CN106645045B - TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy imaging - Google Patents
TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy imaging Download PDFInfo
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- CN106645045B CN106645045B CN201610812435.5A CN201610812435A CN106645045B CN 106645045 B CN106645045 B CN 106645045B CN 201610812435 A CN201610812435 A CN 201610812435A CN 106645045 B CN106645045 B CN 106645045B
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Abstract
The invention provides a TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy imaging, which comprises the following steps: s1 setting trigger window information; s2 enabling the TDI-CCD to realize forward scanning by applying a first level control signal to the TDI-CCD; s3, controlling the three-dimensional precision movement control platform to move forward along the X axis according to the information of the trigger window, and giving a trigger signal according to the movement position of the three-dimensional precision movement control platform to control the TDI-CCD to realize exposure imaging along the forward scanning direction; s4 enabling the TDI-CCD to perform a reverse scan by applying a second level control signal to the TDI-CCD in a direction opposite to that of the first level control signal; s5, controlling the three-dimensional precision movement control platform to move reversely along the X axis according to the trigger window information; and a trigger signal is given according to the moving position of the three-dimensional precision movement control platform to control the TDI-CCD to realize exposure imaging along the reverse scanning direction. The invention can better detect the weak signal of fluorescence, improve the whole imaging speed and shorten the whole fluorescence optical imaging period.
Description
Technical Field
The invention belongs to the technical field of fluorescence optical microscopic imaging, and particularly relates to a TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopic imaging.
Background
A tdi (time Delay integration) CCD, i.e., a time Delay integration CCD, is an imaging device of a line scanning type, developed based on the concept of multiple exposures of the same object, and is generally used to image some high-speed objects. The method has wide application in the fields of industrial monitoring, space detection, space remote sensing and the like.
At present, the TDI-CCD has also developed in the field of fluorescence optical imaging, especially for detecting weak fluorescence signals, a stack of linear array pixels in its frame transfer device is aligned with and synchronized with the motion of the object to be imaged, and as the image moves from one row of pixels to another, the integrated charges also move, so that the moving object is continuously imaged and output in this way, providing a higher resolution than that of a normal line scan camera in weak light. However, in the field of bioluminescence signal imaging, the conventional TDI-CCD for performing unidirectional line scan imaging on a biological tissue sample also requires that the sample moves along a specified direction, and when a larger imaging range needs to be covered, the sample needs to complete return motion along the opposite direction, and after moving for a certain distance along the vertical direction, the sample performs motion imaging along the specified direction to acquire images of adjacent imaging fields. In the above process, the time consumed by the return motion is wasted and cannot be used for imaging. For imaging of large samples, longer periods may be required.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a TDI-CCD-based bidirectional scanning method, which realizes faster imaging of a biological tissue sample and aims to solve the problems of long imaging period and low efficiency of the biological tissue sample by using fluorescence microscopic imaging.
The invention provides a TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy imaging, which comprises the following steps:
s1: setting triggering window information;
s2: the method comprises the steps that a first level control signal is applied to the TDI-CCD, so that the TDI-CCD realizes forward scanning;
s3: controlling the three-dimensional precision movement control platform to move forwards along the X axis according to the trigger window information, and giving a trigger signal according to the movement position of the three-dimensional precision movement control platform to control the TDI-CCD to realize exposure imaging along the forward scanning direction;
s4: enabling the TDI-CCD to realize reverse scanning by applying a second level control signal to the TDI-CCD, wherein the direction of the second level control signal is opposite to that of the first level control signal;
s5: controlling the three-dimensional precision movement control platform to move reversely along the X axis according to the trigger window information; and a trigger signal is given according to the moving position of the three-dimensional precision movement control platform to control the TDI-CCD to realize exposure imaging along the reverse scanning direction.
Further, in step S1, the trigger window information includes: trigger signal frequency, trigger window starting position and trigger window stroke.
Further, the frequency f0 of the trigger signal is v0/s0, where v0 is the moving speed of the three-dimensional precision motion control platform, and s0 is the trigger distance of the three-dimensional precision motion control platform.
Still further, the trigger signal frequency is less than 50 kHz.
