CN110849848B - Method and device for determining fluorescence brightness and computer storage medium - Google Patents
Method and device for determining fluorescence brightness and computer storage medium Download PDFInfo
- Publication number
- CN110849848B CN110849848B CN201911037048.9A CN201911037048A CN110849848B CN 110849848 B CN110849848 B CN 110849848B CN 201911037048 A CN201911037048 A CN 201911037048A CN 110849848 B CN110849848 B CN 110849848B
- Authority
- CN
- China
- Prior art keywords
- image
- brightness
- target channel
- light source
- measured
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/90—Determination of colour characteristics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
- G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
Abstract
A method, a device and a computer storage medium for determining fluorescence brightness comprise the following steps: collecting a first image of a measured object when a light source is in an open state under a target channel; calculating a luminance value of the first image; calculating to obtain brightness relative values of different channels according to the brightness value of the first image, the brightness value of a second image obtained by pre-calculation, the brightness weight of the target channel and the brightness gain weight of the light source; the second image is an image without a measured object when the light source is in an on state under the target channel; and determining the fluorescence brightness of the measured object according to the brightness relative values of the different channels. By adopting the scheme in the application, the fluorescence brightness evaluation of the measured object can be realized, and the method is simple and has strong operability.
Description
Technical Field
The present application relates to fluorescence signal detection technology, and in particular, to a fluorescence brightness determination method, apparatus, and computer storage medium.
Background
Immunofluorescence technology (Immunofluorescence technology) is also called fluorescent antibody technology, is the earliest developed technology in the labeled immunity technology, and is established on the basis of immunology, biochemistry and microscope technology. The immunofluorescence analysis method is to measure the intensity of light emitted by a substance after the substance absorbs light with a certain frequency, and qualitatively or quantitatively analyze the substance by measuring the spectrum and the fluorescence intensity of fluorescence due to the direct relation between the fluorescence intensity and the quantity of the substance. Compared with the radioimmunoassay, the immunofluorescence assay has no radioactive pollution, and is mostly simple and convenient to operate and convenient to popularize.
The principle of immunofluorescence quantitative detection is as follows: a specific fluorescent antibody is firstly fixed in a certain zone of a nitrocellulose membrane, after a sample is dripped at one end of the dried nitrocellulose membrane, the sample moves forwards along the membrane due to capillary action, when the sample moves to a region where the antibody is fixed, the corresponding antigen in the sample is specifically combined with the fluorescent body, the other antibody of the antigen is used for capturing the complex along with further chromatography, the redundant fluorescent antibody is captured by the second antibody, and the positions of two capturing processes are respectively called a quality control line (C line) and a detection line (T line). When the light source irradiates the detection card, the quality control line and the detection line respectively excite the fluorescence with different intensities, the photoelectric converter receives the fluorescence with different intensities to generate electric signals with different sizes, and the intensity of the electric signals reflects the concentration of the object to be detected, so that the specific immunodiagnosis is realized.
Problems existing in the prior art:
at present, the absolute fluorescence brightness of a dyed object is analyzed, although the absolute fluorescence brightness is an important medical clue and has important clinical significance, the absolute fluorescence brightness is difficult to obtain, and the requirement is high.
Disclosure of Invention
The embodiment of the application provides a method and a device for determining fluorescence brightness, a computer storage medium and electronic equipment, so as to solve the technical problems.
According to a first aspect of embodiments of the present application, there is provided a fluorescence brightness determination method, including the steps of:
collecting a first image of a measured object when a light source is in an open state under a target channel;
calculating a luminance value of the first image;
calculating to obtain brightness relative values of different channels according to the brightness value of the first image, the brightness value of a second image obtained by pre-calculation, the brightness weight of the target channel and the brightness gain weight of the light source; the second image is an image without a measured object when the light source is in an on state under the target channel;
and determining the fluorescence brightness of the measured object according to the brightness relative values of the different channels.
According to a second aspect of embodiments of the present application, there is provided a fluorescence brightness determination apparatus including:
the acquisition module is used for acquiring a first image of the object to be detected when the light source is in an open state under the target channel;
a first calculation module for calculating a luminance value of the first image;
the second calculation module is used for calculating and obtaining the brightness relative values of different channels according to the brightness value of the first image, the brightness value of the second image obtained through pre-calculation, the brightness weight of the target channel and the brightness gain weight of the light source; the second image is an image without a measured object when the light source is in an on state under the target channel;
and the determining module is used for determining the fluorescence brightness of the measured object according to the brightness relative values of the different channels.
According to a third aspect of embodiments of the present application, there is provided a computer storage medium having stored thereon a computer program which, when being executed by a processor, carries out the steps of the fluorescence brightness determination method as described above.
According to a fourth aspect of embodiments herein, there is provided an electronic device comprising one or more processors, and memory for storing one or more programs; the one or more programs, when executed by the one or more processors, implement the fluorescence brightness determination method as described above.
