited for imaging 3D cell structures. In confocal imaging, a sample can be illuminated one point or portion at a time. For example, light can be passed through a small aperture, such as a pinhole, positioned in an optically conjugate plane. Point illumination substantially eliminates out-of-focus and background light, thereby increasing the optical resolution and contrast of the image. The complete image is constructed point by point via a scanning function or stitched together, and is very sharp with distinct features. The scanning function can be performed using a spinning disk, also known as a scanning disk or Nipkow disk. 【0032】 Confocal imaging is an imaging modality particularly well-suited for use with spheroids. Using confocal imaging, spheroids can be sectioned layer by layer, and 3D models can be created on a computer for both accurate cell counting and 3D image manipulation, allowing the spheroids to be observed from various angles. 【0033】 Figures 5A and 5B are comparative images of spheroids. Figure 5A shows spheroids acquired at 20x magnification using wide-field imaging. Figure 5B shows spheroids acquired at 20x magnification using confocal imaging. Spheroid size can be evaluated using the image in Figure 5A, but the structure of individual cells and spheroids is only visible in the confocal image in Figure 5B. 【0034】 The resolution advantage attributed to confocal imaging in Figure 5B is provided at the expense of the reduction in light intensity caused by the confocal aperture, which often requires longer exposure times compared to wide-field imaging in Figure 5A. 【0035】 By adding confocal fluorescence imaging to an instrument that includes non-imaging analytical modalities (fluorescence, absorbance, chemiluminescence, etc.) and wide-field fluorescence imaging at the cellular level, combined with a controlled living cell environment, modern researchers are provided with the most versatile single instrument for analyzing microplate-based assay formats, including those intended for 3D cell spheroid studies. 【0036】 In one example, a workflow might exist where wide-field imaging is performed for faster screening, while confocal imaging is performed for publicly available images. 【0037】 Wide-field imaging may be performed for HCS-type assays, in which case throughput is faster with wide-field imaging, and the resulting image analysis remains statistically robust. Confocal imaging can then be employed to obtain representative wells of "hits" compared to "controls" for publication or presentation purposes. 【0038】 In one example, a workflow might exist where wide-field imaging is performed for a faster primary screening of spheroids based on size. Then, confocal imaging is used for a deeper assessment of the size of each "hit" well based on nuclear counting, which would be more accurate with confocal imaging. 【0039】 Typically, wide-field imaging cannot "see" the 3D spheroid well enough to reliably count individual nuclei, but it can still make "hit" decisions based on the total spheroid size. Once a "hit" well is identified with wide-field imaging, the identified well is then imaged with confocal imaging, providing an improved image analysis to count all nuclei within the spheroid, which cannot be done with wide-field imaging alone. 【0040】 In one example, a proliferation assay (3D endothelial cell spheroid assay) may exist to determine candidate wound healing drugs. Primary drug screening can be performed in microplates where small endothelial spheroids are treated with a library of unknown compounds to determine which compounds induce increased cell growth / proliferation. Compounds that cause increased growth may be candidates for further wound healing research. 【0041】 In the analytical workflow, a plate reader can be used to rapidly screen microplates using GFP fluorescence intensity to identify wells containing enlarged spheroids. Wells that meet a GFP intensity threshold (which is statistically determined during assay development) are considered "hits" and selected for further imaging. Control wells are also always imaged as reference wells for comparison with the hit wells. 3D confocal imaging of spheroids can be performed to obtain a 2-channel z-stack image set of the entire spheroid sample (with Hoescht 33342 nuclear markers and GFP markers). Image processing and analysis of the maximum projection of the Z-stack are performed to determine the number of cells in the spheroids and quantify the spheroid size. A visual inspection of the distribution of nuclear masks in the images is performed to determine whether there is cell death within the spheroids. The results from the hit well image analysis are then compared to the control to determine the percentage of growth relative to the control. 【0042】 In one example workflow, a 3D tumoroid cytotoxicity and immunoassay (a 3D tumoroid assay from surgical samples to determine immunotoxic and cytotoxic therapeutic responses) is performed. This assay involves culturing tumoroids obtained from surgical samples derived from animal models or patients. Because these tumoroids are derived from animals / patients, in vitro tumor-derived immune cell responses can be evaluated, enabling analysis of tumor responses to various therapies. This assay can evaluate the efficacy of novel therapeutic agents in a microplate-based format using heterogeneous multicellular tumor models. 【0043】 For example, chumoroid can be stained for nuclear counting (e.g., blue) and for immunocytomarkers (e.g., red). Using a microplate reader, wells with high cytotoxicity, indicated as a low blue signal, and wells with high immune response, indicated as a high red signal, can be evaluated. Wells that meet one or both threshold criteria for cytotoxicity or immune response (the threshold is statistically determined during assay development) are considered "hits" and selected for further imaging. Control wells are also always imaged further for comparison with the hit wells. 3D confocal imaging of the chumoroid is performed to obtain a 2-channel z-stack image set of the entire chumoroid sample (Hoescht 33342 nuclear market and CY5 marker). Image processing and analysis are performed on the maximum projection of the Z-stack, and cell counting of the chumoroid is performed to quantify the cell number. The number of red-positive cells is determined for the immune response. Results from the hit well image analysis are compared to the control to determine the percentage of cytotoxicity or immune response compared to the control. 【0044】 Some of the examples above utilize the capability of a single instrument to perform assays as "hit picking." A first rapid readout identifies specific samples of interest using a fast readout method, which can typically be a fluorescence non-imaging readout or a fluorescence or bright-field wide-field imaging readout performed at lower magnification. Once the wells of interest (called hits) are identified, a second, more time-consuming modality is deployed to determine the results for those specific samples of interest. This process is particularly important when the final result is high-resolution confocal imaging, which requires large data storage, and collecting a vast amount of information on only a small number of samples of interest provides a substantial saving of data storage space. This process also saves processing time during data acquisition and data review, as most samples are not "hits" and are eliminated during the first assay step. A single, integrated device for performing various different processing steps can streamline the analysis. 【0045】 Other applications are possible for the capabilities of a single instrument with diverse functionalities for spheroid studies. Spheroids are typically grown in round-bottomed wells, as shown in Figure 4. Often, for the final imaging step, the spheroid is transferred to a flat-bottomed plate to prevent the rounded well bottom from acting like a lens during imaging, unnecessarily inducing optical aberrations and negatively affecting the resulting image quality. High-quality microscope objective lenses are not designed for bottom lenses of such "rounded wells" in the optical path. After being transferred to another well, dish, or plate for the best image quality, the exact position of the spheroid within the well is no longer known. In a preferred embodiment, wide-field imaging at a lower magnification but larger field of view is performed to image the well to locate the spheroid (region of interest), then the well is positioned to align the location of the found spheroid (region of interest) with the optical axis, and the spheroid is imaged in a confocal modality using a higher magnification objective lens with a smaller field of view, and a Z-stack may be performed by collecting multiple images while the objective lens traverses along the focal axis of the objective lens perpendicular to the bottom surface of the well. The spheroid (region of interest) may be identified by using the instrument's non-imaging analysis modality by performing a fluorescence reading region scan and selecting the region of maximum fluorescence signal for imaging. 【0046】 Figure 6 is a block diagram showing a multi-detection system according to one embodiment. 【0047】 As shown in Figure 6, the multi-detection system comprises a controller 1000, a fluid injection subsystem 1100, an imaging subsystem including a wide-field imaging component 1200 and a confocal imaging component 1500, a non-imaging analysis subsystem 1300, an imaging illumination subsystem 1600 for wide-field imaging, a housing 1900, a microplate 300, a microplate carrier 310, an incubation chamber 320 for incubating samples in the wells 200, an environmental control subsystem 2000, and a confocal imaging subsystem. The multi-detection system may also include an external subsystem 2100. 【0048】 The sample is placed in the wells 200 (e.g., microwells) of the microplate 300. The microplate 300 is transported in and out of the measurement and incubation chamber 320 by the microplate carrier 310. When positioned to be exposed to the external environment of the multi-detection system, the microplate 300 can be made accessible outside the incubation chamber 320 and / or housing 1900 (i.e., the incubation chamber 320 or housing 1900 or both) for access by a technician or robotic arm. Once the microplate 300 is positioned within the chamber, various supported imaging and non-imaging analysis modalities can be performed. 【0049】 The microplate carrier 310 is part of the microplate transport subsystem for positioning the microplate 300 and may include any suitable combination of a belt, platform, microplate holder, motor, and positioning software that runs under hardware control for positioning. Once the microplate 300 is placed in the incubation chamber 320, the entire microplate 300 remains incubated. The incubation system and incubation chamber 320 will be described in detail later. 【0050】 The non-imaging analysis subsystem 1300 may be based on illumination via a flashbulb, a dual-excitation monochromator, a dual-emission monochromator, a photomultiplier tube (PMT), and a silicon detector. The non-imaging analysis subsystem 1300 supports absorbance, fluorescence, and chemiluminescence analysis modalities for detecting corresponding properties of the sample in the well 200. The non-imaging analysis subsystem 1300 can be implemented as a filter-based subsystem or as a hybrid of any or all of the above. 【0051】 The imaging subsystem includes wide-field imaging components 1200 and confocal imaging components 1500, such as objective lenses, lenses, LEDs, filter cubes, spinning disks, cameras, and other components. The imaging illumination subsystem 1600 includes illumination components for wide-field imaging and is capable of providing illumination for brightfield, color brightfield, and phase-contrast imaging modalities. 