Through the technical scheme of the invention, as TDI-CCD is used for carrying out bidirectional scanning imaging on the biological tissue sample, compared with the existing TDI-CCD unidirectional line scanning imaging technology, the TDI-CCD bidirectional line scanning imaging technology can better detect weak fluorescence signals, improve the integral imaging speed and shorten the whole fluorescence optical imaging period.
Drawings
FIG. 1 is a schematic diagram of a fluorescence microscopy imaging system.
Fig. 2 is a schematic diagram of the principle of bidirectional scanning imaging.
Fig. 3 is a flow chart of a procedure of a bidirectional scanning imaging three-dimensional precision movement control platform.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention belongs to the technical field of fluorescence optical microscopic imaging, and relates to a TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopic imaging.
The invention solves the problem that a TDI-CCD camera capable of supporting bidirectional scanning is used in the existing fluorescence optical microscopic imaging system, a bidirectional scanning imaging method is used in combination with external triggering of a three-dimensional precise movement control platform, rapid high-resolution imaging of a biological tissue sample is realized, and the integral imaging speed is improved.
The fluorescence optical microscopy imaging system comprises: the three-dimensional precision motion control platform comprises a three-dimensional precision motion control platform, an acquisition module and an imaging light path part. The high-speed microscopic imaging system is shown in fig. 1, wherein a processing tank 2 can be arranged on a three-dimensional precision mobile control platform 1, the processing tank can be tightly fixed on the three-dimensional precision mobile control platform, and a biological tissue sample 3 is fixed in the processing tank. The cutter 4 can cut the biological tissue sample, and when each cutting is finished, a coronal plane is formed, the three-dimensional precision movement control platform carries the biological tissue sample to move to the position below the objective lens 5 for scanning and imaging. The laser emitted by the laser 9 is adjusted by the illumination light path 8 and then reaches the lower part of the objective lens by the detection light path 6, so that the biological tissue sample is excited. The fluorescence signal excited by the biological tissue sample passes through the detection light path through the objective lens, and is finally collected and imaged by the TDI-CCD7, the TDI-CCD line scanning imaging direction is controlled by the level signal of the control line 19 externally connected with the control line, the image collected by the TDI-CCD is transmitted to the cache of the data acquisition card 17 through the cable 12, the data acquisition card is inserted into the PCI card slot of the workstation 11, and the data acquisition card can be programmed in the workstation to acquire image data. The workstation is also provided with a serial port 16 which is connected with a laser through a serial port line 13, the laser is controlled through self-contained software of the laser, and a 1394 card 15 is inserted into a PCI card slot of the workstation and is connected with a control box 10 of the three-dimensional precise mobile control platform through a cable 14, so that the control box can be programmed in the workstation to be controlled, and meanwhile, the control box can control the three-dimensional precise mobile control platform through a cable 18.
The TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy provided by the invention comprises a TDI-CCD camera and an external trigger. The TDI-CCD camera can use a camera which supports bidirectional scanning imaging, and for external triggering, the requirement on the motion precision of an object is very high due to line scanning delay integral imaging and the application of the TDI-CCD camera in the field of microscopic imaging. The method adopts a control box of a three-dimensional precise movement control platform to carry out programming control on the movement of the platform, and gives a trigger signal to carry out exposure imaging on the TDI-CCD according to the relative position while controlling the movement of the three-dimensional precise movement control platform; when the three-dimensional precision motion control platform moves along different directions, different trigger signals are required to be given to control the TDI-CCD camera to carry out integral imaging in different directions. External trigger signals required by the TDI-CCD camera can be output from the three-dimensional precision mobile control platform, and a control box needs to be programmed.
Compared with the prior art, the invention has many advantages by using bidirectional scanning delay integral imaging:
(1) the invention uses TDI-CCD delay integration device, which has the advantages of high sensitivity, low noise, high stability, etc.
(2) The method uses external trigger to control the exposure of the TDI-CCD, and the trigger signal is given according to the position of the three-dimensional precise movement control platform, so that the aim of synchronously exposing the TDI-CCD camera and an object is fulfilled; meanwhile, when the three-dimensional precision movement control platform moves along different directions, different level signals are given to control the integration direction of the TDI-CCD.
(3) The invention provides a bidirectional scanning method, which greatly improves the imaging speed of fluorescence optical microscopic imaging.
The invention realizes a TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy, which has a basic schematic diagram shown in FIG. 2, and shows a process of using bidirectional scanning imaging by a fluorescence optical microscopy system. Wherein 105 and 106 respectively represent that the three-dimensional precision motion control platform runs along the X axis in the forward direction and the three-dimensional precision motion control platform runs along the X axis in the reverse direction, and 107 represents a timing chart of the trigger signal given by the three-dimensional precision motion control platform. The stroke of the three-dimensional precision motion control platform is divided into three parts 101, 102 and 103, when the three-dimensional precision motion control platform runs along the X axis in the positive direction, the distance of the part 101 is the acceleration distance of the three-dimensional precision motion control platform, the part 102 is the part of the three-dimensional precision motion control platform which gives a trigger signal for triggering TDI-CCD exposure, the part 103 is the deceleration part of the three-dimensional precision motion control platform, and the part 104 is a single period of the trigger signal given by the three-dimensional precision motion control platform. Because the fluorescence weak signal is subjected to microscopic imaging, exposure imaging needs to be carried out when the three-dimensional precision moving control platform moves at a constant speed, so that the exposure time of each pixel of an image obtained by TDI-CCD line scanning is the same, and the condition of uneven brightness is avoided. Also, as can be seen from the schematic diagram, the parts 101, 102 and 103 in the figure constitute the whole moving stroke of the three-dimensional precision movement control platform. When the three-dimensional precision motion control platform moves reversely along the X axis, part 103 in FIG. 2 is the acceleration distance of the three-dimensional precision motion control platform, part 102 is the part of the three-dimensional precision motion control platform that gives the trigger signal for triggering the exposure of TDI-CCD, and part 101 is the final deceleration part of the three-dimensional precision motion control platform.
By using the bidirectional scanning imaging method provided by the invention, the strokes of the part 101 and the part 103 in fig. 2 need to be consistent, so that the image range acquired by the TDI-CCD can be consistent. Meanwhile, before the part 101 and the part 103 start, a specific level signal is required for the TDI-CCD to control the integral imaging direction of the TDI-CCD; in the 102 part, a trigger signal is required to be given according to the real-time movement of the three-dimensional precise movement control platform to control the TDI-CCD to carry out exposure imaging. The level output signal and the real-time TDI-CCD trigger signal of the part are all given from an interface in a control box of the three-dimensional precision mobile control platform, and the specific given numerical value and frequency need to be programmed.
FIG. 3 is a flowchart of the procedure for controlling TDI-CCD exposure imaging by using a three-dimensional precision motion control platform, as follows:
firstly, setting triggering window information, wherein the triggering window information comprises triggering signal frequency, triggering window initial position and triggering window stroke. The main determining factor of the trigger signal frequency is the frame frequency of the TDI-CCD, and the frame frequency of the TDI-CCD used in this example is 50kHz, so the trigger signal frequency must be set to be lower than 50 kHz.
The trigger signal frequency is related to the moving speed of the three-dimensional precision movement control platform and the set trigger distance, and the value f0 of the trigger signal frequency is calculated through the trigger distance s0 of the three-dimensional precision movement control platform and the moving speed v0 of the three-dimensional precision movement control platform, namely f0 is v0/s 0; meanwhile, an acceleration process is needed each time the three-dimensional precision movement control platform moves at a speed of 400 mm/min-800 mm/min, and the size of the initial position of the trigger window is the distance passed by the three-dimensional precision movement control platform in the acceleration process; the stroke of the trigger window refers to a section of stroke of a trigger signal which is given in real time according to the position information in the moving process of the three-dimensional precision moving control platform, and the stroke of the trigger window is behind the acceleration stroke.
Secondly, applying a first level control signal to the TDI-CCD to enable the TDI-CCD to realize forward scanning; before the three-dimensional precision mobile control platform starts to move, a level signal is given out through the control box to serve as a control signal of the integral direction of the TDI-CCD, and the level is directly output through an IO port of the control box.
Thirdly, controlling the three-dimensional precision movement control platform to move forwards along an X axis according to the trigger window information, wherein the X axis refers to the direction in which the three-dimensional precision movement control platform carries the biological tissue sample to move for line scanning imaging, and is shown in figure 2; and a trigger signal is given according to the moving position of the three-dimensional precision movement control platform to control the TDI-CCD to realize exposure imaging along the forward scanning direction. The method comprises the steps of controlling a three-dimensional precise movement control platform to move forwards along an X axis, and simultaneously giving a trigger signal to control TDI-CCD exposure according to the movement position of the three-dimensional precise movement control platform. The part is a key part for performing line scanning delay integral imaging by using a TDI-CCD (time delay integration-charge coupled device), and the type of a trigger signal, namely rising edge triggering or falling edge triggering, needs to be set at first; then the trigger window needs to be set to be a dual trigger, i.e. entering from both ends of the trigger window will give a trigger signal.
Fourthly, applying a second level control signal with the direction opposite to that of the first level control signal to the TDI-CCD to enable the TDI-CCD to realize reverse scanning;
fifthly, controlling the three-dimensional precision movement control platform to move reversely along the X axis according to the trigger window information; and a trigger signal is given according to the moving position of the three-dimensional precision movement control platform to control the TDI-CCD to realize exposure imaging along the reverse scanning direction.
The bidirectional scanning method utilizes the self bidirectional scanning imaging characteristic of the TDI-CCD, combines the hardware characteristic of a three-dimensional precise movement control platform, and realizes combined control through programming. The invention is suitable for a device for performing line scanning imaging by using a TDI-CCD and a three-dimensional precision mobile control platform.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (4)
1. A TDI-CCD-based bidirectional scanning imaging method in fluorescence optical microscopy is characterized in that a biological tissue sample is cut through a cutter, each cutting process is finished by a coronal plane, and a three-dimensional precise movement control platform carries the biological tissue sample to move to a position below an objective lens for scanning imaging;
the scanning imaging specifically comprises the following steps:
s1: setting triggering window information;
s2: the method comprises the steps that a first level control signal is applied to the TDI-CCD, so that the TDI-CCD realizes forward scanning;
s3: controlling the three-dimensional precision movement control platform to move forwards along the X axis according to the trigger window information, and giving a trigger signal according to the movement position of the three-dimensional precision movement control platform to control the TDI-CCD to realize synchronous exposure imaging with the object along the forward scanning direction;
s4: enabling the TDI-CCD to realize reverse scanning by applying a second level control signal to the TDI-CCD, wherein the direction of the second level control signal is opposite to that of the first level control signal;
s5: controlling the three-dimensional precision movement control platform to move reversely along the X axis according to the trigger window information; and a trigger signal is given according to the moving position of the three-dimensional precision movement control platform to control the TDI-CCD to realize synchronous exposure imaging with the object along the reverse scanning direction.
2. The bidirectional scanning imaging method of claim 1, wherein in step S1, said trigger window information includes: trigger signal frequency, trigger window starting position and trigger window stroke.
3. The bidirectional scanning imaging method of claim 2, wherein the frequency f0 of the trigger signal is v0/s0, where v0 is the moving speed of the three-dimensional precision motion control platform and s0 is the trigger distance of the three-dimensional precision motion control platform.
4. A method of bidirectional scanning imaging as in claim 3, wherein said trigger signal frequency is less than 50 kHz.
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| WO2021159285A1 (en) * | 2020-02-12 | 2021-08-19 | 深圳华大智造科技股份有限公司 | Optical imaging system and biochemical substance detection system using same |
| CN113055576B (en) * | 2021-03-31 | 2021-12-31 | 中国科学院长春光学精密机械与物理研究所 | Space camera TDICCD bidirectional push-broom imaging circuit |
| CN114268740B (en) * | 2022-01-05 | 2023-07-18 | 南京大学 | Image sensor based on bidirectional Time Delay Integration (TDI) and imaging method thereof |
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| US5459325A (en) * | 1994-07-19 | 1995-10-17 | Molecular Dynamics, Inc. | High-speed fluorescence scanner |
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| JP2007502419A (en) * | 2003-08-11 | 2007-02-08 | イムニベスト・コーポレイション | Apparatus and method for imaging an object by time delay integration |
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