CN114527126A - Clinical microbial unicellular drug resistance detection instrument and detection method - Google Patents

Abstract

The invention relates to a clinical microorganism unicellular drug resistance detection instrument and a detection method, wherein the instrument comprises an electric displacement platform, an optical tweezers module, an imaging module, an excitation optical module, a micro focusing module, a Raman main optical path and transmission module, a coaxial illumination module and an automatic acquisition control module; the drug resistance detection of the microbial sample in a dry plate and a static and flowing state in a liquid phase is realized by combining the optical tweezers module to capture the cells to be detected in the liquid phase state in the sample chip. The detection instrument has multiple working modes, and drug resistance detection of different requirements is met; the optical box body is integrally cast, has a totally-enclosed design and good light shielding property, prevents bacterial pollution, can greatly improve the stability of the instrument and ensures the accuracy of a single cell detection result.

Description

Clinical microbial unicellular drug resistance detection instrument and detection method

Technical Field

The invention relates to the technical field of microbial detection, in particular to a clinical microbial unicellular drug resistance detection instrument and a detection method.

Background

The traditional clinical detection of the drug resistance of pathogenic bacteria mainly adopts a culture method, and the method has the defects of long detection time, high professional requirements on experiment operators, easy occurrence of false positive and the like. At present, the suppression of the spread of drug resistance not only needs to develop novel antibiotics, but also needs to develop a drug resistance quick detection instrument and method to improve the pertinence and effectiveness of the use of the existing antibiotics, so as to delay and suppress the spread of the drug resistance. At present, one of the most promising directions is the 'single cell' drug-resistant detection technology, namely, skipping the cell culture proliferation, directly aiming at the 'growth' or 'metabolism' phenotype of the original single cell in the sample, and performing the characterization of the single cell precision, thereby realizing the rapid phenotype-based wide-application range target in principle.

Raman spectroscopy is an efficient information identification technology, and through inelastic scattering spectral line analysis of a compound by specific incident light, Raman microscopic spectroscopy can directly detect the molecular vibration or rotation energy level of the compound. Through the analysis of Raman characteristic spectral lines, the information of the molecular composition and the structure of the compound can be obtained. However, the existing instrument using the raman spectroscopy detection technology to detect the drug resistance of the clinical microbial unicells can only realize dry-chip detection and cannot detect the microbial samples in a static state and a flowing state in a liquid phase.

Disclosure of Invention

In view of the above problems, the present invention is to provide an apparatus and a method for detecting drug resistance of single cells of a clinical microorganism, which can detect drug resistance of single cells of the clinical microorganism and can detect drug resistance of a microorganism sample in a static state or a flowing state in a dry sheet or a liquid phase.

In order to achieve the purpose, the invention adopts the following technical scheme:

a clinical microorganism unicellular drug resistance detecting instrument comprises an electric displacement platform, an optical tweezer module, an imaging module, an excitation optical module, a micro-focusing module, a Raman main optical path and transmission module, a coaxial illumination module and an automatic acquisition control module;

the electric displacement platform is used for placing a sample chip;

the optical tweezers module is used for capturing cells to be detected in a liquid phase state in the sample chip and locking the cells to be detected;

the imaging module is used for shooting panoramic information of a sample chip, rapidly positioning the cell to be detected on the sample chip by matching with the electric translation platform and determining the acquisition position of the cell to be detected;

the excitation light module is used for emitting laser;

the micro-focusing module is used for automatically focusing laser to the cell to be detected at the collecting position so as to enable the cell to be detected to generate a Raman signal;

the Raman main light path and transmission module is used for acquiring a Raman spectrum of the cell to be detected;

the coaxial illumination module is used for providing coaxial illumination light for the micro-focusing module;

the automatic acquisition control module is used for controlling the excitation optical module, the Raman main optical path and transmission module, the micro-focusing module, the coaxial illumination module, the imaging module, the optical tweezers module and the electric displacement platform, processing the Raman spectrum of the acquired cell to be detected and realizing the automatic detection of the cell to be detected.

Preferably, the excitation light module comprises a first laser, a first electric shutter, a first beam expander, an electric adjustable attenuator and a first reflector; the micro-focusing module comprises a micro objective lens; the Raman main light path and transmission module comprises a spectroscope, a first dichroic mirror, a second lens, a pinhole, a spectrometer and a detector;

laser emitted by the first laser sequentially passes through the first electric shutter, the first beam expander, the electric adjustable attenuator and the first reflector, passes through the spectroscope and the first dichroic mirror, passes through the microscope objective lens, and is focused on a cell to be detected to generate a Raman spectrum, the Raman spectrum passes through the first dichroic mirror, the spectroscope, the second lens, the pinhole and the spectrometer and is sent to the detector, the Raman information of the cell to be detected is acquired, and the light path mode is a Raman mode.

