CN109342378B - Colony growth state detection device and method based on multi-modal imaging technology - Google Patents
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Abstract
本发明公开了一种基于多模态成像技术的菌落生长状态检测装置与方法，该装置包括：一白光光源、一相干激光光源与一荧光激发光源，作为三类光源在菌落样品斜上方分别进行照射供样品图像采集；一彩色相机、一高速相机与一冷却相机，作为三类相机，在菌落样品上方分别对三类光源各自照射下的菌落样品图像进行采集；一成像系统，包括成像镜头、分光片、滤光片与中继目镜，设置在菌落样品与相机之间，实现三类光源分别照射下的菌落样品图像与三类相机对应成像，且成像参数一致；外加一计算机，接收三路成像信号，完成图像融合处理。采用多模态光学成像与信息融合方法消除了培养基背景影响，提高了菌落计数精度，实现了菌落生长状态的全方面检测与判别。
The invention discloses a colony growth state detection device and method based on multi-modal imaging technology. The device comprises: a white light source, a coherent laser light source and a fluorescent excitation light source, which are used as three types of light sources to be obliquely above the colony sample. Irradiate for sample image acquisition; a color camera, a high-speed camera and a cooling camera, as three types of cameras, collect images of the colony samples under the illumination of the three types of light sources above the colony sample respectively; an imaging system, including imaging lenses, The beam splitter, the filter and the relay eyepiece are set between the colony sample and the camera to realize the corresponding imaging of the colony sample images under the three types of light sources and the three types of cameras, and the imaging parameters are consistent; an additional computer is added to receive three channels Image signal, complete image fusion processing. The multimodal optical imaging and information fusion method eliminates the influence of the medium background, improves the colony counting accuracy, and realizes the comprehensive detection and discrimination of the colony growth state.
Description
Technical Field
The invention relates to the field of optical detection, in particular to a colony growth state detection device and method based on a multi-mode imaging technology.
Background
In the field of food and drug safety, microorganisms are the main reasons for food spoilage, microbial toxins (pathogenic bacteria and parasites) and infectious disease epidemics (hepatitis viruses), and food-borne diseases caused by the microorganisms are the first problems of food safety in China. In the food hygiene management of China, the microbial indicators are divided into total bacterial count (total bacterial count), coliform group, mold, yeast and pathogenic bacteria. The bacterial colony is mainly used as a mark for judging the degree of the food polluted by microorganisms, on one hand, the number of the bacterial colony can be observed as the mark for the degree of the food polluted, and the relevant regulations are also carried out in the national standard GB 4789.2-2010 food safety national standard food microbiology test bacterial colony total number determination; on the other hand, the dynamic of bacteria propagation in food can be observed to provide a basis for hygienic evaluation of the test sample. The over-standard colony count or the over-fast bacterial reproduction can indicate that the sanitation condition of the product can not meet the basic sanitation requirement to a certain extent, the nutrient content of the food can be damaged, the food spoilage is accelerated, and the food loses the edible value. Consumers eat food with serious overproof microorganisms, are easy to suffer from intestinal diseases such as dysentery and the like, possibly cause symptoms such as vomit, diarrhea and the like, and harm the health and safety of human bodies.
Colony counting is the most basic and important work of the conventional detection of microorganisms, and is mainly carried out by adopting a culture medium manual counting mode at present, but in the processes of colony culture and manual counting, the following factors bring errors to accurate quantification.
The spreading growth and pollution directly hinder the counting or inhibit the growth of bacteria in the specimen, and influence the accuracy of the counting result. The colony growth is uneven due to different evenness of the cultured bacterium blocks, and the colony chain growth can be caused to a certain extent due to different dispersity, so that the counting difficulty is caused. Counting errors are caused by particulate impurities in the sample, and solid particles in the sample are very similar to the shape and the colony in size and are difficult to distinguish in shape, so that the counting errors are caused. Colonies grow too slowly, resulting in long culture count times required.