The method and the device for determining fluorescence brightness, the computer storage medium and the electronic device provided in the embodiment of the application provide a fluorescence brightness evaluation mode aiming at a multi-channel object to be measured of a fluorescence microscope, are simple and strong in operability, and can meet the fluorescence brightness evaluation requirement of the object to be measured.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a schematic flow chart illustrating an implementation of a fluorescence intensity determination method according to a first embodiment of the present application;
FIG. 2 is a schematic structural diagram of a fluorescence brightness determining apparatus according to a second embodiment of the present application;
FIG. 3 is a schematic structural diagram of an electronic device in a fourth embodiment of the present application;
FIG. 4 is a schematic structural diagram of a fluorescence microscope in example V of the present application;
FIG. 5 is a schematic structural diagram of an optical device in accordance with a fifth embodiment of the present application;
FIG. 6 is a diagram showing a process of determining fluorescence brightness in example V of the present application;
fig. 7 shows a schematic image of the fluorescence brightness determination process in example five of the present application.
Detailed Description
In the process of implementing the present application, the inventors found that:
when the fluorescence brightness analysis is performed on the medical dyeing cell scanning subsystem, if the existing immunofluorescence technology is adopted, the technical implementation difficulty is high, and the fluorescence brightness evaluation in the complex scene is very complex due to the influence of multiple factors such as a light source, a filter set, the exposure time of a camera, gain and the like possibly involved in the medical dyeing cell scanning environment.
At present, the fluorescence brightness evaluation of the fluorescence microscope in the complex scene of the acquired image is generally to compare the fluorescence brightness of the pictures shot under different scenes and camera parameter conditions, however, the fluorescence brightness evaluation cannot be realized because different scenes and camera parameter conditions have no uniform evaluation standard.
Therefore, considering that different staining positions of different staining substances and different excitation light sources are considered, the embodiment of the application provides a fluorescence brightness determination method, a fluorescence brightness determination device and a computer storage medium for complex scenes of medical stained cell scanning, and the immunodiagnosis of the detected object can be realized only by knowing the relative brightness of several immunofluorescent substances in the same scene.
The scheme in the embodiment of the application can be implemented by adopting various computer languages, such as object-oriented programming language Java and transliterated scripting language JavaScript.
In order to make the technical solutions and advantages of the embodiments of the present application more apparent, the following further detailed description of the exemplary embodiments of the present application with reference to the accompanying drawings makes it clear that the described embodiments are only a part of the embodiments of the present application, and are not exhaustive of all embodiments. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
Example one
Fig. 1 shows a schematic flow chart of an implementation of the fluorescence brightness determination method in the first embodiment of the present application.
As shown in the figure, the fluorescence brightness determination method includes:
102, calculating a brightness value of the first image;
103, calculating to obtain brightness relative values of different channels according to the brightness value of the first image, the brightness value of the second image obtained by pre-calculation, the brightness weight of the target channel and the brightness gain weight of the light source; the second image is an image without a measured object when the light source is in an on state under the target channel;
and step 104, determining the fluorescence brightness of the measured object according to the brightness relative values of the different channels.
In specific implementation, the images are collected in the same channel according to the embodiment of the present application, for example: the image is collected under the target channel, which can refer to the image collection under the conditions of fixed excitation light intensity, fixed camera gain and fixed camera exposure time.
In specific implementation, an image with a light source and a measured object under a target channel can be collected first, then the brightness value of the image is calculated, further the brightness relative values of different channels are calculated, and finally the fluorescence brightness of the measured object is determined.
In one embodiment, the analyte may be an object that is stained with a fluorescent substance.
In one embodiment, the test agent can be a stained cell stained with a fluorescent substance.
In one embodiment, the test object may be an object having stained cells with a fluorescent substance attached thereto.
In one embodiment, the object with the stained cells attached thereto may be a medical probe.
The fluorescence brightness determination method provided in the embodiment of the application provides a fluorescence brightness evaluation mode aiming at the multi-channel object to be measured of the fluorescence microscope, is simple and strong in operability, and can meet the fluorescence brightness evaluation requirement of the object to be measured.
In one embodiment, the process of calculating the brightness weight of the target channel includes:
and calculating the brightness weight of the target channel according to the pre-calculated brightness value of the second image without the measured object in the light source starting state of the target channel and the brightness value of the third image without the measured object in the light source starting state of the non-target channel.
In specific implementation, images without the object to be measured in the light source opening state under different channels can be collected firstly, and the different channels can comprise a target channel and a non-target channel.
Specifically, an image without the object to be measured in the on state of the light source (under the light filter corresponding to the light source) in the channel 1 can be shot; then switching channels, and shooting images without the object to be measured under the opening state of the light source (under the light filter corresponding to the light source) under the channel 2; and then switching channels until no image of the object to be detected exists when the light source on states of all the channels of the microscope are shot.
