CN101788709B - Digital confocal microscope optical section collecting drive device - Google Patents
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Abstract
一种数字共焦显微仪光学切片采集驱动器，由单片机系统、细分驱动器、步进电机和位移驱动装置组成，设置于显微镜，通过计算机控制，驱动载物台，采用图像探测器对载物台上的细胞进行序列光学切片图像采集。该驱动器的光学切片图像采集方法是通过对切片间距、采集切片数等参数设置，对显微镜载物台进行亚微米等间距步进控制。该方法以一种清晰度评价函数值的变化率及其函数值大小作为评判标准，对细胞自动聚焦和上下边缘自动定位，采集完整细胞的序列光学切片图像。
A digital confocal microscope optical slice acquisition driver, which is composed of a single-chip microcomputer system, a subdivision driver, a stepper motor and a displacement drive device, is installed in the microscope, and is controlled by a computer to drive the stage. Sequential optical sectioning image acquisition of the cells. The optical slice image acquisition method of the drive is to control the microscope stage by stepping submicron equal intervals by setting parameters such as the slice distance and the number of slices to be collected. In this method, the rate of change of a sharpness evaluation function value and the value of the function value are used as a judgment standard, the cell is automatically focused and the upper and lower edges are automatically positioned, and a sequence of optical slice images of the complete cell is collected.
Description
One, technical field
The present invention relates to a kind of digital confocal microscope optical section collecting device.This driver is the ingredient of digital confocal microscopy instrument, belongs to collecting image of computer control technology field.This driver drives the equidistant step-wise displacement of microscope stage by computer control, utilizes the optical section image-pickup method of driver, is implemented in the purpose of in the microscope biological cell being carried out the sequence optical section collecting.
Background technology
The Photobiology microscope provides strong instrument for the mankind explore microworld since being born, still be widely used in every field such as biomedical research so far.But because optical diffraction effect and three-dimensional biological specimen defocus the influence of the virtual image, make its resolution not high, sharpness is low, and details is difficult to see clearly.
Numeral confocal microscopy instrument is the micro-new technology that occurs the nineties in last century, this technology is based on ordinary optical microscope, configuration image detector, accurate mobile control gear and robot calculator, adopt digital image processing techniques, the biological specimen micro-image of gathering is handled, to improve the resolution and the sharpness of cell image.Mode by optical section, mobile sample passes through from the focal plane, adopt image detector, as CCD, gather the cell sample sectioning image of a series of different cross sections, adopt Image Restoration Algorithm that the sequence section image is deconvoluted to restore again and handle, reach the purpose that improves image resolution ratio and sharpness.
Before digital confocal technology occurred, early the confocal laser scanning microscope that occurs defocused light by irradiation of scan laser point and pin hole interception, and the interference of removing defocused image improves image resolution ratio, can promote clearness equally.But owing to adopt hardware approachs such as laser instrument and scanister, price is very expensive, and active somatic cell is had detrimental effect, is not suitable for obtaining and the observation of cell image under long-time.Numeral confocal microscopy instrument adopts software approach to improve resolution, to the harmless effect of active somatic cell, but does not domesticly still have a product development.
In order to guarantee to restore algorithm effects, reach the purpose that improves image resolution ratio and sharpness, the sequence optical section spacing that pair cell is gathered requires very little, need reach submicron order.For this reason, the adjust the telescope to one's eyes driving step distance of objective table also needs to reach submicron order.
The optical section collecting driver is that pair cell carries out the ingredient that optical section collecting starts to control making usefulness in the digital confocal microscopy instrument.Its function is to carry out sub-micron by computing machine and Single-chip Controlling microscope stage equidistantly to drive, and realizes the sequence optical section image acquisition of pair cell.Require very for a short time owing to drive step distance, all adopt piezoelectric ceramics as driver element abroad at present, but piezoelectric ceramics costs an arm and a leg, domestic being difficult to applies.