At present, in the fields of food and drug safety detection and the like, the monitoring of colony counting and growth states is developed from manual operation, semi-manual operation to automation, intellectualization and the like. The on-line detection method is required to have the advantages of high resolution, rapid dynamic property, multi-mode property and the like, so that the intellectualization and informatization of the colony detection technology are realized.
Disclosure of Invention
Because the bacterial colony variety that needs to detect is many, and the growth process difference is great, in order to improve detection efficiency and precision, need a detection method that can obtain multiple parameter simultaneously. Aiming at the problems, the invention provides a device and a method for detecting the growth state of the bacterial colony based on a multi-mode imaging technology, which adopt an optical detection technology to achieve the effects of high precision, no damage, high speed and the like in the detection process.
For living colonies, the shape of the colony part can be changed in the growth process, the growth state of the living colonies can be acquired by collecting image characteristics, and the living colonies are counted by using an image processing algorithm. In the growth process of the bacterial colony, the scattering structure and the refractive index of the bacterial colony can be changed to a certain extent, after the coherent laser is incident, light with different directions and scattering angles forms a speckle interference phenomenon in space, when the scattering structure and the refractive index are changed, fluctuation of frequency and intensity of speckle images can be caused to be averaged, and the growth process of the bacterial colony can be quantitatively analyzed through certain processing. Various proteins are generated in the colony growth process, fluorescence is generated under the irradiation of light with certain wavelength and intensity, and in the growth process, the fluorescence intensity changes along with the quantity of the proteins with fluorescence effect generated in the protein, so the colony growth state can be obtained by counting the change of the fluorescence intensity.
The invention provides a colony growth state detection device and method based on a multi-modal imaging technology, and the technical scheme is as follows:
the colony growth state detection device based on the multi-modal imaging technology comprises:
a white light source, a coherent laser source and a fluorescence excitation light source which are used as three types of light sources to respectively irradiate above the colony sample in an inclined manner for sample image acquisition;
a color camera, a high-speed camera and a cooling camera which are used as three types of cameras and respectively collect the colony sample images irradiated by the three types of light sources above the colony sample;
the imaging system comprises an imaging lens, a beam splitter, an optical filter and a relay eyepiece, is arranged between the colony sample and the camera, realizes the corresponding imaging of the colony sample image irradiated by the three light sources and the three cameras respectively, and has consistent imaging parameters;
and a computer is additionally arranged to receive the three types of imaging signals to complete image fusion processing.
Furthermore, the white light source, the imaging lens and the color camera form a digital image acquisition device for acquiring a white light image in the bacterial colony growth process in real time.
Furthermore, the coherent laser light source, the imaging lens and the high-speed camera form a laser coherent speckle imaging device for collecting laser speckle images.
Furthermore, the fluorescence excitation light source, the imaging lens, the optical filter and the cooling camera form a fluorescence intensity imaging device for collecting fluorescence images.
The invention also provides a colony growth state detection method based on the multi-modal imaging technology, which comprises the following steps:
the method comprises the following steps: selecting different kinds of common industrial bacterial colonies for culture;
step two: collecting and recording a white light image, a dynamic speckle image and a fluorescence image in the colony culture process by using a colony growth state detection device;
step three: the white light image, the dynamic speckle image and the fluorescence image enter a computer for image fusion processing through an image acquisition device programming digital signal;
step four: comparing the image signal subjected to image fusion processing with the judgment result of a professional, storing the image signal as a training set, and establishing a topological relation between the measurement parameters and the colony growth state;
step five: and (4) bringing the online measurement result into a training set to judge the growth state of the bacterial colony.