Specifically, the light source of the microscope may further have a filter corresponding to the light source, for example: the red light source and the corresponding red filter lens group. The filter lens set may generally include: collimating mirror, excitation filter, dichroic mirror, and cut filter.
The brightness value of each image can be calculated, and the calculation of the specific brightness value can adopt a brightness evaluation method in the prior art, which is not described herein again.
According to the embodiment of the application, after the images without the measured object in the light source opening states of all the channels are shot, the brightness values of all the images are calculated respectively; the brightness value of the image can be calculated when the image of the measured object does not exist in the light source opening state of each channel after being shot, and the sequence of image acquisition and brightness value calculation is not limited in the application.
In an embodiment, the calculating the brightness weight of the target channel according to the pre-calculated brightness value of the second image without the object to be measured in the light source on state of the target channel and the brightness value of the third image without the object to be measured in the light source on state of the non-target channel is specifically according to the following formula:
wherein alpha is the brightness weight of the target channel, x is the brightness value of the second image, n is the sum of the target channel and the number of non-target channels, and xiIs the luminance value of the third image.
In one embodiment, the process of calculating the light source brightness gain weight of the target channel includes:
and calculating the light source brightness gain weight of the target channel according to the pre-calculated brightness value of the second image without the measured object in the light source opening state of the target channel and the brightness value of the fourth image without the measured object in the light source closing state of the target channel.
In specific implementation, a second image without the object to be measured in the light source on state of the target channel and a fourth image without the object to be measured in the light source off state of the target channel can be respectively collected, and the brightness values of the second image and the fourth image are respectively calculated; and then, calculating the light source brightness gain weight of the target channel according to the brightness values of the second image and the fourth image.
Specifically, if the target channel is the channel 1, the light source may be turned on under the channel 1 (and under the optical filter corresponding to the light source) to capture a second image without the object to be detected, and then the light source may be turned off to capture a fourth image without the object to be detected; or shooting a fourth image without the object to be detected when the light source is in a closed state under the channel 1, and then turning on the light source to shoot a second image without the object to be detected. The acquisition sequence of the second image and the fourth image is not limited in the embodiment of the application, namely the light source can be turned on first for shooting and then turned off for shooting, and the light source can be turned on first for shooting in a light source turning-off state.
The brightness values of the second image and the fourth image may be calculated respectively, and the calculation of the specific brightness value may adopt a brightness evaluation method in the prior art, which is not described herein any further.
According to the embodiment of the application, the brightness values of the second image and the fourth image can be calculated after the second image and the fourth image are shot; or after the second image (or the fourth image) is captured, the brightness value of the second image (or the fourth image) may be calculated, then the fourth image (or the second image) is captured, and then the brightness value of the fourth image (or the second image) is calculated.
In an embodiment, the calculating, according to the pre-calculated brightness value of the second image without the object to be measured in the light source on state of the target channel and the brightness value of the fourth image without the object to be measured in the light source off state of the target channel, the light source brightness gain weight of the target channel is specifically calculated according to the following formula:
wherein β is a light source brightness gain weight of the target channel, x is a brightness value of the second image, and y is a brightness value of the fourth image.
In specific implementation, the light source brightness gain weight in the embodiment of the present application may represent the brightness gain when the light source is turned on and turned off relatively under the target channel.
In an embodiment, the brightness relative values of different channels are calculated according to the brightness value of the first image, the brightness value of the second image obtained by pre-calculation, the brightness weight of the target channel, and the brightness gain weight of the light source, and specifically calculated according to the following formula:
wherein, L is a relative brightness value, x' is a brightness value of the first image, x is a brightness value of the second image, α is a brightness weight of the target channel, and β is a light source brightness gain weight of the target channel.
In one embodiment, L is a fluorescence intensity value of the stained cells in a stained cell image scanning system.
In one embodiment, the luminance value of the image is calculated using the following formula:
wherein δ is a constant, Lum (x, y) is the luminance value of any pixel in the image, and N is the number of pixels in the image.
In one embodiment, δ may be a small constant, for example: δ may be taken to be 0.0001, and δ acts to prevent the logarithm calculation from going to infinity.
The meaning of the above formula is: for each pixel in the image with the brightness value to be calculated, calculating the brightness value Lum (x, y) of the pixel, and then calculating the natural logarithm of the brightness value; then, the logarithm of the luminance values of all the pixels is averaged, and then the natural index value of the average is calculated.
In one embodiment, before the acquiring the first image of the object when the light source is on under the target channel, the method further includes:
controlling a carrier carrying a measured object by an object carrying device to move the carrier of the measured object along the y-axis direction within a predetermined image definition experience range, and acquiring an image of the carrier of the measured object on each focal plane; the y-axis is perpendicular to the focal plane of the microscope;
determining an experience area image of the carrier of the object to be measured, which is to be intercepted from the image of the carrier of the object to be measured, according to the edge of the carrier of the object to be measured in the image of the carrier of the object to be measured;
calculating a contrast value according to the experience area image of the carrier of the measured object;
the microscope focus is placed at the position of the maximum contrast value.