Three, summary of the invention
The purpose of this invention is to provide a kind of digital confocal microscope optical section collecting device.This driver is the ingredient of digital confocal microscopy instrument, mainly solves the problem of obtaining of the two-dimensional sequence sectioning image of cell in biomedical image field.
The present invention achieves the above object by the following technical programs:
A kind of digital confocal microscope optical section collecting device, by Single Chip Microcomputer (SCM) system, subdivision driver, stepper motor and displacement drive are formed, control by computer system, utilize the serial ports of computer system to connect Single Chip Microcomputer (SCM) system, the parallel port output of Single Chip Microcomputer (SCM) system connects subdivision driver, the output of subdivision driver connects stepper motor by driving cable, by stepper motor displacement drive is driven, microscopical objective table is fixed on the displacement drive, the output of image detector connects image pick-up card by vision cable, and the output of image pick-up card connects computer system.
A kind of optical section image-pickup method of digital confocal microscopy instrument optical section driver comprises the steps:
1, the control information that optical section collecting driver receiving computer system sends, move according to its control information driving objective table, allow cell pass through from the equidistant stepping in the focal plane of microscope optical system, gather the different optical section profile image of the vertical degree of depth of cell, the control information of driver foundation, be based on a kind of rate of change of sharpness evaluation function value and functional value size thereof as judgment criteria, this sharpness evaluation function is made of Laplace operator and grey scale difference operator, and it is regional to adopt Flame Image Process mode automatic setting to pass judgment on to the cell image of gathering, judge and pass judgment on when the sharpness functional value is maximum in the zone, find the two dimensional cross-section area maximum of whole sample cell
2,1 described sharpness evaluation function value when the record cell departs from the focal plane fully, get certain functional value variable quantity according to concrete cell, get this sharpness evaluation function value and variable quantity sum edge threshold as cell, utilize the rate of change of sharpness evaluation function value and functional value size thereof as judgment criteria, and it is regional to adopt Flame Image Process mode automatic setting to pass judgment on to the cell image of gathering, by judging in this judge zone whether the sharpness functional value levels off to edge threshold and rate of change size thereof, the work of Control Driver, seek the marginal position up and down of cell
3, the area of section maximum with cell is the center, work with 2 described method Control Driver, driving objective table moves and finds the cell upper marginal position, automatic afterwards continuous acquisition cell section image, seek cell lower limb position simultaneously, after finding the lower limb position, stop to gather and the image of gathering is sent to computer system and store.
The course of work and the principle of described digital confocal microscope optical section collecting device are as follows:
At first, computer system adopts the computer interface software control to send relevant parameters information and gives Single Chip Microcomputer (SCM) system, wherein mainly is meant motion step parameter, segmentation parameter and gathers number of slices.Then, segment by Single Chip Microcomputer (SCM) system control subdivision driver, the control step motor rotates, displacement drive is converted to rectilinear motion to the rotation of stepper motor, it is accurate mobile in Z-direction to drive microscope stage, slice distance is gathered in control, and precision can reach submicron order, the tens of millimeters of maximum Z axle displacement.Utilize the optical section image-pickup method of described driver, search out the two dimensional surface area of section maximum and the upper marginal position of cell, with the two dimensional surface area of section maximum of cell is that the coboundary of center and cell is a starting point, continuous acquisition cell section image is judged simultaneously and is sought cell lower limb position.Like this, between marginal position up and down, can automatically carry out equidistant imaging by microscope to the cell two dimensional cross-section of Z-direction different depth.After gathering by high precision ccd image detector, each width of cloth image information is passed to image pick-up card conversion, receive by computer system at last, and have each width of cloth image sequentially optical section order be stored in the corresponding file according to cell, finish the gatherer process of whole two-dimension optical sectioning image.
Outstanding effect of the present invention is:
1, on the basis of traditional wide field optical microscope, use computer system control, there is not the photobleaching of confocal laser scanning microscope and poisons the cell phenomenon, can under shining and shine for a long time, low light obtain image to living cells fast, can be equipped in common biological optical microscope, be the ingredient of digital confocal microscopy instrument.