【0052】 The external subsystem 2100 can be an external confocal illumination subsystem for confocal imaging, which can be modularly connected to and separated from the imaging subsystem within the housing 1900 via optical fiber to increase the flexibility of the physical placement of the external subsystem 2100 relative to the instrument. Alternatively, the confocal imaging illumination subsystem may be arranged to be integrated within the housing 1900. 【0053】 The fluid injection subsystem 1100 delivers reagents to the wells 200 as required for the assay. The fluid injection subsystem 1100 may include any combination of pumps, reservoirs, lines or tubing, pipettes and tips, and software running under hardware control to deliver fluid to the wells and, if necessary, aspirate fluid from the wells. 【0054】 The environmental control subsystem 2000, located externally to the housing 1900, may include a gas control module that provides control over the atmospheric conditions inside the housing 1900. Other control modules may include modules for controlling temperature, humidity, and other conditions, which can be controlled within the housing 1900 under the control of the environmental control subsystem 2000. The environmental control subsystem may include any combination of pumps, reservoirs, lines or pipes, fans, heating and cooling elements, etc., to control all conditions within the housing 1900. The housing 1900 houses the majority of the subsystem and defines a physical space in which the gas atmosphere contributing to living cells can be effectively maintained and controlled by the environmental control subsystem 2000. 【0055】 The controller 1000 can control all operations of the multi-detection system. The controller 1000 can communicate with each of the various subsystems within the multi-detection subsystem via wired or wireless means. The controller 1000 can include any combination of hardware (e.g., CPU, memory, cables, connectors, etc.) and software for execution by the hardware to control the operation of the multi-detection system. 【0056】 Figure 7 is a block diagram showing a multi-detection system according to one embodiment. 【0057】 Several imaging modalities are made possible by multi-detection systems. Wide-field imaging in fluorescence, brightfield, and phase contrast can be performed in addition to confocal imaging modalities. The optical elements of both confocal and wide-field imaging systems are shown in Figure 7. 【0058】 The microplate 300 can be placed on a microplate carrier 310 that positions the well of interest 200 aligned with the imaging optical axis of the objective lens 1230. The objective lens can be selected from several objective lenses of various magnifications, which are located on the objective lens turret 1232. The relative position of the imaging illumination subsystem 1600 is shown in Figure 7, and the imaging illumination subsystem 1600 can be used for brightfield, color brightfield, and phase contrast imaging of the sample. Many optical elements are shared between the widefield system and the confocal system, and a more detailed description of such sections is provided below in Figures 8 and 9, while some elements in Figure 7 are omitted for clarity. 【0059】 Figure 8 is a block diagram showing a multi-detection system according to one embodiment. 【0060】 The confocal imager is deployed as shown in Figure 8. The wide-field imaging subsystem elements (e.g., LED cube 1201 and filter cube 1210) are automatically removed from the optical path to the sample, and the system shown in Figure 7 is transformed into the confocal optical system shown in Figure 8 for understanding the confocal optical path. 【0061】 A spinning disk confocal system is deployed as an illustrative embodiment of a confocal imaging system. This system is based on utilizing a spinning disk (Figure 10) as the optical path. The disk is positioned in an intermediate image plane conjugate to the sample and the detector surface. Thus, the disk is present in both the excitation and emission optical paths. The disk is typically about 2 mm thick and, in one exemplary embodiment, is made of glass or quartz. The disk may be coated to be opaque, or to have a given transparency or opacity, except for transparent areas left as a pattern of pinholes or slits. Ideally, the disk surface is fabricated so as not to reflect incoming light. The sample to be imaged is illuminated by excitation light transmitted through the pinholes. Only the radiation emitted by the sample, generated from these illuminated spots on the sample, reaches the detector through the pinholes in the disk. The pinholes or slits are numerous but far apart from each other so as to act optically independently. Energy from adjacent pinholes ideally does not affect the sample spot illuminated by a given pinhole. The disk spot pattern is typically arranged in several spirals, as shown in Figure 10. The disk is controlled to rotate continuously, and thus can scan the sample. As the disk rotates, the sample is illuminated one spot at a time, and a complete sample image is detected on the detector and reconstructed as a complete image of the sample. 【0062】 Returning to Figure 8, the confocal light source 1540 can be any light source suitable for confocal microscopy. For example, the confocal light source 1540 can be a solid-state light source such as a light-emitting diode (LED), a solid-state laser, or a semiconductor-based laser (laser diode). In one exemplary embodiment, the output tip of the optical fiber can be a light (emission) source. Since the excitation spectrum can be outside the 380 nm to 630 nm range commonly referred to as light, emission is an embodiment. However, the term “light source” is more commonly used in imaging, and the term “light” is used interchangeably with “emission” in this specification. The input tip of the fiber can be illuminated from an external light source module of the instrument, providing flexibility in selecting the best light source match for the needs regarding the imaging of the sample. The fiber also provides flexibility in branching inputs from multiple external light sources. The output tip of the fiber is imaged by the capacitor 1522 onto or near the intermediate sample image plane where the spinning disk 1504 is located. Light from the fiber may also be sent through the excitation filter 1531, then reflected by the dichroic mirror 1533, and focused onto the spinning disk 1504 by the tube lens 1520. The term “lens” can refer to a single lens or a group of lenses here and throughout this description, depending on the embodiment and function, as will be understood by those skilled in the art. As described, the disk has a helical pattern of slit holes. The field lens 1519 minimizes light loss and directs the light exiting the disk so that it is collected by the tube lens 1250. The tube lens 1250 directs the excitation radiation to the objective lens 1230 via the mirror 1220. The objective lens 1230 illuminates a small spot on the sample near the bottom of the well. The sample components are stained with dyes corresponding to the excitation wavelength. These components are excited by the incoming radiation and emit radiation, typically having longer wavelengths. This synchrotron radiation is directed to the detector as follows: 【0063】 Light emitted by the sample is collimated by the objective lens 1230, reflected by the mirror 1220, and collected onto the spinning disk 1504 by the tube lens 1250 and the field lens 1519. An intermediate image of the sample in synchrotron radiation is formed on the surface of the spinning disk 1504. The tube lens 1520 and lens 1521 invert the image to form a sample image in the detector 1560. The detector 1560 is typically a pixelated digital camera such as a charge-coupled device (CCD) camera or a complementary metal-oxide-semiconductor (CMOS) camera. The sample image is captured by the camera and can be stored in the memory of a multi-detection system or an external computing system, enhanced and analyzed for various characteristics, and / or presented to the user on a visual display (i.e., enhanced and analyzed, or presented, or all of the above). 【0064】 A confocal cube 1530 (e.g., a confocal excitation / dichroic mirror / emission cube) is shown between the tube lens 1520 and lens 1521, which is the arrangement for fluorescence microscopy. The filters and dichroic can be thin film coatings on glass. The excitation filter 1531 forms a band-pass for excitation, the emission filter 1532 forms a band-pass for emission, and the dichroic mirror 1533 separates excitation and emission so as to make full use of the available energy and suppress the scale of excitation light reflected from multiple optical surfaces as the excitation light travels toward the sample, including the disk surface, and reaches the detector. Lens 1521 (e.g., the emission filter) provides most of the excitation light suppression, but the dichroic mirror 1533 also plays a role in suppression. Alternative arrangements of the described cube can be several filter wheels carrying the excitation filter, emission filter, and dichroic. In the illustrated embodiment, the cube is a method of arranging the described elements, which allows for very easy replacement by the user when imaging needs change. Some filter cubes (e.g., confocal cube 1530) can be mounted on an electric slider and identified by software setup performed by the user, or by electronic or optical labeling with a code that is automatically read via a barcode or some other automatically available method. 【0065】 The surface of the spinning disk is imaged onto the detector along with the sample. Therefore, any dust particles adhering to the disk surface may appear as artifacts in the image, such as streaks of light due to disk rotation. Small particles can easily adhere to the disk surface with enough force to counteract centrifugal force. The spinning disk 1504 and disk drive motor 1509 are part of the disk module 1553. The disks within the module are typically assembled in a clean environment, such as a cleanroom, and sealed from the surrounding environment to prevent dust particles from accumulating on the disk. Windows 1551 and 1550 within the module allow light to pass through but not dust. Ideally, these dust-protection windows should be positioned as far away as possible from the mid-image plane so that dust that may accumulate on the window glass does not introduce artifacts into the image. The disks are completely housed within the disk modules 1502 and 1553. Therefore, the user should not open the module to avoid introducing dust particles onto the disk. 【0066】 Figure 8 shows two disk modules 1553 and 1502 installed in a multi-detector. The disks can be moved to position one disk or the other within the optical path. Alternatively, both disks may be moved outside the optical path, aligning space 1501 along the optical axis. This allows for the performance of wide-field imaging modalities such as fluorescence imaging, bright-field imaging, or phase-contrast imaging. 