In specific implementation, the predetermined image clarity experience range may be a distance range from the image acquisition device to the object to be measured or the carrying device. In particular, the image sharpness experience range may be one or more continuous values.
In one embodiment, the range of image sharpness experience is different for different acquisition channels. And after the acquisition channels are switched, determining the distance to be moved under the current acquisition channel according to the position of the object carrying device when the previous acquisition channel is focused and the image clarity experience range of the current acquisition channel.
After the image of the carrier of the measured object is obtained, the embodiment of the application can further determine the edge position of the carrier of the measured object in the image of the carrier of the measured object, determine the experience area image of the carrier of the measured object according to the edge position, and then intercept the experience area image from the image of the carrier of the measured object.
And after obtaining the experience area image of the carrier of the measured object, calculating a contrast value according to the experience area image of the carrier of the measured object.
In one embodiment, the calculating the contrast value according to the experience area image of the carrier of the measured object includes:
sequentially calculating the brightness difference between two adjacent pixels in the experience area image of the carrier of the measured object obtained at each focus;
and calculating the brightness difference value to obtain the contrast value of the empirical region image of the carrier of the measured object obtained at each focus.
In a specific implementation, the calculating the luminance difference value may be performed according to the following formula:
wherein, m and n are the number of pixels in the transverse direction and the longitudinal direction of the experience area image; the X, Y is a coordinate value of a pixel point; and F is the contrast value of the empirical region image.
In one embodiment, the placing the microscope focus at the position focus where the contrast value is largest according to the position focus includes:
comparing the size of the contrast value of the empirical region image of the carrier of the measured object obtained at each focus, and determining the position of the focus corresponding to the image with the maximum contrast value;
and driving the image acquisition device to place the focus at the position of the focus with the maximum contrast value.
Because the embodiment of the application only needs to carry out contrast type focusing on the empirical region image of the carrier of the measured object, the calculation amount of focusing scanning and contrast value calculation is greatly reduced, and the focusing time is shortened, so that the focusing of the carrier of the measured object can be completed quickly.
According to the microscope focusing method provided by the embodiment of the application, the clear experience range of the image is predetermined, the carrier of the measured object is controlled to move along the focusing axis (y axis) direction according to the experience range, the experience area image of the carrier of the measured object is obtained after the image of the carrier of the measured object is obtained, the contrast value is calculated according to the experience area image, the focus of the microscope is placed on the focus of the position according to the position with the maximum contrast value, therefore, focusing can be completed only through few operations of the air pulling box, and the focusing efficiency is greatly improved.
In one embodiment, the object carrier is a cylindrical object, the side surface of the object carrier is a curved surface, and the object is attached to the side surface of the object carrier.
In specific implementation, the measured object carrier is a cylindrical object, the side surface of the measured object carrier is a curved surface with a certain curvature, and when the measured object carrier with the curved surface structure is focused, if a focusing mode in the prior art is adopted, the curved surface structure influences the focusing process, so that a shot image is not clear; by adopting the focusing method provided by the embodiment of the application, the influence caused by the curved surface structure can be well filtered, the image of the experience area of the carrier of the measured object is selected for focusing, the focusing process is fast, and the shot image is clear.
In one embodiment, the microscope is a fluorescence microscope, the carrier of the test object is a medical probe, the test object is a stained cell, the medical probe adsorbs the stained cell, and the stained cell is stained with a fluorescent substance.
The embodiment of the application provides a focusing method aiming at the problem that the image scanning by a microscope of a medical probe is difficult, and the probe is extremely fine and has a certain curvature, so that the scanning of stained cells on the probe by adopting the existing focusing method is very difficult.
In specific implementation, in order to perform medical diagnosis or observation, only the image of the stained cells needs to be scanned, and the carrier of the stained cells, that is, the medical probe, may be of a uniform specification, and only a new medical probe of the specification needs to be used to extract the stained cells each time the extraction of the stained cells is performed, so that the medical probe can have a clear experience range and an experience area of the image.
In a specific implementation, for better medical diagnosis or observation, the stained cells adsorbed on the medical probe may be stained with a fluorescent substance, and the microscope may be a fluorescence microscope, and the fluorescence microscope may emit fluorescence to excite the fluorescent substance stained by the stained cells, so that images of the stained cells may be clearly presented.
Example two
Based on the same inventive concept, the embodiment of the application provides a fluorescence brightness determination device, the principle of the device for solving the technical problem is similar to that of a fluorescence brightness determination method, and repeated parts are not repeated.