Preferably, the coaxial lighting module comprises an LED light source and a half-transmitting and half-reflecting mirror; the imaging module comprises a CCD camera and a first lens;

white light emitted by the LED light source enters the micro-focusing module through the reflection of the semi-transparent semi-reflector to illuminate cells to be detected, reflected light carrying image information of the cells to be detected enters the imaging module to obtain image information of the sample chip, and the light path mode is an imaging mode.

Preferably, the optical tweezers module comprises a second laser, a second electric shutter, a second attenuator, a second beam expander, a second reflector and a second dichroic mirror;

and the laser emitted by the second laser passes through a second electric shutter, a second attenuator, a second beam expander, a second reflector and a second dichroic mirror and then passes through the microscope objective lens to capture the cells, so as to obtain the image information of the cells in a liquid phase state, wherein the light path mode is an optical tweezers mode.

Preferably, the automatic microorganism Raman drug sensitive rapid detection instrument comprises four working modes:

the imaging mode is used for acquiring image information of the cell to be detected;

the imaging mode-Raman mode is used for realizing the acquisition of Raman information of the cells to be detected;

an optical tweezers mode-imaging mode for acquiring image information of cells in a liquid phase state;

the optical tweezers mode-Raman mode is used for realizing the acquisition of Raman information of cells in a liquid phase state.

Preferably, the junction of the raman main light path and transmission module and the imaging module is provided with the spectroscope, the junction of the optical tweezers module and the raman main light path and transmission module is provided with a dichroic mirror, and the imaging mode, the imaging mode-raman measurement mode, the optical tweezers mode-imaging mode and the optical tweezers mode-raman measurement mode are switched by controlling the movement of the spectroscope and/or the dichroic mirror.

Preferably, the detection apparatus further comprises a sterilization module for eliminating microorganisms in the environment to prevent cross-contamination.

Preferably, the excitation light module, the raman main light path and transmission module, the coaxial illumination module, the imaging module and the optical tweezers module are fixed inside an optical box body, and the optical box body is of a fully-closed structure.

The sample detection method of the clinical microorganism unicellular drug resistance detection instrument comprises imaging detection, imaging-Raman detection, optical tweezers-imaging detection or optical tweezers-Raman detection;

imaging detection: white light emitted by the LED light source enters the micro-focusing module through the reflection of the semi-transparent semi-reflector to illuminate cells to be detected, reflected light carrying image information of the cells to be detected enters the imaging module, and the acquisition position of the cells is positioned after the image information of the sample chip is processed;

imaging-raman detection: the LED light source of the coaxial illumination module is closed, the second electric shutter of the optical tweezers module is closed, the spectroscope is moved into the main light path, the first electric shutter of the excitation light module is opened, laser emitted by the first laser enters the micro focusing module through the spectroscope to illuminate a sample to be detected, according to a cell image obtained by imaging detection, the three-dimensional electric displacement platform moves a cell to a focusing point of a micro objective lens so as to generate a Raman signal, the generated Raman signal and stray light are collected by the micro objective lens, collimated and returned, and are reflected by the spectroscope to enter the Raman main light path and the transmission module, and are projected onto a detector through the first dichroic mirror, the second lens, the pinhole and the spectrometer in sequence, so that Raman information of the cell to be detected of the sample chip is acquired;

optical tweezers-imaging detection: changing the placing mode of a sample to be detected, moving the second dichroic mirror into a main light path, closing a first electric shutter of the excitation light module, moving the spectroscope into the main light path, further acquiring image information of the sample to be detected through the imaging detection mode, finding cells needing to be captured by the optical tweezers, and enabling laser emitted by the optical tweezers module to sequentially pass through the second electric shutter, a second attenuator, a second beam expander, a second reflecting mirror and a second dichroic mirror; capturing the cells after passing through a microscope objective to obtain image information of the cells in a liquid phase state in the sample chip;

optical tweezers-raman detection: changing the placing mode of a sample to be detected, moving the second dichroic mirror into a main optical path, closing the first electric shutter of the excitation optical module, moving the spectroscope into the main optical path, obtaining image information of the sample to be detected through the imaging detection mode, finding out cells needing to be captured by the optical tweezers, opening the second electric shutter, enabling the optical tweezers module to emit laser to capture the cells, opening the first electric shutter, enabling the laser emitted by the first laser to enter the micro focusing module through the spectroscope to illuminate the sample to be detected, and obtaining Raman spectrum information of the cells through the Raman main optical path and the transmission module, wherein the Raman spectrum information is used for realizing the acquisition of Raman information of the cells in a liquid phase state in the sample chip.

Due to the adoption of the technical scheme, the invention has the following advantages:

1. the clinical microbial unicellular drug resistance detection instrument and the detection method provided by the invention are combined with the optical tweezers module to realize rapid drug resistance detection of the microbial sample in a static and flowing state in a dry plate and a liquid phase.

2. The clinical microorganism unicellular drug resistance detection instrument and the detection method provided by the invention have multiple working modes, realize cell imaging, Raman detection and cell optical tweezers capture, and meet drug resistance detection of different requirements.