2, described digital confocal microscope optical section collecting device and its optical section image-pickup method combine and not only can drive microscope stage, to the equidistant scanning imagery of biological sample, and the analysis of fluorescence faultage image and the visual foundation that provides of 3 d data field can be provided, for follow-up two dimensional image recovery and 3 dimensional drawing reconstruct facilitate.
Four, description of drawings
Fig. 1 is the The general frame of whole digital confocal microscopy instrument, wherein is that block diagram is realized in the inside of optical section collecting driver of the present invention in the frame of broken lines.
Fig. 2 is the algorithm pattern of searching of sharpness evaluation function that driver software adopted evaluation and test.
Fig. 3 carries out the depiction of automated optical slice imaging for optical section collecting driver drives microscope stage of the present invention.
Fig. 4 is the general structure connection layout of whole digital confocal microscopy instrument, is the inner structure connection layout of optical section collecting driver of the present invention in the frame of broken lines wherein.
Five, embodiment
Below by accompanying drawing and example digital confocal microscope optical section collecting device of the present invention is described in further detail.
Optical section collecting driver of the present invention, connect to form by Single Chip Microcomputer (SCM) system 2, subdivision driver 3, stepper motor 4 and displacement drive 5, be arranged at microscope 6, Control Software by computer system 1 is controlled, adopt the adjust the telescope to one's eyes sequence optical section image of the biological cell after 6 imagings of image detector 7 to gather, transmit computer systems 1 by image pick-up card 8 conversion backs and show and storage.Computer system 1 possesses the function that information transmission, file operation, driver setting, image acquisition and image show according to the content of the optical section image-pickup method of described driver.
The principle of work of described digital confocal microscope optical section collecting device and process are:
By serial ports the Single Chip Microcomputer (SCM) system 2 of computer system 1 with described optical section collecting internal drive coupled together, send control information to Single Chip Microcomputer (SCM) system 2 by computer system 1, be convenient to the step parameter of Single Chip Microcomputer (SCM) system 2 control step motors 4, so that finally control slice distance, the parallel port output of Single Chip Microcomputer (SCM) system 2 is connected to subdivision driver 3, segment the back by subdivision driver 3 output segmentation parameter and kinematic parameter are sent to stepper motor 4 by driving cable, displacement drive 5 is fixed on the stepper motor 4, the objective table fixed processing of microscope 6 is on displacement drive 5, stepper motor 4 receives the step angle output information that comes from subdivision driver 3, the objective table that correspondingly drives microscope 6 by displacement drive 5 is in the equidistant step-wise displacement of Z-direction, change the position of sample in the focal plane, realized the submicron order displacement, carry out imaging by 6 pairs of target samples of microscope, 7 imaging ends of image detector at microscope 6, gather the imaging of 6 pairs of target samples of microscope, the output of image detector 7 is connected to image pick-up card 8 by vision cable, sends computer system 1 after the image that image pick-up card 8 collects image detector 7 is changed to and stores and show.
A kind of optical section image-pickup method of digital confocal microscopy instrument optical section driver, its specific implementation process is:
1, the control information that optical section collecting driver receiving computer system 1 sends, the objective table that drives microscope 6 is accurate mobile in Z-direction, allow cell pass through from the equidistant stepping in the focal plane of microscope optical system, gather the different optical section profile image of the vertical degree of depth of cell, the control information of driver foundation, be based on a kind of rate of change of sharpness evaluation function value and functional value size thereof as judgment criteria, this sharpness evaluation function is made of Laplace operator and grey scale difference operator, and it is regional to adopt Flame Image Process mode automatic setting to pass judgment on to the cell image of gathering, find the two dimensional cross-section area maximum of whole sample cell, at this moment sharpness functional value maximum.Shown in the C position of Fig. 2.