【0067】 A major advantage of providing users with both confocal and wide-field imaging options in the same instrument is the ability to superimpose images from various imaging modalities, such as the same image in both the wide-field and confocal imaging modalities. Alternatively, a bright-field image may be used to locate the region of interest, which can then be confocally imaged. For this configuration to produce a proper image, the magnifications of both modalities must match precisely; otherwise, the images will not superimpose properly. The light in the section between tube lenses 1520 and 1250 is not parallel. In the confocal modality, several flat windows, namely confocal disks and dust protection windows, are present in the optical path in this section. These windows are not necessary in the wide-field modality. However, to match the optical path lengths in the non-parallel optical paths, a glass 1505 is added to the space 1501 between the confocal disks, through which wide-field imaging is performed. This ensures that the sample remains in focus relative to the fixed objective lens position when the imaging modality changes. This ensures that the magnification matches in confocal and wide-field imaging modes. The thickness of glass 1505 should match the sum of the flat windows of the disk used in confocal imaging (window 1551, spinning disk 1504, and window 1550). Glass 1505 should be positioned as far away from the mid-image plane as possible so that dust that may accumulate on the glass does not introduce artifacts into the image. 【0068】 The pinhole size on the confocal disk is ideally selected based on the parameters of the imaging objective lens 1230. In one embodiment, the size of the disk pinhole image formed on the sample can be matched to the distance between the first two minimum values ​​of the Airy diffraction pattern of the objective lens. The formula for the disk pinhole size, as presented in Zeiss's "Introduction to Spinning disk microscopy," is as follows: 【0069】 Disk pinhole diameter = 1.2 × objective lens magnification × radiation wavelength / numerical aperture of the objective lens 【0070】 Both the numerical aperture (NA) and magnification of the objective lens are part of the formula. If the pinhole is too small, light loss becomes too great, and the time required to acquire an image increases. If the pinhole is too large, the confocal effect may be reduced or completely lost. Most commercially available spinning disk microscopes feature a non-replaceable spinning disk with a pinhole in the range of 50 μm to 70 μm. This works reasonably well as a compromise with the range of high-magnification objective lenses typically deployed in confocal microscopy. However, it is preferable that a disk with an appropriate pinhole be adaptable to the objective lens being used. 【0071】 Some embodiments of spinning disks do not have a circular hole spiral pattern, but instead employ slit openings. Slit openings can provide relatively brighter illumination and stronger emission signals to the sample, while pinhole openings can provide relatively better axial resolution. Therefore, in some imaging applications, including biological fluorescence applications, slits may be preferable to reduce image acquisition time, which is another reason to change the disk even with a fixed objective lens. 【0072】 Multiple disks may be deployed within the imaging device so that selection from among the disks can be performed by the user or automatically by a multi-detection system. 【0073】 Figure 8 shows examples of two disk modules 1502 and 1553 used in a multi-detector. All disk modules can be configured to be replaceable by the user. Modules can be identified by user-controlled software setup to enable automatic configuration by the multi-detector system, or they can be electronically or optically labeled with a code that can be automatically read via a barcode or any other available method. 【0074】 One additional advantage of the modular disk module is that when the disk module is removed from the device and both windows are easily accessible, the user can clean windows 1551 and 1550, which can provide dust protection. 【0075】 Module identification enables automated software setup and allows for automatic resetting and calibration of the module axial position in the optical path. In a spinning disk confocal imaging system, the disk surface, the detector sensing element surface, and the sample surface should be conjugate to each other. This means that, following the radiation from the sample, the image of the sample surface coincides with the disk surface, and the images of the disk surface and the sample surface coincide with the detector surface. The sensing tip surface of the detector 1560 is fixed by the camera design. The objective lens 1230 can move along the focusing axis to sharpen the sample image on the detector. In this case, the disk should ideally be positioned with an intermediate surface conjugate to both the detector and the intermediate sample image surface, such that all three surfaces are conjugate. In the proposed embodiment, the disk axial position is maintained very close to the ideal conjugate position by the disk module design, but the final position of the disk surface can be automatically adjusted by observing the disk pattern on the detector and sharply focusing this pattern on the detector. Multiple image-based focusing methods are available and known in the industry. Once the optimal disk surface position is found, this position is stored in software and memory and can be associated with the disk module. If a disk module is removed and reinstalled, the software can automatically restore the correct disk position. When a new disk module is introduced, the system alternatively performs a disk focus routine to select the best axial position for the new disk module. Therefore, the user may be freed from tracking which disk modules are deployed in the equipment and their various positions. 【0076】 Alternatively, if only a small number of disk modules are expected to be used, the user can set up the disk modules via the setup screen in the calibration section of the user interface of the software included in the multi-detection system. 【0077】 The two concepts of user-replaceable disk modules and automated axial disk positioning work best together, but may be implemented separately. If automated axial disk positioning is unavailable, disk modules can be configured to be interchangeable for disk position and some data on the module to ensure proper placement within the instrument. The concept of easily replaceable disk modules, which do not require user opening and are therefore not exposed to the environment, remains applicable and beneficial to users who desire the flexibility of having multiple disks best suited to the deployed imaging objective lens and sample. 【0078】 Even when the number of disk modules is limited to one or two within the device, automatic axial alignment can be used to reduce the need for precise control over the position of the sensing surface of the image sensor of the detector within the detector 1560 (e.g., camera). This allows for maximum user flexibility in camera selection and also enables camera upgrades within a multi-detection system. If the sensor surface moves after camera replacement, the disk surface can be automatically repositioned to be conjugate with the sensor surface via an image-based autofocus routine. 【0079】 Figure 9 is a block diagram showing a multi-detection system according to one embodiment. 【0080】 Figure 9 shows a wide-field imaging setup deployed in one exemplary embodiment. As described above, the optical section (having elements labeled 15xx) enables both confocal imaging (having spinning disks 1504 or 1503 in the optical path) and wide-field imaging (through the space 1501 between disks). However, there may be drawbacks to using this optical component, as well as the confocal light source 1540 and confocal cube 1530, for wide-field modalities that researchers may want to deploy in a single multi-purpose instrument. In the case of confocal imaging, the excitation radiation should be directed onto the disk via multiple optical elements positioned in front of the disk surface (e.g., dichroic mirror 1533, tube lens 1520, window 1551). After the disk, the excitation radiation is guided to the sample via more optical elements (e.g., window 1550, field lens 1519, tube lens 1250, mirror 1220, objective lens 1230). In the case of confocal imaging, this approach is not an option. However, at every opposing surface, some of the excitation light is reflected back. A good design then relies on careful ray tracing to ensure that the reflected light is kept away from the detector as much as possible, and on the emission filter 1532 to suppress undesirable reflections. Optical elements in front of the disk surface, such as the tube lens 1520 and window 1551, as well as the surface of the spinning disk 1504, are exposed to very strong levels of excitation radiation that are partially reflected. Dust particles may also be excited and fluoresce. Despite the designer's best intentions, some of the light reaches the detector, reducing the signal-to-noise ratio. Therefore, non-fluorescent samples that should appear very dark in the image may not appear very dark. This can be due to a prominent background signal caused by reflected light, i.e., an effect that tends to be uniform across the entire image. Wide-field microscopy using the confocal section excitation elements described above in Figure 8 involves significant compromises in image quality and system capabilities. 【0081】 In one exemplary embodiment, an alternative subsystem is provided within the same instrument that can be used for wide-field fluorescence imaging. The confocal cube 1530 of the confocal subsystem is positioned out of the way, and the spinning disk module is positioned in space 1501 for wide-field imaging. This transforms the configuration of Figure 7 into the configuration of Figure 9. Dedicated wide-field section elements are the LED cube 1201 and the wide-field excitation / emission / dichroic imaging filter cube 1210. The excitation filter 1211, dichroic mirror 1212, and emission filter 1213 are typically mounted within the filter cube, which would be matched with the LED cube 1201 for best signal-to-noise performance. Some of these cube pairs corresponding to the specific chemistry being investigated can be provided on a slider. 【0082】 This design has several advantages. 【0083】 Firstly, the LED excitation optical component is very close to the sample, and therefore the excitation light encounters fewer optical surfaces on its way to the sample. Consequently, reflections from these surfaces that can reach the detector are greatly reduced, improving the signal-to-noise ratio of the image. 【0084】 Secondly, while the various LEDs used in the commercially available LED cube 1201 may not be powerful enough for use in a confocal optical tube, they can deliver sufficient excitation when placed closer to the sample, as shown in Figure 9. 【0085】 Thirdly, particularly important when the sample must be excited in the UV range, some objective lenses are evaluated as UV objective lenses, transmitting UV light and exhibiting very low fluorescence when excited by UV. However, generally, commercially available optical elements for the rest of the optical tube, such as tube lenses, are not guaranteed to be non-fluorescent when illuminated by UV light. When wide-field images of samples stained with common DAPI nuclear stains are required, a common technique in confocal optical tubes is to use a wavelength of approximately 400 nm, thereby avoiding strong excitation of the optical elements in addition to the sample. However, shifting the excitation from 360 nm, the ideal wavelength for DAPI staining excitation, towards 400 nm results in a significant decrease in synchrotron radiation. Researchers need to place a higher concentration of dye in the sample or increase the detector gain, and thus reduce the signal-to-noise ratio of the image. Ideally, the excitation of a DAPI-stained sample is performed at 360 nm, but the UV excitation light does not pass through optical elements that may emit fluorescence. The LED cube 1201 and filter cube 1210 enable precisely such an optimal option in one exemplary embodiment. UV excitation enters only the objective lens 1230, which can be selected not to fluoresce. Synchrotron radiation returns to the detector through multiple optical elements common to confocal and wide-field tubes, but because the synchrotron radiation is within the visible spectral range, the optical elements that the light confronts typically do not fluoresce at levels that would fluoresce under UV light. 