Further, the acquisition and processing of the white light image comprises:
denoising the image, and processing the original image by using a Gaussian template to improve the signal-to-noise ratio of the image;
colony features are extracted by adopting an expansion corrosion algorithm, the growth trend and length of hyphae in a single colony are obtained in different growth periods, and the ratio of the number of hypha pixels to the total area is calculated to obtain the colony density;
analyzing pixel points one by one based on the obtained hypha growth characteristic image to obtain the ratio of the number of the link pixel points to the total number of the pixel points to obtain the hypha growth connectivity;
comparing colony white light image characteristics at different times, wherein the image characteristics comprise shape characteristics and color characteristics, and calculating colony characteristic variability according to pixel-by-pixel intensity.
Further, the dynamic speckle image processing includes:
on the basis of obtaining colony scattering characteristics, calculating the dynamic speckle power spectrum of the measured speckle image by adopting a model-based parameter spectrum estimation method, and obtaining colony growth activity parameters, wherein the colony scattering characteristics comprise colony morphology, refractive index, scattering structure, scattering intensity and angle distribution.
Further, the fluorescence image processing includes:
metabolism produces different proteins among the bacterial colony growth process, adopts monochromatic light excitation, produces fluorescence signal, judges the bacterial colony kind according to fluorescence signal wavelength, obtains bacterial colony growth activity according to fluorescence intensity measurement, carries out correlation analysis to fluorescence signal intensity and obtains the biggest time point of bacterial colony metabolic activity, judges bacterial colony growth state.
Furthermore, the three images are fused by a multi-mode information fusion method for different state parameters in the colony growth process of the white light image, the dynamic speckle image and the fluorescence image reaction, and the colony growth state is comprehensively and quantitatively evaluated.
The invention adopts a multi-modal optical imaging method to monitor the growth state of the colony culture, can effectively eliminate the background influence of the culture medium and improve the counting precision of the colony;
by adopting a multi-mode measurement method, not only can the colony technology be realized, but also the growth activity of the colony can be detected according to dynamic speckles;
the method can detect specific protein produced in the growth process of the bacterial colony by adopting a fluorescence detection method, simultaneously judges the type of the bacterial colony according to the characteristic fluorescence of the protein, and obtains the growth state of the bacterial colony according to the fluorescence related signal of the bacterial colony;
the multi-modal information fusion method can be used for comprehensively detecting and distinguishing the growth state of the bacterial colony.
Drawings
FIG. 1 is a schematic diagram of a colony growth state detection device based on a multi-modal imaging technology according to an embodiment of the invention;
FIG. 2 is a flow chart of an algorithm for calculating a dynamic speckle power spectrum in an embodiment of the present invention;
FIG. 3 is a flowchart of a method for detecting the growth status of colonies based on a multi-modal imaging technique according to an embodiment of the present invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The embodiment of the invention provides a colony growth state detection device based on a multi-modal imaging technology, wherein, referring to fig. 1, the device comprises:
a white light source 1, a coherent laser light source 2 and a fluorescence excitation light source 3 which are used as three types of light sources to respectively irradiate above the colony sample in an inclined manner for sample image acquisition;
a color camera, a high-speed camera and a cooling camera as three cameras, wherein the images of the colony samples under the respective irradiation of the three light sources are respectively collected above the colony samples;
the imaging system comprises an imaging lens, a beam splitter, an optical filter and a relay eyepiece, is arranged between the colony sample and the camera, realizes the corresponding imaging of the colony sample image under the respective irradiation of the three light sources and the three cameras, and has consistent imaging parameters;
and a computer is additionally arranged to receive the three types of imaging signals to complete image fusion processing.
Further, the white light source, the imaging lens and the color camera form a digital image acquisition device for acquiring a white light image in the growth process of the bacterial colony in real time;
in this embodiment, the digital image acquisition device: the device mainly comprises a white light source 1, an imaging lens and a color camera, and can acquire a color image in the growth process of a bacterial colony in real time.
Furthermore, a coherent laser light source, an imaging lens and a high-speed camera form a laser coherent speckle imaging device for collecting laser speckle images;
in this embodiment, the laser coherent speckle imaging apparatus: the system mainly comprises a coherent laser light source 2, an imaging lens and a high-speed camera, and mainly has the function of realizing the acquisition of laser coherent speckle images.
Furthermore, the fluorescence excitation light source, the imaging lens, the optical filter and the cooling camera form a fluorescence intensity imaging device for collecting fluorescence images;
in this embodiment, the fluorescence intensity imaging device: the device mainly comprises a fluorescence excitation light source 3, an imaging lens, a filter wheel and a high-sensitivity cooling camera, and has the main function of realizing fluorescence excitation signal acquisition.
The embodiment of the present invention further provides a method for detecting a growth state of a bacterial colony based on a multi-modal imaging technology, please refer to fig. 3, which includes:
the method comprises the following steps: selecting different kinds of common industrial bacterial colonies for culture;
step two: collecting and recording a white light image, a dynamic speckle image and a fluorescence image in the colony culture process by using a colony growth state detection device;
step three: the white light image, the dynamic speckle image and the fluorescence image enter a computer for image fusion processing through an image acquisition device programming digital signal;
step four: comparing the image signal subjected to image fusion processing with the judgment result of a professional, storing the image signal as a training set, and establishing a topological relation between the measurement parameters and the colony growth state;
step five: and (4) bringing the online measurement result into a training set to judge the growth state of the bacterial colony.
Further, the acquisition and processing of the white light image comprises:
denoising the image, and processing the original image by using a Gaussian template to improve the signal-to-noise ratio of the image;
colony features are extracted by adopting an expansion corrosion algorithm, the growth trend and length of hyphae in a single colony are obtained in different growth periods, and the ratio of the number of hypha pixels to the total area is calculated to obtain the colony density;
analyzing pixel points one by one based on the obtained hypha growth characteristic image to obtain the ratio of the number of the link pixel points to the total number of the pixel points to obtain the hypha growth connectivity;
comparing colony white light image characteristics at different times, wherein the image characteristics comprise shape characteristics and color characteristics, and calculating colony characteristic variability according to pixel-by-pixel intensity.
In this embodiment, the color image processing method for the growth state of the colony includes:
and for the colony color image processing, processing the collected white light image according to the growth characteristics of different strains, extracting image characteristics including shape characteristics and color characteristics, calculating colony area and connectivity, and comparing shape variation within a period of time.
A digital image processing mode is adopted for extracting leaf vein density characteristics, firstly, denoising is carried out on a picture, a Gaussian template is used for processing an original image, and the signal to noise ratio of the image is improved; then, extracting colony shape characteristics by adopting an expansion corrosion algorithm, then respectively obtaining the growth trend and length of hyphae in a single colony for different growth periods, and calculating the ratio of the number of hypha pixel points to the total area to obtain colony density; the calculation of hypha growth connectivity is based on the hypha growth characteristic image that obtains, and pixel point carries out the analysis one by one, obtains linking the ratio between pixel point quantity and the total pixel point quantity, obtains hypha growth connectivity, compares colony white light image characteristic in a period of time respectively, according to pixel intensity one by one, calculates the variability of colony characteristic.
Further, the dynamic speckle image processing includes:
on the basis of obtaining colony scattering characteristics, calculating the dynamic speckle power spectrum of the measured speckle image by adopting a model-based parameter spectrum estimation method, and obtaining colony growth activity parameters, wherein the colony scattering characteristics comprise colony morphology, refractive index, scattering structure, scattering intensity and angle distribution.
In this embodiment, the method for processing a dynamic speckle image of a colony growth state includes:
the measured speckle images are analyzed by a dynamic speckle processing method based on parameter estimation, so that dynamic speckle signals can be obtained, and the growth state of the colony is quantitatively analyzed based on the dynamic speckle signals. The dynamic speckle signals are caused by the scattering structure and the refractive index change in the colony growth process, and the intensity fluctuation and the frequency change of the coherent speckle signals in space and time reflect the colony growth activity. The change frequency and intensity of the laser speckle signal can be measured by using the acquired speckle image in relation to the distribution power spectral density function. The dynamic speckle image obtained by the apparatus of fig. 1 in the present invention can be expressed in a matrix form, and can be expressed in consideration of noise during measurement
Y＝HC+n，
Where Y is the measurement signal, H is the dynamic scattering function, C is the volume fraction representing different flow rates, n represents the noise present in the measurement, and the signal and noise are independent of each other.
The dynamic scattering function is mainly related to colony morphology, refractive index and scattering structure size, and the Mie scattering method can be adopted to extract the scattering structure according to the hypha geometric shape displayed by a colony white light image and acquire information such as scattering intensity and angle distribution. On the basis of obtaining colony scattering characteristics, the invention utilizes mathematical methods such as optimization and the like to process laser coherent speckle signals in a recursive iteration mode to obtain power spectrum estimation of dynamic speckle signals, and the principle is as follows:
the covariance matrix of noise and interference for k frequencies is first expressed as:
the covariance matrix of the signal samples is represented as:
in the case where the measured values and the dynamic scattering function are known, the estimate for x can be given by a weighted least squares criterion:
please refer to fig. 2 for the algorithm flow.
Further, the fluorescence image processing includes:
metabolism produces different proteins among the bacterial colony growth process, adopts monochromatic light excitation, produces fluorescence signal, judges the bacterial colony kind according to fluorescence signal wavelength, obtains bacterial colony growth activity according to fluorescence intensity measurement, carries out correlation analysis to fluorescence signal intensity and obtains the biggest time point of bacterial colony metabolic activity, judges bacterial colony growth state.
In this embodiment, the fluorescence image processing method of the colony growth state includes:
in the invention, monochromatic light with a certain wavelength (ultraviolet band) is adopted to irradiate a colony sample, and a narrow-band filter is adopted in image acquisition to remove the influence of exciting light on an acquired fluorescence image. The fluorescence wavelength characteristic that different bacterial colonies produced is different, and the fluorescence signal intensity that the bacterial colony growth process produced is relevant rather than producing protein concentration, and when bacterial colony metabolic activity changed, fluorescence signal intensity also can produce certain fluctuation, and bacterial colony fluorescence signal intensity can be expressed as x (n) in the assumption period, utilizes correlation analysis can obtain the maximum time point of bacterial colony metabolic activity to judge the bacterial colony growth state, the autocorrelation analysis formula can be expressed as:
according to the calculated fluorescence intensity autocorrelation function, the time point at which the colony growth state is most active can be judged according to the maximum value of the autocorrelation function.
Furthermore, the three images are fused by a multi-mode information fusion method for different state parameters in the colony growth process of the white light image, the dynamic speckle image and the fluorescence image reaction, and the colony growth state is comprehensively and quantitatively evaluated.
In this embodiment, the image fusion and state monitoring method includes:
the invention utilizes the multi-mode optical imaging system and the corresponding processing method to obtain the white light image, the dynamic speckle image and the fluorescence image which can reflect different state parameters in the colony growth process. In the invention, a multi-mode information fusion method is further adopted to fuse images measured by various different methods, so that the colony growth state can be comprehensively and quantitatively evaluated.
In the embodiment, different types of common industrial bacterial colonies are selected for culture, bacterial colony images in different periods are obtained by using the multi-mode imaging device and method, after information fusion processing is carried out, the bacterial colony images are compared with judgment results of professionals and stored as a training set, and a topological relation between a measurement parameter and the bacterial colony growth state is established; and then, substituting the online measurement result into the established classification fitting model, thereby realizing the judgment of the growth state of the bacterial colony.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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| CN204116228U (en) * | 2014-09-09 | 2015-01-21 | 青岛佳明测控科技股份有限公司 | The online fluorescence detection device of microorganism |
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