Fig. 2 is a schematic structural diagram of a fluorescence brightness determination apparatus according to a second embodiment of the present application.
As shown in the figure, the fluorescence brightness determination apparatus includes:
the acquisition module 201 is used for acquiring a first image of a measured object when a light source is in an on state under a target channel;
a first calculating module 202, configured to calculate a brightness value of the first image;
the second calculating module 203 is configured to calculate brightness relative values of different channels according to the brightness value of the first image, the brightness value of the second image obtained through pre-calculation, the brightness weight of the target channel, and the light source brightness gain weight; the second image is an image without a measured object when the light source is in an on state under the target channel;
and the determining module 204 is used for determining the fluorescence brightness of the measured object according to the brightness relative values of the different channels.
The fluorescence brightness determination device provided in the embodiment of the application provides a fluorescence brightness evaluation mode aiming at the multi-channel object to be measured of the fluorescence microscope to acquire images, is simple and strong in operability, and can meet the fluorescence brightness evaluation requirement of a fluorescence object to be measured.
In one embodiment, the apparatus further comprises:
and the third calculation module is used for calculating the brightness weight of the target channel according to the pre-calculated brightness value of the second image without the measured object in the light source starting state under the target channel and the brightness value of the third image without the measured object in the light source starting state under the non-target channel.
In an embodiment, the third calculating module specifically calculates the luminance weight of the target channel according to the following formula:
wherein alpha is the brightness weight of the target channel, x is the brightness value of the second image, n is the sum of the target channel and the number of non-target channels, and xiIs the luminance value of the third image.
In one embodiment, the apparatus further comprises:
and the fourth calculation module is used for calculating the light source brightness gain weight of the target channel according to the pre-calculated brightness value of the second image without the measured object in the light source opening state of the target channel and the brightness value of the fourth image without the measured object in the light source closing state of the target channel.
In an embodiment, the fourth calculating module specifically calculates the light source brightness gain weight of the target channel according to the following formula:
wherein β is a light source brightness gain weight of the target channel, x is a brightness value of the second image, and y is a brightness value of the fourth image.
In one embodiment, the second calculating module calculates the relative brightness values of the different channels according to the following formula:
wherein, L is a relative brightness value, x' is a brightness value of the first image, x is a brightness value of the second image, α is a brightness weight of the target channel, and β is a light source brightness gain weight of the target channel.
In one embodiment, the apparatus further comprises:
the image brightness calculation module is used for calculating the image with the brightness value to be calculated by adopting the following formula:
wherein δ is a constant, Lum (x, y) is the luminance value of any pixel in the image whose luminance value is to be calculated, and N is the number of pixels in the image whose luminance value is to be calculated.
EXAMPLE III
Based on the same inventive concept, embodiments of the present application further provide a computer storage medium, which is described below.
The computer storage medium has a computer program stored thereon, which, when being executed by a processor, implements the steps of the fluorescence brightness determination method according to an embodiment.
The computer storage medium provided in the embodiment of the application provides a fluorescence brightness evaluation mode aiming at the collected image of a multi-channel object to be measured of a fluorescence microscope, is simple and strong in operability, and can meet the fluorescence brightness evaluation requirement of the fluorescence object to be measured.
Example four
Based on the same inventive concept, the embodiment of the present application further provides an electronic device, which is described below.
Fig. 3 shows a schematic structural diagram of an electronic device in the fourth embodiment of the present application.
As shown, the electronic device includes memory 301 for storing one or more programs, and one or more processors 302; the one or more programs, when executed by the one or more processors, implement the fluorescence intensity determination method of embodiment one.
The electronic equipment provided in the embodiment of the application provides a fluorescence brightness evaluation mode which can be used for acquiring images of a multi-channel object to be measured of a fluorescence microscope, is simple and strong in operability, and can meet the fluorescence brightness evaluation requirement of the fluorescence object to be measured.
EXAMPLE five
To facilitate the practice of the present application, the embodiments of the present application are described with reference to a fluorescence microscope system scanning medical probe as an example.
The embodiment of the application provides a fluorescence microscope system, which comprises a fluorescence microscope and a controller, wherein the controller comprises a fluorescence brightness determination device,
fig. 4 shows a schematic structural diagram of a fluorescence microscope in example five of the present application.
As shown in the figure, the fluorescence microscope comprises a base 1, a carrying device 2 arranged on the base 1, a driving device 3 for driving the carrying device 2 to move, a light-emitting device 4, an optical device 5 and an image acquisition device 6; wherein the content of the first and second substances,
the object carrying device 2 is used for clamping the medical probe, stained cells are attached to the medical probe, and the object carrying device is positioned on the object carrying side of the optical device 5; the light-emitting device 4 is positioned at the light source side of the optical device 5, corresponds to the light source interface of the optical device 5, and is used for mapping the image of the medical probe onto the image acquisition device 6; the optical device 5 is internally provided with a lens and an optical filter and is used for changing the light transmission direction and carrying out reflection, projection, filtering and other treatments on the light; the image acquisition device 6 is located on the imaging side of the optical device 5 and corresponds to the image acquisition interface of the optical device 5.