【0086】 Figure 9 shows the relative positions of the imaging illumination subsystem 1600 for wide-field imaging in non-fluorescent modalities. This can be a brightfield, a color brightfield with three color LEDs that can be switched one at a time, or a phase-contrast illumination system with a ring aperture that matches the phase-contrast objective lens. 【0087】 Figure 11 shows a confocal disk imaging module according to one embodiment. 【0088】 The disk drive motor 1509 is a DC brushless motor in one exemplary embodiment, capable of high rotational speeds of several thousand RPM at a constant speed, and is mounted on the housing base 1800. The spinning disk 1504 is secured on the motor shaft by hub portions 1820 and 1830. A cover 1810 is mounted on the housing base 1800, completing a dust-free environment for the disk. There is no user access to the disk. Optical windows 1550 and 1551 allow light to pass through while keeping the inside of the module dust-free. From an imaging perspective, it is advantageous to keep both windows as far away from the disk surface as possible within the overall spatial constraints to avoid dust particles on the windows affecting the image. The disk modules can be identified via barcode labels, simple binary code labels, or some other instrument-readable means, so that a multi-detection system can automatically identify which disk modules are present and available at any time. 【0089】 Referring to Figure 10, it is necessary to closely correlate the disk speed and the confocal image exposure time. As shown in Figure 10, multiple helices are provided on the disk, and as the disk rotates, the sample is swept by the pinhole pattern. There is a minimum angle of disk rotation required to illuminate the sample continuously and completely in one go. For many commercially available disks and the disk in one exemplary embodiment, this angle is 30 degrees. If the exposure time is not a multiple of the time it takes to move the disk 30 degrees, some artifact, such as stripes, will appear in the image. This is a known problem in the industry. In one exemplary embodiment, the disk rotation speed is set to 2400 rpm, and the exposure time is set to a multiple of one rotation (e.g., 25 milliseconds, 50 milliseconds, 75 milliseconds, etc.). This method has been found to provide a good compromise between image quality and the minimum time to capture an image. Furthermore, this method using full rotation time exposure increments resulted in minimizing image artifacts caused by potential non-concentricity between the disk helical pattern and the disk rotation axis. 【0090】 Figure 12 shows a disc replacement mechanism and a disc focusing mechanism according to one embodiment. 【0091】 Referring to Figure 12, the disk replacement mechanism and the disk focusing mechanism can be implemented in one exemplary embodiment. However, the configuration of the disk replacement mechanism and the disk focusing mechanism is not limited to this. 【0092】 Base 1701 supports all elements of the mechanism. A linear way rail 1705, such as part of an IKO or HTK guide system, is mounted to base 1701. The linear way carriage 1706 supports bracket 1710. Bracket 1710 is translated by motor 1715 via timing belt 1717 in a direction perpendicular to the optical axis. This movement allows either disk module 1502 or disk module 1553 or space 1501 to be positioned in alignment with the imaging optical axis. Other mechanical embodiments are also possible, and the main advantage of the timing belt is the rate of change achievable in this particular method. An axis homing sensor and / or possible encoding (i.e., axis homing sensor or possible encoding or both) is not explicitly shown. 【0093】 Bracket 1710 holds the linear way rail 1720 and motor 1725. In one exemplary embodiment, the motor shaft is formed as a lead screw. The motor translates a support 1730, attached to the linear way carriage 1721 via a lead nut 1727, in the direction of the optical axis, providing the axial focus of the confocal disk. An axial homing sensor and / or possible encoding is not shown. 【0094】 The disk module is mounted directly to the support 1730 and accessible by the user. Mounting can be via fasteners or magnets for easy removal. Alternatively, the disk module can be mounted to the guide 1732 and then slip-fitted and secured to the support 1730 for easy removal by the user from the device. 【0095】 As those skilled in the art will understand, other mechanisms can be provided to achieve the functions of disk module access, positioning, and disk focusing. 【0096】 Figure 13 shows a non-imaging analysis subsystem according to one embodiment. 【0097】 Referring to Figure 13, a non-imaging analysis subsystem 1300 of the multi-detection system is provided. 【0098】 The analytical modalities of the non-imaging analysis subsystem 1300 can be absorbance, top and bottom fluorescence, and chemiluminescence. The Xe flashbulb 13001 emits radiation in the range of 200 nm to 1000 nm. Two stages 13002 and 13003 of the fluorescence excitation / absorbance dual monochromator select narrow bandpass of the radiation. The radiation is guided towards the sample by fiber optic cables via fiber 13030 to the absorbance channel, via 13005 to the top fluorescence, or via 13033 to the bottom fluorescence. Since only one fiber is acting at a time, there is no optical crosstalk between the various analytical modes. Absorbance is measured by a silicon detector 13060 via lenses 13040 and 13050. 【0099】 The pickup of upper fluorescence excitation and emission is performed via lens 13020, which can move up and down to adapt to various microplate and fluid levels. Bottom fluorescence is performed in a similar manner using lens 13055. Both upper and bottom emission are guided by optical fiber cables to the first stage of emission dual monochromators 13010 and 13011, and then to photomultiplier tube 13012. A chemical luminescence fiber 13021 is directly connected to the photomultiplier tube and can provide very weak light measurements by bypassing the monochromator. 【0100】 The fluid injection subsystem 1100 can inject reagents via fluid lines 1112 and 1111, providing researchers with the ability to rapidly measure the injection results by the analysis subsystem, further increasing the range of tests that can be performed on the instrument. 【0101】 Figure 14 shows an injection subsystem according to one embodiment. 【0102】 Referring to Figure 14, an optional injection subsystem is provided. The injection subsystem 1100 can be located on top of the multi-detection instrument, as shown in Figure 15, and fluid lines 1112 and 1111 can be supplied through a partition access at the top of the housing. Reagents are delivered to the microwells by a pump in the fluid injection subsystem 1100 via fluid lines 1111 and 1112, which can be PTFE lines, as shown in Figure 14, and delivered into the wells via injection needles 1102 and 1101. 【0103】 Referring to Figure 13, environmental control can be deployed in a multi-detection system. 【0104】 The microplate carrier 310, as shown in Figure 13, supports the microplate 300 and is located within the incubation chamber 320. This ensures that the microplate 300 is maintained at the desired temperature at all positions within the microplate carrier 310 in the incubation chamber 320. The incubation chamber 320 can be constructed from a material well suited to maintaining a constant temperature, such as a continuous aluminum sheet, while still providing access to the optical elements through a small opening. The incubation chamber 320 is typically insulated. Such chamber designs are known to those skilled in the art and are known from many multi-detection instruments. A typical controlled temperature range can be room temperature to 65 degrees Celsius. 【0105】 Figure 15 shows a multi-detection system according to one embodiment. 【0106】 For live cells, the temperature is typically 37 degrees Celsius, but in addition, gas control around the sample is required. Control is achieved by filling the complete housing 1910 of the apparatus shown in Figure 15 with an appropriate gas mixture. This design avoids attempting to confine the gas-controlled environment to only the measurement chamber or separation partition. The aim of this design is to homogenize the atmosphere within the housing 1910. Therefore, the housing 1910 is designed to be as airtight as possible by avoiding gaps within the housing and using soft gasket material around the user access door. 【0107】 Figure 16 shows a gas control subsystem according to one embodiment. 【0108】 Referring to Figure 16, the environmental control subsystem 2000 (e.g., a gas control subsystem) can be located outside the instrument. The environmental control subsystem 2000 allows the user to set the CO2 and / or O2 (i.e., CO2 or O2 or both) concentration levels in the chamber to be different from the normal atmosphere, i.e., higher CO2 and lower O2. A gas sampling line connects the environmental control subsystem 2000 inside the instrument housing. Based on the composition of the gas sampled or extracted from the instrument via the sampling line, the control system can adjust the flow of CO2 or N2 gas supplied to the instrument, for example, by dispersing the incoming gas with a small fan. This allows all gas sensors and valves to be located outside the main instrument, while maintaining the complexity and reliability of gas control within an external gas controller. 【0109】 The combination of the incubation chamber surrounding the XY carrier transfer zone and the gas control of the atmosphere inside the housing and, consequently, around the microplate, provides users with the ability to perform long-term live-cell experiments. 【0110】 Referring to Figure 15, an external view of the entire apparatus and elements receiving user interaction with the apparatus as implemented in the exemplary embodiment is shown. The microplate carrier 310 presents itself to the user (shown on the right), and the microplate 300 is placed on the microplate carrier 310, for example, by the user or a robotic arm, and then positioned within the multi-detection system. Access to the confocal cube 1530, wide-field LED cube 1201, and wide-field filter cube 1210, confocal disk module, and objective lens 1230 is via the front of the apparatus through the door 1905. Thus, it is easy for the user to access most of the user-variable elements at once. 【0111】 According to certain embodiments, the objective lens of the present disclosure (for example, objective lens 1230 or objective lens 2210) can be an immersion objective lens. 