3. The clinical microbial unicellular drug resistance detection instrument and the detection method provided by the invention have the advantages that the optical box body is integrally cast, the totally-enclosed design is realized, the light shading performance is good, the bacterial pollution is prevented, the instrument stability can be greatly improved, and the accuracy of a unicellular detection result is ensured.

Drawings

FIG. 1 is a schematic diagram of an optical path of a detecting apparatus provided in embodiment 1 of the present invention;

FIG. 2 is a schematic structural diagram of an optical box of a detection instrument according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a cell location according to an embodiment of the present invention;

FIG. 4 is a schematic diagram showing the variation of the Raman spectrum of single cells of helicobacter pylori in heavy water medium with different concentrations according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that the terms "upper", "lower", "front", "rear", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the system or component in question must have a specific orientation, be constructed in a specific orientation, and be operated, and thus should not be construed as limiting the present invention.

In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "assembled", "disposed" and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a detachable connection, or an integral connection; can be mechanically or electrically connected; the two components can be directly connected or indirectly connected through an intermediate medium, and the two components can be communicated with each other. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

The clinical microbial unicell drug resistance detection instrument and the detection method provided by the invention can be used for carrying out drug resistance detection on the clinical microbial unicells, and realize the automatic rapid drug resistance detection of the microbial samples in a static and flowing state in a dry sheet and a liquid phase through the mutual cooperation of the excitation optical module, the micro-focusing module, the Raman main optical path and transmission module, the coaxial illumination module, the imaging module, the optical tweezers module, the electric displacement platform and the automatic acquisition control module.

The following describes the apparatus and method for detecting single cell drug resistance of a microorganism in clinical practice in detail with reference to the accompanying drawings.

Example 1

As shown in fig. 1, the clinical microbial unicell drug resistance detection instrument provided in this embodiment includes an electric displacement platform, an optical tweezer module, an imaging module, an excitation optical module, a micro-focusing module, a raman main optical path and transmission module, a coaxial illumination module, and an automatic acquisition control module;

the electric displacement platform is used for placing a sample chip;

the optical tweezers module is used for capturing cells to be detected in a liquid phase state in the sample chip and locking the cells to be detected;

the imaging module is used for shooting panoramic information of the sample chip, obtaining position information of each cell, forming position correction information obtained by comparing each cell with a preset cell, rapidly positioning the cell to be detected on the sample chip by matching with the electric translation platform, and determining the acquisition position of the cell to be detected;

the exciting light module is used for emitting laser;

the micro-focusing module is used for automatically focusing laser to the cell to be detected at the collecting position so as to enable the cell to be detected to generate a Raman signal;

the Raman main light path and transmission module is used for acquiring the Raman spectrum of the cell to be detected and transmitting the Raman spectrum information of the cell to the automatic acquisition control module;

the coaxial illumination module is used for providing coaxial illumination light for the micro-focusing module;

and the automatic acquisition control module is used for controlling the excitation light module, the Raman main light path and transmission module, the micro-focusing module, the coaxial illumination module, the imaging module, the optical tweezers module and the electric displacement platform, and processing the Raman spectrum of the cell to be detected so as to realize automatic detection of the cell to be detected.

In this embodiment, the excitation light module includes a first laser 1, a first electrically-operated shutter 2, an electrically-operated adjustable attenuator 3, a first beam expander mirror 4, and a first mirror 5; the micro-focusing module comprises an objective turntable and a micro objective 8; the electric displacement platform is a three-dimensional electric displacement platform 9; the Raman main light path and transmission module comprises a spectroscope 7, a first dichroic mirror 6, a second lens 14, a pinhole 15, a spectrometer 16 and a detector 17; the sterilization module comprises an ultraviolet lamp.

Laser emitted by the first laser 1 sequentially passes through the first electric shutter 2, the first beam expander 4, the electric adjustable attenuator 3 and the first reflector 5, the spectroscope 7 and the first dichroic mirror 6 and the microscope objective 8 to focus a cell to be detected to generate a Raman spectrum, the Raman spectrum passes through the first dichroic mirror 6, the spectroscope 7, the second lens 14, the pinhole 15 and the spectrometer 16 and then is sent to the detector 17, the Raman information of the cell to be detected is acquired, and the light path mode is a Raman mode.

More specifically, the first laser 1 may be a 532nm or 633nm or 785nm or 1064nm single longitudinal mode solid-state laser for serving as a laser source for exciting a raman signal; the electric shutter 2 can be electrically controlled to be opened and closed and is used for controlling the physical on-off of the first laser 1 in the optical path; the beam expanding ratio of the first beam expander 3 is 3:1, 4:1 or 5: 1; the electric adjustable attenuator 4 has different attenuation rates, and the attenuation effect can be adjusted through the automatic acquisition control module; the attenuation effect is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, and the laser emitted by the first laser 1 is attenuated in different proportions; the first reflector 5 is used for making a 45-degree turn on the laser light emitted by the first laser 1.