2, continue to pass judgment on the sharpness evaluation function that Laplace operator and grey scale difference operator constitute, described sharpness evaluation function value when the record cell departs from the focal plane fully, get certain variation delta S according to concrete cell, getting described sharpness evaluation function value is the edge threshold of cell with concrete variable quantity sum, utilize the rate of change of sharpness evaluation function value and functional value size thereof as judgment criteria, and it is regional to adopt Flame Image Process mode automatic setting to pass judgment on to the cell image of gathering, the sharpness functional value levels off to described edge threshold and rate of change when very little in passing judgment on the zone, find the upper marginal position of cell, be stored in computer system 1 inside.Shown in the A position of Fig. 2.
3, the objective table of control microscope 6 is the center with the area of section maximum of cell, upper marginal position with cell is a starting point, move lower limb position toward cell, and the lower limb position of searching cell, the two-dimension optical sectioning image of automatic continuous acquisition cell before finding the lower limb position of cell, the image of collection is sent to computer system 1 and stores.The lower limb position of cell is shown in the B position of Fig. 2, and the optical section depiction as shown in Figure 3.
Whole courses of work of described driver are as follows:
At first, computer system 1 adopts the interface software programming to send the Single Chip Microcomputer (SCM) system 2 that relevant parameters information is given the optical section collecting internal drive, and described parameter information is meant motion step parameter and segmentation parameter.Then, segment by Single Chip Microcomputer (SCM) system 2 control subdivision drivers 3 again, the rotation of control step motor 4, displacement drive 5 is converted into rectilinear motion to the rotation of stepper motor 4, the objective table that drives microscope 6 is accurate mobile in Z-direction, precision can reach submicron order, and maximum Z axle displacement can reach tens of millimeters.According to the optical section image-pickup method of described optical section collecting driver,, search out the two dimensional surface area of section maximum and the upper marginal position of cell, and be stored in the computer system 1 by the control of interface software.Two dimensional surface area of section maximum with cell is the center, is starting point with the upper marginal position of cell, judges and also seeks cell lower limb position, continuous acquisition cell two-dimension optical sectioning image automatically before finding cell lower limb position.At up and down between the marginal position of cell, just can carry out equidistant scanning imagery by the cell two dimensional cross-section of 6 pairs of Z-direction different depths of microscope, gather the imaging of microscope 6 pair cell two dimensional cross-section by image detector 7, the image information that image detector 7 collects is passed to image pick-up card 8 and is changed, receive by computer system 1 at last, and there is each width of cloth image sequentially optical section order according to cell show and is stored in the corresponding file, finished whole by computer system 1 control, utilize the objective table of optical section collecting driver drives microscope 6, gather the gatherer process of the serial two-dimension optical sectioning image of intact cell.
Claims (1)
1. the optical section image-pickup method of a digital confocal microscopy instrument optical section driver is characterized in that, comprises the steps:
1.1 send control information by computer system; The control objective table moves; Cell is passed through from the focal plane of microscope optical system; Adopt a kind of rate of change of sharpness evaluation function and functional value size thereof as judgment criteria; This sharpness evaluation function is made of Laplace operator and grey scale difference operator; In system interface software; Adopt the judge zone of image processing method formula automatic setting cell image; When sharpness function value maximum and rate of change are very little in the judgement judge zone; Find the two dimensional cross-section area maximum of whole sample cell
The described sharpness evaluation function value of step 1.1 when 1.2 the record cell departs from the focal plane fully; Get certain variable quantity according to concrete cell; Getting described sharpness evaluation function value is the edge threshold of cell with concrete variable quantity sum; Utilize the rate of change of sharpness evaluation function and functional value size thereof as judgment criteria; In system interface software; Adopt the judge zone of image processing method formula automatic setting cell image; By judging in this judge zone whether the sharpness function value levels off to edge threshold and rate of change size thereof; Seek the up and down marginal position of cell
1.3 the area of section maximum with cell is the center, execution in step 1.2 control objective tables move and find the cell upper marginal position, judge afterwards and seek cell lower limb position, before finding the lower limb position of cell, automatically gather continuous cell section image simultaneously, the image of collection is sent to computer system and stores.
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