The base 1 is provided with a connecting line interface which can comprise a power interface and a data interface. The power supply interface is connected with each component and used for supplying power to each component through a power supply; the data interface is connected to the controller for controlling the operation of the components and for transmitting images of stained cells on the medical probe.
The carrying device 2 comprises a rotating shaft, an x axis and a y axis, wherein the y axis is a focusing axis and is vertical to the direction of a focal plane of the fluorescence microscope. The x-axis can be understood as the left-right translation direction, the y-axis as the front-back translation direction, and the rotation axis as the direction in which the medical probe is held for rotation.
The image acquisition device comprises a camera and the like.
The optical means may comprise components such as an objective lens, a cut-off filter, a dichroic mirror, a tube lens, a collimator mirror, a laser filter, etc
The light emitting device may include a fluorescent LED lamp or the like.
Fig. 5 shows a schematic structural diagram of an optical device in the fifth embodiment of the present application.
As shown, the optical device 5 includes: a first light shielding housing, a disc-shaped holder assembly 51 disposed within the first light shielding housing, a rotary shaft 52, a lens barrel 53, and a plurality of element holders 54. Wherein the content of the first and second substances,
the first shading shell is used for packaging internal components, and light of the external environment is prevented from interfering with internal optical elements.
The rotating shaft 52 is inserted through the center of the disc-shaped bracket assembly 51, and both ends of the rotating shaft extend out of the first shading shell. The disc-shaped holder assembly 51 is rotatably supported by a rotary shaft 52, thereby achieving rotational adjustment and selection of optical elements mounted on an element holder 54 of the disc-shaped holder assembly 51.
A plurality of component supports 54 are mounted on the circumferential direction of the disc-shaped support assembly 51, and the component supports 54 may be uniformly distributed or randomly distributed on the circumferential direction of the disc-shaped support assembly 51.
The element holder 54 is used for mounting an optical element such as a filter, and is provided with a cavity for mounting the optical element therein, and a light inlet and a light outlet communicating with the cavity, so that light passes through the light inlet and then exits from the light outlet through the optical element.
The first shading shell is provided with a first light hole corresponding to the position of the light inlet hole and a second light hole corresponding to the position of the light outlet hole, and the central axis of the first light hole is perpendicular to the central axis of the second light hole. By adjusting the rotating shaft 52, the first light-transmitting hole is opposite to the light-entering hole of the component holder 54 and the second light-transmitting hole is opposite to the light-exiting hole on one side of the component holder 54, so as to form an optical channel.
The lens barrel 53 is fixedly mounted on the inner side surface of the shading shell and is opposite to the second light-transmitting hole, and the lens barrel 53 is used for mounting light-transmitting elements such as lenses and objective lenses.
The light emitting device 4 may include: the second light-shading shell and the light source are positioned in the second light-shading shell. One side of the second shading shell facing the optical device 5 is provided with a light hole, and the light source emits light towards the light hole.
The second shading shell can be internally provided with a plurality of light sources, and the characteristics of the light emitted by each light source, such as wavelength, intensity and the like, have certain differences. A plurality of light sources may be disposed on a rotating bracket, which is driven to rotate by a rotating motor to select one of the light sources to emit light toward the optical device 5. The structure of the rotating bracket can refer to the implementation of the disc-shaped bracket assembly 51 described above.
It is assumed that the fluorescence microscope comprises a Nucleus-Hoechst channel and an EpCAM/CKs-FITC channel.
FIG. 6 is a diagram showing the process of determining the fluorescence brightness in example five of the present application.
Fig. 7 shows a schematic image of the fluorescence brightness determination process in example five of the present application.
As shown in the figure, the fluorescence brightness determination process provided in the embodiment of the present application includes:
under the nucleous-Hoechst channel
1. Turning on the excitation light source, collecting background images of the target area without stained cells (fig. a 1);
obtaining the brightness value x by using an averaging method0。
2. Turning off the excitation light source, and collecting a background image of the target area without staining cells (fig. a 2);
by averaging to obtain brightnessValue y0。
3. Turning on the excitation light source, collecting the background image of the stained cells in the target area (FIG. a3, the cell position is blue);
the cell area (FIG. a4, blue cell position) was extracted by averaging to obtain the brightness value x0’。
Under EpCAM/CKs-FITC channel
1. Turning on the excitation light source, collecting background images of the target area without stained cells (fig. b 1);
obtaining the brightness value x by using an averaging method1。
2. Turning off the excitation light source, and collecting a background image of the target area without stained cells (fig. b 2);
obtaining the brightness value y by using an averaging method1。
3. Turning on the excitation light source, collecting the background image of the stained cells in the target region (FIG. b3, the cell position is green);
the cell area (FIG. b4, cell position is green) is intercepted by averaging to obtain the brightness value x1’。
Then:
1) determination of the fluorescence intensity of the stained cells of the Nucleus-Hoechst channel:
α0＝x0/x0+x1；
β0＝x0/y0；
L0＝(x′0-x0)α0/β0。
wherein L is0The fluorescence (blue light) intensity of the stained cells in the nucleous-Hoechst channel was reflected.