More specifically, the microscope objective 8 is arranged on the objective turntable and used for focusing laser and facilitating imaging of a sample to be detected; the objective turntable is an electric or manual objective turntable with 4 holes, 5 holes or 6 holes and is used for switching objectives with different multiplying powers or different NA values; the micro objective 8 can be a 4-fold, 10-fold, 50-fold or 100-fold double/semi-apochromatic objective or a water immersion objective; the three-dimensional electric displacement platform 9 is used for placing a sample chip to be detected and can control the three-axis movement through the automatic acquisition control module; the three-dimensional electric displacement platform 9 is provided with a sample clamp, a sample to be detected is placed in the sample clamp, three-dimensional movement can be controlled through the automatic acquisition control module, the minimum step value of movement is 0.1 mu m, the repeated positioning precision in the movement process is 0.5 mu m, and the three-dimensional electric displacement platform 9 and the left, right and rear three groups of support frames of the optical box body 30 are arranged on the same optical bottom plate.

More specifically, the spectroscope 7 is a broadband reflecting mirror, the spectroscope 7 is mounted on a one-dimensional electric displacement table and used for moving the spectroscope 7 into or out of an optical axis between the microscope objective 8 and the half-transmitting and half-reflecting mirror 11, the bandwidth range of the spectroscope 7 covers the excitation light of the first laser 1 and the excitation light of the second laser 18, and the reflectivity is higher than 90%; the beam splitter 7 may also be a total reflection mirror, which totally reflects the raman signal. The first dichroic mirror 6 is a high-pass low-reflection mirror, and the first dichroic mirror 6 forms an included angle of 45 degrees with the optical axis, and has the function of enabling Raman excitation laser. For example, laser light at 532nm is reflected and raman signals above 532nm are transmitted, both reflectance and transmittance being above 90%. The second lens 14 is used for focusing the transmitted raman signal light at the pinhole of the pinhole 15; pinhole 15 has different sizes, and the accessible automatic acquisition control module is adjusted, prevents that the unnecessary signal from reaching the detector beyond the microscope focal plane, plays the effect of spatial filtering, improves the spatial resolution of instrument for block stray light's signal under the confocal condition. The spectrometer 16 is a grating spectrometer, and may be equipped with gratings of different lines, so as to perform different degrees of light splitting on the raman light, and the raman signal after light splitting is imaged and received by the detector 17. The detector 17 is a linear array or an area array CCD or an EMCCD, and can obtain signals of raman scattered light of different resolutions in cooperation with the spectrometer 16.

In this embodiment, the coaxial illumination module includes an LED light source 10 and a half-mirror 11, and the imaging module includes a CCD camera and a first lens; the imaging module includes a CCD camera 13, a first lens 12.

More specifically, white light emitted by the LED light source 10 is reflected by the half-mirror 11 and enters the micro-focusing module to illuminate cells to be measured, and reflected light carrying image information of the cells to be measured enters the imaging module to obtain image information of the sample chip, where the light path mode is an imaging mode.

More specifically, the LED light source 10 is a micro-lighting light source, and the emitted white light is collimated; the half-transmitting and half-reflecting mirror 11 is arranged at an angle of 45 degrees with the optical axis, the transmission inverse ratio is 1:1, the half-transmitting and half-reflecting mirror 11 can reflect white light into the microscope objective 8, and white light imaging of cells can be reflected into the microscope objective 8 so as to penetrate through the half-transmitting and half-reflecting mirror 11.

More specifically, the image signal reflected and collected by the microscope objective 8 passes through the half mirror 11 and enters the first lens 12 to be focused on the CCD camera 13.

In this embodiment, the optical tweezers module comprises a second laser 18, a second electrically operated shutter 19, a second attenuator 20, a second beam expander lens 21, a second reflector 22, and a second dichroic mirror 23.

The laser emitted by the second laser 18 passes through the second electric shutter 19, the second attenuator 20, the second beam expander 21, the second reflector 22 and the second dichroic mirror 23, and then passes through the microscope objective 8 to capture the cells, and image information of the cells in the liquid phase state is obtained, wherein the light path mode is an optical tweezers mode.