【0112】 One way to improve the optical performance of a microscope is to use an immersion objective lens. In optical microscopy, an immersion objective lens is a specially designed objective lens used to increase the resolution of the microscope. According to embodiments of this disclosure, the optical system is an inverted microscope, meaning that the objective lens is positioned below the sample and the sample is observed from below. In the inverted microscope configuration of this disclosure, when fluid immersion is performed, a droplet of fluid (e.g., water or another fluid) is placed on the objective lens and held in place by the surface tension of the fluid. The objective lens is then brought to the sample, and the droplet is sandwiched between the sample and the objective lens. In this way, the light passing through to the sample and from the sample to the objective lens does not pass through the air. If the refractive index of the fluid is higher than that of air, the numerical aperture increases. This increases the resolution and increases the signal level. According to embodiments, the objective lens may be brought to the sample, and then the droplet of fluid is placed on the objective lens. 【0113】 In addition to water immersion objective lenses, the objective lenses of this disclosure may be equipped with other types of fluids to increase the numerical aperture. Some examples of fluids include, for example, oil and glycerol. In embodiments of this disclosure, the fluid may be water, oil, glycerol, or any other type of fluid that will increase the refractive index. 【0114】 Referring to Figures 19A and 19B, an immersion objective lens according to an embodiment of the present disclosure is described below. According to one embodiment, the objective lens 1330 may include a sleeve 1332 fitted onto the objective lens 1330. The sleeve 1332 may be configured to provide fluid pathways inside and outside the sleeve 1332. In addition, the sleeve 1332 helps to hold fluid droplets 33 in place. According to one embodiment, the sleeve 1332 has a port for pumping fluid inward and a port for pumping fluid outward. According to one embodiment, as shown in Figures 19A and 19B, the inlet and outlet ports may be the same port 31. Referring to Figure 19B, excess droplets 34 can exit the sleeve 1332 through the port 31. In one exemplary embodiment, the sleeve 1332 may be formed from, for example, anodized aluminum, plastic, or other material. 【0115】 According to an embodiment, a fluid pump system may be provided, referring to Figure 20. The fluid pump system may include a first pump 1336, a second pump 1337, a first reservoir 1338 (source reservoir), and a second reservoir 1339 (waste reservoir). Fluid can be pumped by the first pump 1336 from the first reservoir 1338 to the head of the objective lens 1330. As shown in Figure 20, the first pump 1336 may be a syringe pump. The fluid is then removed from the objective lens 1330 via the second pump 1337, which pumps the fluid to the second reservoir 1339. The second pump 1337 may also be called a waste pump and may also be a syringe pump, as shown in Figure 20. The first pump 1336 and the second pump 1337 may be other types of pumps that achieve the same or similar functions. The sleeve 1332 is fitted onto the objective lens 1330 and can guide the fluid to the top of the objective lens 1330, helping to hold the fluid droplets in place. The sleeve 1332 may also have a waste port, which may be configured to allow the fluid to be removed from the sleeve 1332. The objective lens 1330 may be a specially designed objective lens optimized for fluid (e.g., water) immersion applications. In Figure 20, the first reservoir 1338 and the second reservoir 1339 are shown as separate source reservoir and waste reservoir, respectively. However, according to the embodiment, instead of two separate reservoirs, a single reservoir from which the fluid can be reused may be provided. Furthermore, the pump may be multipurpose. For example, the BioTek C 10 product has a fluid dispensing module that can be used to dispense reagents into a sample. This same dispensing module may be configured to have additional purposes (including the purposes of the first pump 1336 and / or the second pump 1337 (i.e., the first pump 1336 or the second pump 1337 or both)) in order to reduce costs. 【0116】 Referring further to Figure 20, the objective lens 1330 may be attached to the objective lens turret 1232 by an objective lens connector 1334. A description of the objective lens connector 1334 is provided below with reference to Figure 21. 【0117】 As shown in Figure 21, the objective lens connector 1334 may include a kinematic connection 1334A and a magnet 1334B configured to connect the objective lens 1330 and the objective lens turret 1232 to each other. For example, the objective lens 1330 may have at least one convex or concave portion as a first part of the kinematic connection 1334A, and the objective lens turret 1232 may have at least one other convex or concave portion as a second part of the kinematic connection 1334A corresponding to the first part. The magnet 1334B may comprise one or more of the objective lens 1330 and the objective lens turret 1232. According to the embodiment, both the objective lens 1330 and the objective lens turret 1232 may have a magnet 1334B configured to correspond to each other and connect to each other via magnetic force. In other embodiments, the magnet 1334B can be provided on only one of the objective lens 1330 and the objective lens turret 1232, and this magnet can be configured to connect to a magnetic material (e.g., metal) provided on the other of the objective lens 1330 and the objective lens turret 1232. 【0118】 According to comparative embodiments, the objective lens may be screwed into the objective lens turret. However, the use of sleeves and tubes with the objective lens can make it difficult to screw the objective lens into the objective lens turret, at least in some embodiments. The use of the objective lens connector 1334, which includes a kinematic connector 1334A and a magnet 1334B, as in embodiments of the present disclosure, allows for easy installation of the objective lens with sleeves and tubes. 【0119】 According to the embodiment, as shown in Figures 22A to 25C, the objective lens 1330 and the sleeve 1332 can have various configurations. According to the embodiment, the sleeve 1332 is sometimes called a cap. 【0120】 Figure 21 is a diagram showing the objective lens coupling portion according to one embodiment. Figure 22A is a perspective view showing the immersion objective lens according to the first embodiment. Figure 22B is a top view showing the immersion objective lens according to the first embodiment. Figure 22C is a cross-sectional view along line AA in Figure 22B showing the immersion objective lens according to the first embodiment with a liquid valve provided. Figure 22D is a second cross-sectional view along line AA in Figure 22B showing the immersion objective lens according to the first embodiment with a microplate provided on top. Figure 23A is a top view showing the immersion objective lens according to the second embodiment. Figure 23B is a first cross-sectional view along line BB in Figure 23A showing the immersion objective lens according to the second embodiment with a liquid valve provided. Figure 23C is a second cross-sectional view along line BB in Figure 23A showing the immersion objective lens according to the second embodiment with a microplate provided on top. Figure 24A is a top view showing the immersion objective lens according to the third embodiment. Figure 24B is a first cross-sectional view along line CC in Figure 24A, showing an immersion objective lens according to a third embodiment with a liquid valve. Figure 24C is a second cross-sectional view along line CC in Figure 24A, showing an immersion objective lens according to a third embodiment with a microplate on top. Figure 25A is a top view showing an immersion objective lens according to a fourth embodiment. Figure 25B is a first cross-sectional view along line DD in Figure 25A, showing an immersion objective lens according to a fourth embodiment with a liquid valve. Figure 25C is a second cross-sectional view along line DD in Figure 25A, showing an immersion objective lens according to a fourth embodiment with a microplate on top. 【0121】 In the following descriptions of Figures 22A to 25C, the same or similar feature parts are given the same or similar reference numerals. For clarity, redundant descriptions of the same or similar feature parts can be omitted. 【0122】 Referring to Figures 22A to 22D, the upper surface 10A of the sleeve 1332A can be made coplanar with the upper surface 11A of the objective lens 1330A, and the sleeve 1332A can be configured to clamp onto the objective lens 1330A. 【0123】 The sleeve 1332A may include, for example, an upper portion 50A, an intermediate portion 60A, and a lower portion 70A. According to the embodiment, the upper portion 50A, the intermediate portion 60A, and the lower portion 70A may be provided separately or integrally with each other to constitute a single body or a plurality of bodies. According to the embodiment, two of the upper portion 50A, the intermediate portion 60A, and the lower portion 70A may be provided integrally to constitute a single body, and the other one of the upper portion 50A, the intermediate portion 60A, and the lower portion 70A may be provided separately as a separate body configured to be attached to the other two. According to the embodiment, the upper portion 50A, the intermediate portion 60A, and / or the lower portion 70A (i.e., the upper portion 50A, the intermediate portion 60A, or the lower portion 70A, or all of them) may be further divided into separate bodies and / or additional bodies may be provided (i.e., further division, or additional bodies may be provided, or both). According to this embodiment, any number of upper portions 50A, intermediate portions 60A, and lower portions 70A can be formed from aluminum. 【0124】 According to the embodiment, any number of upper portions 50A, intermediate portions 60A, and lower portions 70A may be formed to exhibit substantially rotational symmetry about the central axis of the objective lens 1330A. The central axis may be, for example, the optical axis of the objective lens 1330A. 【0125】 The intermediate portion 60A may be located above the lower portion 70A. The intermediate portion 60A may include an inlet port 62 and an outlet port 63. Fluid may be pumped into the sleeve 1332A through the inlet port 62 and out of the sleeve 1332A through the outlet port 63 by a fluid pump system (see, for example, Figure 20). The inlet port 62 and the outlet port 63 may be located apart from each other, with the sleeve 1332A in between. However, the positions of the inlet port 62 and the outlet port 63 are not limited to this configuration and can be varied in various ways. According to one embodiment, the inlet port 62 and the outlet port 63 may be composed of a single port. 【0126】 The intermediate portion 60A may further include a tapered portion 64A that follows the contour of the objective lens 1330A. For example, the tapered portion 64A may extend upward and radially inward from the outer portion of the intermediate portion 60A. The tapered portion 64A may be formed to substantially exhibit rotational symmetry about the central axis of the objective lens 1330A. According to the embodiment, the tapered portion 64A may have a shape other than tapered, as long as its shape follows the contour of the objective lens 1330A. The shape of the tapered portion 64A (e.g., an inverted V shape along the contour of the objective lens 1330A) allows the droplet 90 on the objective lens 1330A to be shaped as desired for immersion. According to the embodiment, the tapered portion 64A may alternatively be referred to as a “projection”. 【0127】 According to one embodiment, the inlet port 62 may include a passage that extends through the tapered portion 64A to the inside of the tapered portion 64A, so as to be configured to supply liquid for the droplet 90 into the space between the objective lens 1330A and the tapered portion 64A. 【0128】 The upper portion 50A may include a main body. For example, the main body may include a side wall 52A extending upward from the intermediate portion 60A and a top wall 53A extending radially inward from the side wall 52A. The side wall 52A and the top wall 53A may extend from each other at substantially 90-degree angles. However, the angles are not limited thereto and can be varied depending on the embodiment. The main body, including the side wall 52A and the top wall 53A, may be formed to exhibit substantially rotational symmetry about the central axis of the objective lens 1330A. 【0129】 A groove 84 may be formed between the upper portion 50A and the intermediate portion 60A. For example, the groove 84 may be defined by the inner surface of the upper wall 52, the inner surface of the side wall 53, and the outer surface of the tapered portion 64A. According to one embodiment, the groove 84 may be formed to exhibit substantially rotational symmetry about the central axis of the objective lens 1330A. The groove 84 may be configured to receive and contain an excess amount of liquid. According to one embodiment, the groove 84 may communicate with an outlet port 63 such that the excess amount of liquid in the groove 84 exits the sleeve 1332A through a passage of the outlet port 63 communicating with the groove 84. 【0130】 Referring to Figures 22C and 22D, at least the upper surface of the upper wall 53A can constitute the upper surface 10A of the sleeve 1332A, which is coplanar with the upper surface 11A of the objective lens 1330A. According to this embodiment, the upper surface of the tapered portion 64 may also be coplanar with the upper surface 11A of the objective lens 1330A. 【0131】 According to the embodiment, one or more O-rings 32 may be provided between the sleeve 1332A and the objective lens 1330A. For example, the O-ring 32 may be provided between the intermediate portion 60A and the objective lens 1330A. The O-ring 32 can be configured to seal the bottom side of the space in which liquid is received between the objective lens 1330A and the tapered portion 64A. 【0132】 Referring to Figure 22D, a microplate 80 holding a sample in at least one well 82 may be positioned directly above the sleeve 1332A and the objective lens 1330A. A droplet 90 on the lens of the objective lens can contact the bottom surface of the microplate 80 at a position directly below the well 82. The microplate 80 may correspond to, for example, the microplate 300 described herein, or other microplates. 【0133】 Referring to Figures 23A to 23C, the upper surface 10B of the sleeve 1332B may be above the upper surface 11B of the lens of the objective lens 1330B, and the sleeve 1332B may be configured to clamp (fix) to the objective lens 1330B. 【0134】 The sleeve 1332B may include, for example, an upper portion 50B, an intermediate portion 60B, and a lower portion 70B. 【0135】 The intermediate portion 60B may include a tapered portion 64B, and the upper portion 50B may include a body including a side wall 52B and an upper wall 53B. At least the upper surface of the upper wall 53B may constitute the upper surface 10B of the sleeve 1332B, which is above the upper surface 11B of the lens of the objective lens 1330B. According to the embodiment, the upper surface of the tapered portion 64B may also be above the upper surface 11B of the lens of the objective lens 1330B and coplanar with the upper surface of the upper wall 53B. 【0136】 Referring to Figures 24A to 24C, the upper surface 10C of the sleeve 1332C may be below the upper surface 11C of the lens of the objective lens 1330C, and the sleeve 1332C may be configured to clamp onto the objective lens 1330C. 【0137】 The sleeve 1332C may include, for example, an upper portion 50C, an intermediate portion 60C, and a lower portion 70C. 【0138】 The intermediate portion 60C may include a tapered portion 64C, and the upper portion 50C may include a body including a side wall 52C and an upper wall 53C. At least the upper surface of the upper wall 53C may constitute the upper surface 10C of the sleeve 1332C, which is located below the upper surface 11C of the lens of the objective lens 1330C. According to the embodiment, the upper surface of the tapered portion 64C may also be located below the upper surface 11C of the lens of the objective lens 1330C, or it may be coplanar with the upper surface of the upper wall 53C. 【0139】 Referring to Figures 25A to 25C, the upper surface 10D of the sleeve 1332D may be coplanar with the upper surface 11D of the lens of the objective lens 1330D, and the sleeve 1332D may be configured to be screwed onto the objective lens 1330D. 【0140】 According to one embodiment, the inner surface of the sleeve 1332D and the outer surface of the objective lens 1330D may include corresponding and engaging threads so that the sleeve 1332D and the objective lens 1330D can be attached to and detached from each other by the rotational movement of at least one of the sleeve 1332D and the objective lens 1330D. 【0141】 The sleeve 1332D may include, for example, a first portion 60D and a second portion 50D. 【0142】 The first portion 60D may include a tapered portion 64D, and the second portion 50D may include a body including a side wall 52C and an upper wall 53C. At least the upper surface of the upper wall 53D may constitute the upper surface 10D of the sleeve 1332D, which is coplanar with the upper surface 11D of the lens of the objective lens 1330D. According to the embodiment, the upper surface of the tapered portion 64D may also be coplanar with the upper surface 11D of the lens of the objective lens 1330D. 【0143】 According to one embodiment, the inner surface of the first portion 60D may include threads. 【0144】 According to the embodiment, the upper surface 10D of the sleeve 1332D may be above or below the upper surface 11D of the objective lens 1330D. For example, the upper surface of the upper wall 53D may be above or below the upper surface 11D of the objective lens 1330D, and the upper surface of the tapered portion 64D may be coplanar with the upper surface of the upper wall 53D. 【0145】 Various embodiments of confocal microscopy may be provided alternatively or additionally according to embodiments of the present disclosure. For example, a laser point scanning confocal system may be provided. Laser point scanning confocal microscopy may involve focusing a single point of laser light through a small aperture (pinhole) and sequentially scanning the sample point by point in a zigzag pattern. The sample fluoresces, and the light is sent back through the optical system. The light is then read point by point by a detector, which may be a photomultiplier tube (PMT), but can also be detected using other optical measuring sensors. The signal from the sensor may be recorded point by point, and each point may constitute a single pixel in the image. Laser point scanning systems have advantages and disadvantages over spinning disk confocal systems. Laser point scanning systems are typically slower than spinning disk confocal systems and are therefore often unsuitable for high-throughput applications or live cell imaging. On the other hand, laser point scanning confocal systems penetrate deeper into the sample and provide better axial and transverse resolution. Recently, improvements have been made to laser point scanning systems to increase speed, and thus they are beginning to rival spinning disk speeds while still offering increased penetration depth. The speed of a laser point scanning confocal system is limited by the scanning speed of the motor that drives the system's scanning mirror. 【0146】 According to embodiments, the confocal subsystem of the present disclosure may comprise both laser point scanning confocal and spinning disk confocal systems. The spinning disk confocal system may be used for raw sample imaging and high-throughput applications, while the laser point scanning confocal system may be used for deeper penetration into a sample with increased resolution. As with how wide-field imaging or other measurement modalities can be used to provide a "hit," embodiments of the present disclosure may implement spinning disk confocal to rapidly scan through a 3D sample and locate a certain point of interest. A laser point scanning system can then be used to acquire a more detailed image of the region of interest. Both laser point scanning confocal systems and spinning disk systems are commercially available as two separate instruments. However, there are several problems with using two separate instruments in this manner. For one, the cost of both the spinning disk and laser confocal microscopes makes such workflows impractical. Furthermore, there are technical problems with repositioning the region of interest on an alternative microscope. With both a laser point scanning confocal system and a spinning disk system implemented in the same instrument, a "hit" can be detected, and then the optics can be switched without moving the stage to scan the region of interest. Finally, there is the problem of studying living cells where the sample changes over time. Moving the sample to a different instrument takes too long compared to the rate of biological change. When moving the sample to a different instrument, the region of interest of the "hit" may have changed and may no longer be relevant. 【0147】 Another advantage of having both laser spot scanning confocal and spinning disk confocal systems in the same instrument is that the laser spot scanning confocal system can be used not for imaging, but for targeting specific areas of a sample to photobleach. The laser spot scanning confocal system, along with specific controls for the XY scanning mirrors within it, allows for targeting of very small, specific areas of a sample using a laser. This may be a single spot or a block defined by a zigzag scan. Once photobleaching occurs, the instrument can then quickly switch to spinning disk confocal to monitor fluorescence recovery after photobleaching (FRAP). Some specific applications include (a) analysis of intracellular molecular diffusion (e.g., studying F-actin diffusion in primary dendritic cells after photobleaching of regions of interest), (b) quantification of biological membrane fluidity (e.g., membrane fluidity in C. elegans), and (c) analysis of protein binding (e.g., monitoring of dynamic binding of chromatin proteins in vivo). 【0148】 The pinpoint accuracy of a laser point scanning confocal system combined with the imaging speed of a spinning disk system, as embodied in this disclosure, addresses an unmet market need in FRAP assays. 【0149】 Referring to Figures 22A and 22B, a configuration according to an embodiment of this disclosure, which includes a laser point scanning confocal system, a spinning disk confocal system, and a wide-field function within a single device, is described below. However, embodiments of this disclosure may include any combination of the above systems and functions. 【0150】 Figure 22A shows the instrument configured in a laser point scanning confocal (LSC) modality. Figure 22B shows the instrument configured in a wide-field or spinning disk confocal modality. According to embodiments, a mechanism may be provided for switching between the LSC system and the wide-field or spinning disk confocal system. As shown in Figures 22A and 22B, elements within block 2220 are movable and enable switching between laser point scanning optics and spinning disk / confocal. For example, block 2220 may consist of multiple disk modules that can be moved for selection between disks (and thus modalities), as described in this disclosure. 【0151】 Referring to Figure 22A, embodiments of the present disclosure may include a laser point scanning confocal system. Typically, light from a laser source enters such a system in an optical input device 2201. The optical input device 2201 may be, for example, a fiber-coupled input or a directly coupled laser without a fiber. The light is then collimated as it passes through a lens 2202. The light then strikes a long-pass dicroic 2203. The long-pass dicroic 2203 is designed to reflect the input light and allow the passage of high-wavelength emitted light. It is typical for the light source to have multiple input wavelengths. 【0152】 Embodiments of the present disclosure can support automated means for switching the long-path dichroic 2203 to adapt to the input wavelength. The light is then reflected by a scanning mirror 2204. The scanning mirror 2204 can be controlled using two-axis motors 2205 and 2206. In some embodiments, both motors are Galvo-type motors, and in other embodiments, one motor is driven by a Galvo and the other is a resonant scanner. Resonant scanners are much faster than Galvo motors but offer less controllability for positioning. Both types of motors are known to those skilled in the art. According to embodiments, the scanning mirror 2204 may be configured as a plurality (e.g., two) separate scanning mirrors. For example, the plurality of separate scanning mirrors may include a first mirror configured for x-scanning and a second mirror configured for y-scanning, where the positioning of each separate scanning mirror can be controlled, for example, by their respective motors. 【0153】 After the light is reflected from the scanning mirror 2204, it passes through the focusing lens 2207 and then the tube lens 2208. The light then proceeds through the reflecting mirror 2209, the objective lens 2210, and finally to the sample 2211, where the spot illuminated on the sample can be minute. Assuming the sample is fluorescent, the light then proceeds backward through the laser spot scanning system to the long-pass dichroic 2203. If the synchrotron radiation is within the passband of the long-pass dichroic 2203, the synchrotron radiation passes through the focusing lens 2213 and then through the pinhole 2214. The pinhole 2214 may be a single-size pinhole or may be of variable size. Size variation can be achieved by having multiple pinholes on a selector wheel or a variable iris. The light then passes through lens 2215 and then to the dichroic 2216. 【0154】 The arrangement shown in Figure 22A includes a dual PMT2218 configuration that allows for simultaneous measurement of multiple emission wavelengths. This configuration can be extended to include an additional number of PMT2218s. It may also be a single PMT2218 configuration, in which case the emission wavelength is selected via a dichroic 2216 and EM filter 2219 including a switching mechanism. The switching mechanism may be a cube and slider or multiple wheels, both of which will be understood by those skilled in the art. 【0155】 In laser point scanning systems, the position of the optical input device 2201 may require precise alignment with the pinhole 2214. This makes the implementation, installation, and maintenance of the laser point scanning system difficult. Typically, after shipment or maintenance, adjustments may be necessary to realign the pinhole 2214 to the fiber position. A solution to this problem is that both the optical input device 2201 (e.g., optical fiber input) and the pinhole 2214 are on motorized axes, and the equipment (e.g., its controller) can automatically align the optical input device 2201 and the pinhole 2214 by controlling the corresponding motors. Such an embodiment can provide benefits after shipment, after maintenance, or even with respect to thermal changes within the equipment. In addition, the fiber input position may be smaller than the pinhole size, allowing for some margin in the design. By using automated alignment, the pinhole size can be reduced, and therefore the confocality of the system can be increased, thereby increasing resolution and sample penetration. 【0156】 Figure 17 is a functional block diagram showing the control of modalities in the device according to this embodiment. 【0157】 The operation of the modality may be controlled by a central control unit (e.g., a processor, CPU, microprocessor, etc.). In some embodiments, the central control unit may also be referred to as a controller (e.g., controller 1000). 【0158】 The central control unit 900 may be connected to communicate with and control elements of the embodiments of the present disclosure. For example, the central control unit 900 may be connected to communicate with and control elements of the sample environment 90A, elements of the sample selection and positioning 90B, elements of the monochromator module 90C, elements of the imaging device module 90D, the external light source module 932, and the injection module 934. 【0159】 The elements of the controlled sample environment 90A can provide the temperature control 902 and gas control 904 described above. 【0160】 Sample selection and positioning 90B can be controlled through the use of a motor 906 for positioning the sample in any X direction and a motor 908 for positioning it in the Y direction. 【0161】 The elements of the controlled monochromator module 90C may include a monochromator excitation 910, a monochromator emission 912, a monochromator PMT 916, an optical fiber selector 918, and a light source such as a flash lamp 914. 【0162】 The elements of the controlled imaging device module 90D may include an objective lens selector 930, an image acquisition device such as a camera 920, a focus drive 924 for the objective lens, an LED and filter cube selector 922 for wide-field imaging, a confocal cube selector 928, a spinning disk module and control 926 (e.g., selection and focusing), and a laser scanning confocal module control 927. 【0163】 Figure 18 is a flowchart of a control method for a multi-detection system according to one exemplary embodiment. 【0164】 The control of the device may be coordinated through the use of a controller, for example, as described above with respect to Figure 17 and / or Figures 26A to 26B (i.e., Figure 17, or Figures 26A to 26B, or all of them). Input to the device (step S1805) may be achieved via the device's local user interface, such as a touchpad or graphical display, or via communication with the device via a wired or wireless connection, such as a network. 【0165】 In the case of input to a device, the input may be performed through the use of a user interface or graphical user interface displayed on a computer or other terminal running the control application. 【0166】 The input may also be user input such as settings and parameters for controlling the device. 【0167】 In response to receiving input, control of the instrument may be achieved, for example, through various elements of the instrument as discussed above with respect to Figures 17 and / or 26A to 26B. For example, in response to receiving user input, the instrument includes a gas control procedure for the gas module (step S1810), a sample positioning control procedure for controlling the positioning of the sample (step S1820), a monochromator control procedure for controlling the operation of the monochromator (step S1830), an imaging device control procedure for controlling the imaging device (step S1840), and outputting the results of the control of the elements of the instrument (step S1850). 【0168】 The control is presented as shown in Figure 18, but the elements may be controlled individually in any order, and control of all elements is not required. Therefore, multiple modalities of the instrument can be controlled in a single assay. 【0169】 The control method shown in Figure 18, and other functions described herein that can be performed by the controller, may be implemented through the execution of a processing unit (e.g., a CPU) that controls elements of the device by executing one or more control programs. The programs may be stored in memory (i.e., RAM, ROM, flash, etc.) or other computer-readable media (i.e., CD-ROM, disk, etc.). The programs may be executed locally by the device or by a control device, such as a computer, that transmits commands to be executed by the device. 【0170】 Referring to Figure 27, embodiments of the present disclosure may include a display, and the controller may be further configured to display a user interface on the display. Figure 27 shows an example of a user interface when the instrument has various optical mode combinations. Element 2300 is a sample image. Element 2301 is a drop-down menu for selecting magnification. Element 2302 is a selection box for enabling / disabling water immersion. When selected and configured to immerse the objective lens, the controller may automatically pump water into the objective lens and automatically remove the water when imaging is complete or when the checkbox in element 2302 is deselected. Element 2303 is a drop-down list for EM wavelength selection. Figure 27 shows that a selection between four different EM wavelengths may be provided, but any number of EM wavelength selections may be provided. Element 2304 is a drop-down list for EX wavelength selection. Figure 27 shows that a selection between four different EX wavelengths may be provided, but any number of EX wavelength selections may be provided. Element 2305 is a drop-down menu that allows selection between various modes of instruction. Figure 27 illustrates the selection between modalities, and the system includes spinning disk, laser scanning, and wide-field modalities. According to the embodiment, the modalities enumerated in element 2305 may depend on the modalities present in the system. The system may have, for example, any combination of the modalities described above (and / or additional modalities) (i.e., the modalities described above, or additional modalities, or both), or only a single modality. If only a single modality is provided, element 2305 may not be provided. According to the embodiment, elements 2301, 2302, 2303, 2304, and 2305 may not be limited to drop-down menus and selection boxes, but may present options for selection in any manner known to those skilled in the art. 【0171】 According to embodiments, the interface may include a display element that allows a user to select multiple modalities to be automatically executed in a sequence. For example, based on one or more user inputs to the interface, the controller may be configured to control the sequence to be executed automatically. The sequence may include any order of modality operations, including the order of modality operations described herein. For example, an operation using a spinning disk or a wide-field imaging system may be performed, followed by an operation using a laser point scanning confocal system. 【0172】 The embodiments of this disclosure are described for illustrative purposes only, and those skilled in the art will understand that various modifications, additions, and substitutions are possible without departing from the scope and spirit of this disclosure. The claims as originally filed are as follows: Claim 1: A receptacle support configured to support a microplate having microplate wells configured to hold a sample, A confocal imaging subsystem configured to image the sample at the cellular level, A non-imaging analysis subsystem configured to analyze the sample at the well level, comprising: a first measurement modality for measuring the absorbance of the sample; a second measurement modality for measuring the fluorescence of the sample; and a third measurement modality for measuring the chemiluminescence of the sample. A device that analyzes one or more samples. Claim 2: The device according to claim 1, further comprising a positioning subsystem common to both the confocal imaging subsystem and the non-imaging analysis subsystem, configured to position the receptacle support relative to the non-imaging analysis subsystem that analyzes the sample and the confocal imaging subsystem that images the sample. Claim 3: The device according to claim 2, further comprising an incubation chamber configured for incubating the sample. Claim 4: The device according to claim 3, further comprising a housing that forms the exterior of the device and accommodates the non-imaging analysis subsystem, the confocal imaging subsystem, and the positioning subsystem. Claim 5: The device according to claim 4, wherein the non-imaging analysis subsystem is configured to provide the first measurement modality, the second measurement modality, and the third measurement modality. Claim 6: The device according to claim 5, further comprising a temperature control subsystem configured to control the temperature around the sample. Claim 7: The device according to claim 6, further comprising a gas control subsystem configured to control the composition of the atmosphere surrounding the sample. Claim 8: The device according to claim 7, further comprising a processor configured to control the operation of the non-imaging analysis subsystem, the confocal imaging subsystem, the positioning subsystem, the temperature control subsystem, and the gas control subsystem. Claim 9: The device according to claim 8, further comprising a user interface configured to receive user input for controlling the operation of the non-imaging analysis subsystem, the confocal imaging subsystem, the positioning subsystem, the temperature control subsystem, and the gas control subsystem. Claim 10: A temperature control subsystem configured to control the ambient temperature around the sample, A gas control subsystem configured to control the ambient composition surrounding the sample, Furthermore, The device according to claim 1, wherein the aforementioned atmospheric composition contributes to cell viability. Claim 11: (i) the confocal imaging subsystem and the non-imaging analysis subsystem, and (ii) the receptacle support, share a commonly controlled atmospheric composition, the device according to claim 1. Claim 12: The device according to claim 1, wherein the confocal imaging subsystem, the non-imaging analysis subsystem, and the receptacle support share a commonly controlled atmospheric composition. Claim 13: The device according to claim 1, wherein the confocal imaging subsystem comprises an objective lens for imaging the sample. Claim 14: The device according to claim 1, wherein the confocal imaging subsystem comprises a plurality of objective lenses mounted on a turret, the plurality of objective lenses mounted on the turret being selectable to selectively image the sample. Claim 15: The aforementioned confocal imaging subsystem includes an imaging light source, The device according to claim 1, wherein the non-imaging analysis subsystem comprises at least one analysis light source separate from the imaging light source. Claim 16: The device according to claim 1, further comprising a processor configured to control the operation of the non-imaging analysis subsystem and the confocal imaging subsystem. Claim 17: The device according to claim 1, further comprising a user interface configured to receive user input for controlling the operation of the non-imaging analysis subsystem and the confocal imaging subsystem. Claim 18: The device according to claim 1, further comprising a housing that forms the exterior of the device and accommodates the non-imaging analysis subsystem and the confocal imaging subsystem. Claim 19: The device according to claim 1, wherein the non-imaging analysis subsystem is configured to provide the first measurement modality, the second measurement modality, and the third measurement modality. Claim 20: The device according to claim 13, wherein the objective lens is an immersion objective lens. Claim 21: A receptacle support configured to support a microplate having microplate wells configured to hold a sample, An objective lens configured to image the aforementioned sample, A laser point scanning confocal system configured to image the sample through the objective lens, A spinning disk or wide-field imaging system configured to image the sample through the objective lens, Equipped with, A device for analyzing a sample, wherein at least a portion of both the laser point scanning confocal system and the spinning disk or wide-field imaging system is movably mounted such that the laser point scanning confocal system and the spinning disk or wide-field imaging system are configured to be selectively aligned with the objective lens for imaging the sample. Claim 22: The device according to claim 21, wherein the objective lens is an immersion objective lens. Claim 23: The immersion objective lens is provided with a sleeve attached to the upper end of the objective lens. The aforementioned sleeve is A projection in the immersion objective lens that follows the contour of the lens toward the upper surface of the lens, wherein a space is provided between the projection and the lens of the immersion objective lens, and the space is configured to receive the liquid supplied to the upper surface of the lens for immersion, An upper wall, wherein a groove is provided between the upper wall and the protrusion, and the groove is configured to receive a portion of the liquid after a portion of the liquid has been supplied to the upper surface of the lens, and The device according to claim 22, comprising: Claim 24: The device according to claim 23, wherein the upper surface of the sleeve is coplanar with the upper surface of the lens. Claim 25: The display and A controller configured to control the laser point scanning confocal system and the spinning disk or wide-field imaging system, and further configured to display an interface on the display that allows the user to choose between using the laser point scanning confocal system and using the spinning disk or wide-field imaging system. The device according to claim 21, further comprising the following: Claim 26: The device according to claim 25, wherein the controller is further configured to perform an operation using the laser point scanning confocal system based on the user's input to the interface, and then perform an operation using the laser point scanning confocal system.

Claims

[Claim 1] A device for analyzing one or more samples, A receptacle support configured to support a microplate having microplate wells configured to hold a sample, A confocal imaging subsystem configured to image the sample at the cellular level, wherein light from an imaging light source is reflected by a dichroic mirror and emitted onto the sample via a spinning disk, and the light from the sample reaches a detector via the spinning disk and the dichroic mirror, A non-imaging analysis subsystem configured to analyze the sample at the well level, comprising at least one analytical light source separate from the imaging light source, and configured to provide at least one of a first measurement modality for measuring the absorbance of the sample, a second measurement modality for measuring the fluorescence of the sample, and a third measurement modality for measuring the chemiluminescence of the sample, The spinning disk is housed in a disk module that is movable relative to the optical path and has a dustproof window through which light can pass, so that the spinning disk can be positioned inside or outside the optical path, The disk module is a device having a glass optical element in the space located within the optical path when the spinning disk is positioned outside the optical path, for matching the optical path length in a non-parallel optical path. [Claim 2] The device according to claim 1, further comprising a positioning subsystem common to both the confocal imaging subsystem and the non-imaging analysis subsystem, configured to position the receptacle support relative to the non-imaging analysis subsystem that analyzes the sample and the confocal imaging subsystem that images the sample. [Claim 3] The device according to claim 2, further comprising an incubation chamber configured for incubating the sample. [Claim 4] The device according to claim 3, further comprising a housing that forms the exterior of the device and accommodates the non-imaging analysis subsystem, the confocal imaging subsystem, and the positioning subsystem. [Claim 5] The device according to claim 4, wherein the non-imaging analysis subsystem is configured to provide the first measurement modality, the second measurement modality, and the third measurement modality. [Claim 6] The device according to claim 5, further comprising a temperature control subsystem configured to control the temperature around the sample. [Claim 7] The device according to claim 6, further comprising a gas control subsystem configured to control the composition of the atmosphere surrounding the sample. [Claim 8] The device according to claim 7, further comprising a processor configured to control the operation of the non-imaging analysis subsystem, the confocal imaging subsystem, the positioning subsystem, the temperature control subsystem, and the gas control subsystem. [Claim 9] The device according to claim 8, further comprising a user interface configured to receive user input for controlling the operation of the non-imaging analysis subsystem, the confocal imaging subsystem, the positioning subsystem, the temperature control subsystem, and the gas control subsystem. [Claim 10] A temperature control subsystem configured to control the ambient temperature around the sample, A gas control subsystem configured to control the ambient composition surrounding the sample, Furthermore, The device according to claim 1, wherein the aforementioned atmospheric composition contributes to cell viability. [Claim 11] The device according to claim 1, wherein (i) one of the confocal imaging subsystem and the non-imaging analysis subsystem and (ii) the receptacle support share a commonly controlled atmospheric composition. [Claim 12] The device according to claim 1, wherein the confocal imaging subsystem, the non-imaging analysis subsystem, and the receptacle support share a commonly controlled atmospheric composition. [Claim 13] The device according to claim 1, wherein the confocal imaging subsystem comprises an objective lens for imaging the sample. [Claim 14] The device according to claim 1, wherein the confocal imaging subsystem comprises a plurality of objective lenses mounted on a turret, and the plurality of objective lenses mounted on the turret are selectable to selectively image the sample. [Claim 15] The device according to claim 1, further comprising a processor configured to control the operation of the non-imaging analysis subsystem and the confocal imaging subsystem. [Claim 16] The device according to claim 1, further comprising a user interface configured to receive user input for controlling the operation of the non-imaging analysis subsystem and the confocal imaging subsystem. [Claim 17] The device according to claim 1, further comprising a housing that forms the exterior of the device and accommodates the non-imaging analysis subsystem and the confocal imaging subsystem. [Claim 18] The device according to claim 1, wherein the non-imaging analysis subsystem is configured to provide the first measurement modality, the second measurement modality, and the third measurement modality. [Claim 19] The device according to claim 13, wherein the objective lens is an immersion objective lens.

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