2) Determination of fluorescence intensity of stained cells for EpCAM/CKs-FITC channel:
α1＝x1/x0+x1；
β1＝x1/y1；
L1＝(x′1-x1)α1/β1。
wherein L is1The fluorescence (green light) intensity of stained cells at the EpCAM/CKs-FITC channel was reflected.
By adopting the scheme provided by the embodiment of the application, the fluorescence brightness of the stained cells in each channel can be determined.
As will be appreciated by one skilled in the art, embodiments of the present application may be provided as a method, system, or computer program product. Accordingly, the present application may take the form of an entirely hardware embodiment, an entirely software embodiment or an embodiment combining software and hardware aspects. Furthermore, the present application may take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, and the like) having computer-usable program code embodied therein.
The present application is described with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the application. It will be understood that each flow and/or block of the flow diagrams and/or block diagrams, and combinations of flows and/or blocks in the flow diagrams and/or block diagrams, can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.
These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means which implement the function specified in the flowchart flow or flows and/or block diagram block or blocks.
These computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.
While the preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all alterations and modifications as fall within the scope of the application.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present application without departing from the spirit and scope of the application. Thus, if such modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is intended to include such modifications and variations as well.
Claims (5)
1. A method for determining fluorescence brightness, comprising:
controlling a carrier carrying a measured object by an object carrying device to move the carrier of the measured object along the y-axis direction within a predetermined image definition experience range, and acquiring an image of the carrier of the measured object on each focal plane; the y-axis is perpendicular to the focal plane of the microscope;
determining an experience area image of the carrier of the object to be measured, which is to be intercepted from the image of the carrier of the object to be measured, according to the edge of the carrier of the object to be measured in the image of the carrier of the object to be measured;
calculating a contrast value according to the experience area image of the carrier of the measured object;
placing the microscope focus on the position focus according to the position with the maximum contrast value;
collecting a first image of a measured object when a light source is in an open state under a target channel;
calculating a luminance value of the first image;
calculating to obtain brightness relative values of different channels according to the brightness value of the first image, the brightness value of a second image obtained by pre-calculation, the brightness weight of the target channel and the brightness gain weight of the light source; the second image is an image without a measured object when the light source is in an on state under the target channel;
determining the fluorescence brightness of the measured object according to the brightness relative values of the different channels, wherein the calculation process of the brightness weight of the target channel comprises the following steps:
calculating the brightness weight of the target channel according to the pre-calculated brightness value of the second image without the object to be measured in the light source on state of the target channel and the brightness value of the third image without the object to be measured in the light source on state of the non-target channel,
calculating the brightness weight of the target channel according to the pre-calculated brightness value of the second image without the measured object in the light source on state of the target channel and the brightness value of the third image without the measured object in the light source on state of the non-target channel, specifically according to the following formula:
wherein alpha is the brightness weight of the target channel, x is the brightness value of the second image, n is the sum of the target channel and the number of non-target channels, and xiIs the luminance value of the third image,
calculating to obtain brightness relative values of different channels according to the brightness value of the first image, the brightness value of the second image obtained by pre-calculation, the brightness weight of the target channel and the brightness gain weight of the light source, specifically according to the following formula:
wherein L is a relative brightness value, x' is a brightness value of the first image, x is a brightness value of the second image, α is a brightness weight of the target channel, β is a light source brightness gain weight of the target channel,
calculating the brightness value of the image by adopting the following formula:
wherein δ is a constant, Lum (x, y) is the luminance value of any pixel in the image whose luminance value is to be calculated, and N is the number of pixels in the image whose luminance value is to be calculated.
2. The method according to claim 1, wherein the calculating process of the light source brightness gain weight of the target channel includes:
and calculating the light source brightness gain weight of the target channel according to the pre-calculated brightness value of the second image without the measured object in the light source opening state of the target channel and the brightness value of the fourth image without the measured object in the light source closing state of the target channel.
3. The method according to claim 2, wherein the light source brightness gain weight of the target channel is calculated according to a pre-calculated brightness value of a second image without the object to be measured in a light source on state of the target channel and a pre-calculated brightness value of a fourth image without the object to be measured in a light source off state of the target channel, specifically according to the following formula:
wherein β is a light source brightness gain weight of the target channel, x is a brightness value of the second image, and y is a brightness value of the fourth image.