More specifically, the second laser 18 is a 1064nm solid-state laser with a maximum power of 2W, a laser source for generating optical tweezers force; the electric shutter 19 can be electrically controlled to be opened and closed and is used for controlling the physical on-off of the second laser 18 in the optical path; the beam expansion ratio of the second beam expander 21 is 3:1, 4:1 or 5:1, and the transmittance of the second beam expander to 1064nm laser is greater than 85%; the second attenuator 20 has different attenuation rates, and the attenuation effect can be adjusted through the automatic acquisition control module; the attenuation effect is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%; the second reflector 22 is arranged behind the second beam expander 21, forms an angle of 45 degrees with the main optical axis, and is used for turning the laser after beam expansion by 45 degrees; the second dichroic mirror 23 is installed on the one-dimensional electric displacement platform, and forms an included angle of 45 degrees with the optical axis, the cut-off wavelength of the second dichroic mirror 23 is greater than the wavelength of the laser excited by the first laser 1 by more than 100nm, and the reflectivity of 1064nm is higher than 90%. The second dichroic mirror 23 forms an angle of 45 degrees with the optical axis, and can reflect the 1064nm laser light reflected by the second reflecting mirror 22 to the spectroscope 7 to enter the microscope objective lens 8; the one-dimensional electric displacement platform can be electrically controlled to enable the dichroic mirror 23 to leave the main light path, so that the optimal Raman signal is obtained; the spectroscope 7 can also be moved in and out of the main light path by a fixed one-dimensional electric displacement platform, so that each working mode is switched; further, the moving direction of the one-dimensional electric displacement platform under the second dichroic mirror 23 is perpendicular to the moving direction of the one-dimensional electric displacement platform under the dichroic mirror 7, and is used for moving the second dichroic mirror 23 into or out of the optical axis between the dichroic mirror 7 and the first dichroic mirror 6.

In this embodiment, the detecting instrument may further include a sterilization module including an ultraviolet lamp disposed around the microscope objective for performing sterilization operation to prevent the influence of bacterial contamination on the detection result of drug resistance.

In this embodiment, the automatic microorganism raman drug sensitive rapid detection instrument includes four working modes, which are an imaging mode, an imaging mode-raman measurement mode, an optical tweezers mode-imaging mode, and an optical tweezers mode-raman mode, respectively, where the imaging mode is used to obtain image information of a sample chip; the imaging mode-Raman measurement mode is used for realizing the acquisition of Raman information of cells to be detected of the sample chip; the optical tweezers mode-imaging mode is used for acquiring image information of cells in a liquid phase state in the sample chip; the optical tweezers mode-Raman mode is used for realizing the acquisition of cell Raman information in a liquid phase state in the sample chip.

More specifically, a spectroscope 7 is arranged at the intersection of the raman main light path, the transmission module and the imaging module, a second dichroic mirror 23 is arranged at the intersection of the optical tweezers module, the raman main light path and the transmission module, and an imaging mode, an imaging mode-raman measurement mode, an optical tweezers mode-imaging mode and an optical tweezers mode-raman mode are switched by controlling the movement of the spectroscope 7 and the second dichroic mirror 7.

As shown in fig. 2, in this embodiment, the excitation optical module, the raman main optical path and transmission module, the coaxial illumination module, the imaging module, and the optical tweezer module are fixed inside the same optical box 30, and all the modules are arranged in a planar common optical axis manner, that is, the excitation optical module, the raman main optical path and signal transmission module, the coaxial illumination module, and the camera observation module are fixed in the optical box in a planar layout manner, and optical axes of all optical elements are all in the same horizontal plane, so that the modules are relatively independent, that is, the debugging is convenient, and the mutual interference between the modules is reduced. All perpendicular with the optical axis or become 45 and place on the optical element overall arrangement of each module, each module debugging and system integration of being convenient for have also greatly reduced system later maintenance cost simultaneously. More specifically, the optical path of the optical tweezers module is parallel to the main raman optical path and the main optical path of the transmission module.

The optical box 30 includes a support bracket 31, which may include three groups, left, right, and rear, such that the optical box 30 has a predetermined height. The micro-focusing module is positioned at the lower part of the optical box body 30; the Raman main light path and the transmission module are positioned on one side in the box body and are positioned on the same reference surface with the micro-focusing module. The optical box body 30 is integrally cast and formed, and is designed in a totally enclosed manner, so that complete light shielding is realized, and the interference of an external stray light signal is avoided; on the other hand, the whole rigidity is excellent, reliable physical support can be provided for each module optical element, the interference of external force can be effectively blocked, the requirement that the whole machine can be used without re-debugging after moving or long-distance transportation can be met, and meanwhile, the whole machine can work stably for dozens of hours after continuous measurement; and finally, most of the optical part supporting and fixing mechanisms in the box body are locked after debugging is completed, the curing degree of the whole device is high, the vibration and impact resistance is improved, and the device has very high stability.

Example 2

The sample method of the clinical microorganism unicellular drug resistance detection instrument in the embodiment comprises the following steps of imaging detection, imaging-Raman detection, optical tweezers-imaging detection or optical tweezers-Raman detection:

the imaging detection is that the spectroscope 7 and the second dichroic mirror 23 are moved out of a main light path, the coaxial illumination module starts the LED light source 10, white light emitted by the LED light source 10 is reflected by the semi-transparent semi-reflective mirror 11 to enter the microscope objective 8, the microscope objective 8 focuses to illuminate a sample to be detected, reflected light carrying image information of the sample to be detected enters the imaging module, the microscope objective 8 collects collimation and returns to penetrate through the semi-transparent semi-reflective mirror 11, the light is focused on the CCD camera 13 through the first lens 12, the three-dimensional electric displacement platform 9 is adjusted to enable an observed image to move to the center of a view field, the brightness of the LED light source 10 is adjusted, CCD parameters enable the whole observed image to be clear, the imaging module obtains image information of a sample chip, and the acquisition position of a cell is positioned after the image information of the sample chip is obtained.

imaging-Raman detection, based on the imaging detection, a sample needing Raman detection is found, an LED light source 10 of a coaxial illumination module is closed, a second electric shutter 19 of an optical tweezers module is closed, the spectroscope 7 is moved into a main light path through a one-dimensional electric displacement platform at the spectroscope 7, a first electric shutter 2 of an excitation optical module is opened, laser emitted by a first laser 1 is attenuated by a first electric adjustable attenuator 3 and then enters a first beam expander 4, the laser is reflected to a first dichroic mirror 6 through a first reflector 5, the laser is reflected to the spectroscope 7 at 45 degrees and then vertically enters a microscope objective 8, and the laser is focused on the sample to be detected by the microscope objective 8. The three-dimensional electric displacement platform 9 judges the position information of the cell according to the cell image acquired by the imaging detection mode, and automatically moves the cell to the focusing point of the microscope objective 8 so as to generate a Raman signal, thereby realizing the purpose of automatic detection; the generated Raman signal and Rayleigh stray light are collected by the microscope objective and return in a collimated manner, are reflected by the spectroscope 7 to penetrate through the first dichroic mirror 6, are focused on the pinhole 15 by the second lens 14, are subjected to spatial filtering by the pinhole 15 and enter the spectrometer 16 for light splitting, Raman spectrum information is obtained by the detector 17, the Raman information of the cell to be detected of the sample chip is collected, and finally whether the cell is resistant to the drugs is analyzed by the automatic collection control module, and a drug resistance detection result is output.

Optical tweezers-imaging detection, wherein the placement mode of a sample to be detected is changed based on imaging detection, and specifically, the cell sample bacterial liquid after being cleaned is placed into a microfluidic chip, or a water immersion objective lens is adopted for observation; moving the second dichroic mirror 23 into the main optical path, closing the first electric shutter 2 of the excitation optical module, moving the spectroscope 7 into the main optical path, further acquiring image information of a sample to be detected through an imaging detection mode, finding a cell needing to be captured by the optical tweezers, opening the second electric door 19, capturing the cell by 1064nm laser emitted by the second laser 18, acquiring the image information of the cell in a liquid phase state in the sample chip, and then automatically moving the cell to a specified position by the three-dimensional electric displacement platform 9.

Optical tweezers-raman detection, based on imaging detection, changing the placement mode of a sample to be detected, specifically, placing a cell sample bacteria liquid into a microfluidic chip, or observing by using a water immersion objective lens, moving a second dichroic mirror 23 into a main optical path, closing a first electric shutter 2 of an excitation optical module, moving a spectroscope 7 into the main optical path, further obtaining image information of the sample to be detected by an imaging detection mode, finding a cell needing to be captured by optical tweezers, opening a second electric shutter 19, capturing the cell by 1064nm laser emitted by a second laser 18, closing an LED light source 10, opening the first electric shutter 2, allowing the laser emitted by a first laser 1 to enter a micro focusing module through the spectroscope 7 to illuminate the sample to be detected, detecting a cell raman signal, and obtaining raman spectrum information of the cell through the raman main optical path and a transmission module, as shown in fig. 3, and closing the electric shutter 2, acquiring image information of cells in a liquid phase state in the sample chip, analyzing whether drug resistance exists or not through the automatic acquisition control module, and then moving the three-dimensional electric displacement platform 9 to automatically move the drug-resistant cells to a specified position. The optical tweezers-Raman mode aims at partial cells with flagella or cells with cell position shift caused by Brownian motion, optical tweezers are adopted for capturing, Raman laser is used for exciting Raman signals to acquire Raman spectra, and accuracy of cell signal acquisition is guaranteed.

Example 3

In the above embodiment, the automatic acquisition control module includes a data analysis processing device, and the data analysis processing device may be a computer.

In this embodiment, the automatic acquisition control module controls the microscope objective 8 to automatically focus on the sample to be measured, and the control method includes:

and (4) focusing evaluation, namely introducing an evaluation function into the image observed by the CCD camera 13 to quantitatively evaluate the image, wherein the performance of the evaluation focusing function is calculated and judged based on specific data by generally using methods of definition ratio and fluctuation amount of a gentle region. The existing methods such as definition and the like are easy to generate a 'multi-peak' phenomenon when a picture has noise, so that the method is improved. Firstly, selecting a region to be selected, wherein the center position, width and length of a picture are 1/3 of an original picture; then, Sobel is used for carrying out edge extraction on the to-be-selected area in eight directions of 0 degree, 45 degrees, 90 degrees, 135 degrees, 180 degrees and 270 degrees, and the direction with the largest gradient is selected as the final direction; and finally, performing gradient calculation on the edge image, and drawing a definition evaluation function.

And (3) searching a focusing position, setting an initial large step length of focusing search of the three-dimensional electric displacement platform 9 according to a definition evaluation function, acquiring a digital image through the CCD camera 13 in each step, calculating a definition evaluation value of a position point through the focusing evaluation function, judging the defocusing degree of the image according to an image definition evaluation result, and finally finishing the optimal search of the system normal focal position.

In this embodiment, the automatic collection control module automatically locates and collects the cell spectrum, and the control method includes:

cell segmentation, namely adding a full Convolution network (DCN) network on the basis of a void Convolution network (FCN) (full Convolution network) cell segmentation method, wherein the DCN network replaces the common Convolution with the void Convolution to increase the size of a receptive field, improve the richness of obtained information and make up for errors caused by information loss of the traditional FCN cell segmentation method. The void rate of the void convolution is 2, and the field of view is extended from 3 × 3 to 7 × 7 in the case of normal convolution.

And (3) cell collection, wherein the divided cells are subjected to coordinate conversion, the two-dimensional position of each cell is recorded, and automatic spectrum collection is carried out on each cell by combining a three-dimensional electric displacement platform 9.

In this embodiment, the data analysis and processing device of the automatic acquisition control module performs drug sensitivity analysis on the cells, and the analysis step includes:

the number of spectral measurements was estimated dynamically, taking CDR (CD-Rario) calculation as an example, measurement of raman spectra of single cells, randomly obtaining an initial data set Xn with 30 measured spectra out of 1000 times, and then calculating the difference between the average CDR and CDRn. In 1000 statistics, the nth CDR is CDRN, n is an integer, the value range is 1 to 1000, and the relative error between the CDRN and the CDR group is less than 5% of probability P; p is the probability that the sample characteristic value tends to be stable when the number of samples is 30, and P > 95% is generally considered to be reliable. When P is less than 95%, continuing to recalculate P for the sample until P is more than 95%;

single cell resistance assay, the metabolic activity of individual cells is expressed with CDR (C-D/(C-D + C-H)) as the main parameter, and the mean value of CDR is used to measure the resistance of the strain. And aiming at different types of microorganisms, different CDR thresholds are determined, and the drug sensitivity analysis of the single cell drug resistance is realized.

In this example, C-D and C-H are Raman spectra as shown in FIG. 4, and the collected Raman spectra are subjected to background removal, baseline normalization and maximum normalization, and then the C-D peak area (2050-^-1Region) and C-H area (2800 and 3050cm in Raman spectra)^-1) And calculating the CDR at different drug concentrations.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (9)

1. A clinical microorganism unicellular drug resistance detection instrument is characterized by comprising an electric displacement platform, an optical tweezers module, an imaging module, an excitation optical module, a micro focusing module, a Raman main optical path and transmission module, a coaxial illumination module and an automatic acquisition control module;

the electric displacement platform is used for placing a sample chip;

the optical tweezers module is used for capturing cells to be detected in a liquid phase state in the sample chip and locking the cells to be detected;

the imaging module is used for shooting panoramic information of a sample chip, rapidly positioning the cell to be detected on the sample chip by matching with the electric translation platform and determining the acquisition position of the cell to be detected;

the excitation light module is used for emitting laser;

the micro-focusing module is used for automatically focusing laser to the cell to be detected at the collecting position so as to enable the cell to be detected to generate a Raman signal;

the Raman main light path and transmission module is used for acquiring a Raman spectrum of the cell to be detected;

the coaxial illumination module is used for providing coaxial illumination light for the micro-focusing module;

the automatic acquisition control module is used for controlling the excitation optical module, the Raman main optical path and transmission module, the micro-focusing module, the coaxial illumination module, the imaging module, the optical tweezers module and the electric displacement platform, processing the Raman spectrum of the acquired cell to be detected and realizing the automatic detection of the cell to be detected.

2. The clinical microbial unicell drug resistance detection instrument according to claim 1, wherein the excitation light module comprises a first laser, a first electric shutter, a first beam expander, an electric adjustable attenuator and a first reflector; the micro-focusing module comprises a micro objective lens; the Raman main light path and transmission module comprises a spectroscope, a first dichroic mirror, a second lens, a pinhole, a spectrometer and a detector;

laser emitted by the first laser sequentially passes through the first electric shutter, the first beam expander, the electric adjustable attenuator and the first reflector, passes through the spectroscope and the first dichroic mirror, passes through the microscope objective lens, and is focused on a cell to be detected to generate a Raman spectrum, the Raman spectrum passes through the first dichroic mirror, the spectroscope, the second lens, the pinhole and the spectrometer and is sent to the detector, the Raman information of the cell to be detected is acquired, and the light path mode is a Raman mode.

3. The apparatus for detecting single cell drug resistance in clinical microbes according to claim 2,

the coaxial illumination module comprises an LED light source and a semi-transparent semi-reflecting mirror; the imaging module comprises a CCD camera and a first lens;

white light emitted by the LED light source enters the micro-focusing module through the reflection of the semi-transparent semi-reflector to illuminate cells to be detected, reflected light carrying image information of the cells to be detected enters the imaging module to obtain image information of the sample chip, and the light path mode is an imaging mode.

4. The clinical microbial unicell drug resistance detection instrument of claim 3, wherein the optical tweezers module comprises a second laser, a second electric shutter, a second attenuator, a second beam expander, a second mirror, and a second dichroic mirror;

and the laser emitted by the second laser passes through a second electric shutter, a second attenuator, a second beam expander, a second reflector and a second dichroic mirror and then passes through the microscope objective lens to capture the cells, so as to obtain the image information of the cells in a liquid phase state, wherein the light path mode is an optical tweezers mode.

5. The apparatus for detecting single cell drug resistance of clinical microorganism according to claim 4, wherein the automated apparatus for rapid Raman drug-sensitive detection of microorganism comprises four operation modes:

the imaging mode is used for acquiring image information of the cell to be detected;

the imaging mode-Raman mode is used for realizing the acquisition of Raman information of the cells to be detected;

an optical tweezers mode-imaging mode for acquiring image information of cells in a liquid phase state;

the optical tweezers mode-Raman mode is used for realizing the acquisition of Raman information of cells in a liquid phase state.

6. The apparatus of claim 5, wherein the spectroscope is disposed at the intersection of the main Raman optical path and transmission module and the imaging module, the dichroic mirror is disposed at the intersection of the optical tweezer module and the main Raman optical path and transmission module, and the imaging mode, the imaging mode-Raman measurement mode, the optical tweezer mode-imaging mode, and the optical tweezer mode-Raman measurement mode are switched by controlling the movement of the spectroscope and/or the dichroic mirror.

7. The apparatus of claim 1, further comprising a sterilization module for eliminating environmental microorganisms to prevent cross contamination.

8. The clinical microorganism unicell drug resistance detection instrument according to claim 1, wherein the excitation light module, the raman main light path and transmission module, the coaxial illumination module, the imaging module and the optical tweezer module are fixed inside an optical box body, and the optical box body is a fully closed structure.

9. The sample detection method of the clinical microorganism unicellular drug resistance detection instrument based on claim 6, characterized by comprising imaging detection, imaging-Raman detection, optical tweezers-imaging detection or optical tweezers-Raman detection;

imaging detection: white light emitted by the LED light source enters the micro-focusing module through the reflection of the semi-transparent semi-reflector to illuminate cells to be detected, reflected light carrying image information of the cells to be detected enters the imaging module, and the acquisition position of the cells is positioned after the image information of the sample chip is processed;

imaging-raman detection: the LED light source of the coaxial illumination module is closed, the second electric shutter of the optical tweezers module is closed, the spectroscope is moved into the main light path, the first electric shutter of the excitation light module is opened, laser emitted by the first laser enters the micro focusing module through the spectroscope to illuminate a sample to be detected, according to a cell image obtained by imaging detection, the three-dimensional electric displacement platform moves a cell to a focusing point of a micro objective lens so as to generate a Raman signal, the generated Raman signal and stray light are collected by the micro objective lens, collimated and returned, and are reflected by the spectroscope to enter the Raman main light path and the transmission module, and are projected onto a detector through the first dichroic mirror, the second lens, the pinhole and the spectrometer in sequence, so that Raman information of the cell to be detected of the sample chip is acquired;

optical tweezers-imaging detection: changing the placing mode of a sample to be detected, moving the second dichroic mirror into a main light path, closing a first electric shutter of the excitation light module, moving the spectroscope into the main light path, further acquiring image information of the sample to be detected through the imaging detection mode, finding cells needing to be captured by the optical tweezers, and enabling laser emitted by the optical tweezers module to sequentially pass through the second electric shutter, a second attenuator, a second beam expander, a second reflecting mirror and a second dichroic mirror; capturing the cells after passing through a microscope objective to obtain image information of the cells in a liquid phase state in the sample chip;

optical tweezers-Raman detection: changing the placing mode of a sample to be detected, moving the second dichroic mirror into a main optical path, closing the first electric shutter of the excitation optical module, moving the spectroscope into the main optical path, obtaining image information of the sample to be detected through the imaging detection mode, finding out cells needing to be captured by the optical tweezers, opening the second electric shutter, enabling the optical tweezers module to emit laser to capture the cells, opening the first electric shutter, enabling the laser emitted by the first laser to enter the micro focusing module through the spectroscope to illuminate the sample to be detected, and obtaining Raman spectrum information of the cells through the Raman main optical path and the transmission module, wherein the Raman spectrum information is used for realizing the acquisition of Raman information of the cells in a liquid phase state in the sample chip.

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