4. A computer storage medium, on which a computer program is stored which, when being executed by a processor, carries out the steps of the method according to any one of claims 1 to 3.
5. An electronic device comprising one or more processors, and memory for storing one or more programs; the one or more programs, when executed by the one or more processors, implement the method of any of claims 1 to 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911037048.9A CN110849848B (en) | 2019-10-29 | 2019-10-29 | Method and device for determining fluorescence brightness and computer storage medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911037048.9A CN110849848B (en) | 2019-10-29 | 2019-10-29 | Method and device for determining fluorescence brightness and computer storage medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110849848A CN110849848A (en) | 2020-02-28 |
| CN110849848B true CN110849848B (en) | 2022-04-29 |
Family
ID=69599001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911037048.9A Active CN110849848B (en) | 2019-10-29 | 2019-10-29 | Method and device for determining fluorescence brightness and computer storage medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110849848B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112311953A (en) * | 2020-09-28 | 2021-02-02 | 北京临近空间飞行器系统工程研究所 | Reversible image information hiding method and device, electronic equipment and medium |
| CN114466477A (en) * | 2021-12-31 | 2022-05-10 | 珠海雷特科技股份有限公司 | Constant-current dimming method of multichannel light source, computer device and computer readable storage medium |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108174118A (en) * | 2018-01-04 | 2018-06-15 | 珠海格力电器股份有限公司 | Image processing method, device and electronic equipment |
| CN108629738A (en) * | 2017-03-16 | 2018-10-09 | 阿里巴巴集团控股有限公司 | A kind of image processing method and device |
| CN112268879A (en) * | 2020-09-17 | 2021-01-26 | 中国科学院生物物理研究所 | Fluorescence microscopic imaging system |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5672796B2 (en) * | 2010-01-13 | 2015-02-18 | 株式会社ニコン | Image processing apparatus and image processing method |
| US8982237B2 (en) * | 2011-12-09 | 2015-03-17 | Htc Corporation | Portable electronic device with auto-exposure control adaptive to environment brightness and image capturing method using the same |
-
2019
- 2019-10-29 CN CN201911037048.9A patent/CN110849848B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108629738A (en) * | 2017-03-16 | 2018-10-09 | 阿里巴巴集团控股有限公司 | A kind of image processing method and device |
| CN108174118A (en) * | 2018-01-04 | 2018-06-15 | 珠海格力电器股份有限公司 | Image processing method, device and electronic equipment |
| CN112268879A (en) * | 2020-09-17 | 2021-01-26 | 中国科学院生物物理研究所 | Fluorescence microscopic imaging system |
Non-Patent Citations (1)
| Title |
|---|
| "TDI-fMOST 系统的图像预处理方法研究";贾瑶;《中国优秀硕士学位论文全文数据库 信息科技辑》;20190415(第4期);摘要，正文第8-30页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110849848A (en) | 2020-02-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7003241B2 (en) | Paste slide judgment system | |
| CN110849848B (en) | Method and device for determining fluorescence brightness and computer storage medium | |
| US11086118B2 (en) | Self-calibrating and directional focusing systems and methods for infinity corrected microscopes | |
| KR100924117B1 (en) | Lens inspection apparatus | |
| JP3678397B2 (en) | Shooting system | |
| US20110226965A1 (en) | Increased resolution microscopy | |
| JP6985506B2 (en) | Real-time autofocus focusing algorithm | |
| CA3061440C (en) | Optical scanning arrangement and method | |
| US20100214430A1 (en) | Method and system for imaging samples | |
| CN102156976A (en) | Arithmetically operating device, arithmetically operating method, arithmetically operating program, and microscope | |
| JP6605716B2 (en) | Automatic staining detection in pathological bright field images | |
| JP7021348B2 (en) | Safety light curtain that disables the rotation of the carousel | |
| CN109120819B (en) | Imaging wells of a microplate | |
| JP4599941B2 (en) | Automatic focus detection apparatus and microscope system including the same | |
| JP2022084889A (en) | Slide inventory and reinsertion system | |
| JP6869435B2 (en) | Slide rack carousel | |
| CN110850576A (en) | Microscope image scanning control method and device and computer storage medium | |
| JP2020535478A (en) | Double-pass macro image | |
| JP2013516650A (en) | Method and apparatus for high-speed focus imaging of biological specimen | |
| CN110727096B (en) | Microscope focusing method and device and microscope equipment | |
| US9772540B2 (en) | Autofocus for imaging system | |
| JP7119085B2 (en) | Impact rescan system | |
| JP3102954U (en) | Fluorescence observation system with fiber pipette as excitation light source and built-in light intensity detector | |
| JP5239472B2 (en) | Fluorescence imaging device | |
| US20220373464A1 (en) | Control system and method for determining an illumination intensity in a fluorescence microscope and corresponding microscope system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |