阅读说明：本技术 提供概览图像的方法 (The method of general view image is provided ) 是由 J.西本摩根 于 2019-05-17 设计创作，主要内容包括：本发明涉及一种提供在样品平面中布置的物体的概览图像的方法,并且该方法包括以下步骤：生成光片,捕获来自样品平面的检测光,通过检测器将捕获的检测光以至少一个捕获的像的图像数据的形式成像在检测平面中,其中捕获的像在相对于样品平面倾斜的像平面中延伸,捕获物体中的至少一个区域的倾斜的堆栈的形式的多个像,并且将倾斜的堆栈变换到归一化Z堆栈,其中将相对于参考轴线的正确取向分配给捕获的像的图像数据。根据本发明的方法以归一化Z堆栈的最大强度投射为特征,其中通过将选择的像点中的每一个作为虚拟投射成像到平行于检测器的像平面的投射平面中,生成得到的概览图像。(The present invention relates to a kind of methods of the general view image of object for providing and arranging in sample plane, and method includes the following steps: generating mating plate, capture the detection light from sample plane, the detection light of capture is imaged in detection plane in the form of the image data of at least one picture captured detector, the picture wherein captured is inclined as extending in plane relative to sample plane, multiple pictures of the form of the inclined storehouse at least one region in captures object, and inclined storehouse is transformed into normalization Z storehouse, correct orientation relative to axis of reference is wherein distributed to the image data of the picture of capture.According to the method for the present invention characterized by the maximum intensity for normalizing Z storehouse projects, wherein by will each of the picture point that select as virtual projection imaging into the projection plane as plane for being parallel to detector, the general view image that generates.)

1. a kind of method for the general view image (OV) for providing the object (1) arranged in sample plane (5), comprising the following steps:

Step A: it generates mating plate (8), wherein the mating plate (8) is along primary optic axis (A1) Lai Shengcheng and at least partly exists On the sample plane (5),

Step B: coming from the detection light of the sample plane (5) along the second optical axis (A2) capture,

Wherein the primary optic axis (A1) and second optical axis (A2) intersect in the sample plane (5) and enclose together At right angle, and

The primary optic axis (A1) and second optical axis (A2) and the axis of reference (B) for being orthogonal to the sample plane (5) are each Surround the angle (α 1, α 2) being not zero.

Step C, detector by the detection light captured in the form of the image data (9) of at least one picture captured at As in detection plane, wherein the picture captured is inclined as extending in plane relative to the sample plane (5),

Step D: by being moved in the sample plane (5) in the relative movement of the mating plate (8) and the object (1) The mating plate (8) captures multiple pictures at least one region in the object (1), wherein the picture captured is relative to described Sample plane tilts and is formed inclined storehouse,

The inclined storehouse is transformed to normalization Z storehouse (10), wherein will be relative to the axis of reference (B) by step E Correct orientation distribute to the image data (9) of captured picture,

The method is characterized in that,

In step F, maximum intensity projection is carried out in the normalization Z storehouse (10), wherein all picture points are along described the Two optical axises (A2) are located at behind another along in parallel one, axis, comment about the corresponding intensity of all picture points Estimate all picture points, and there is the picture point of maximum intensity along the selection of each axis, and

By by each of picture point selected as virtual projection imaging to be parallel to the detector as plane throwing It penetrates in plane (3), the general view image (OV) generated.

2. the method according to claim 1, which is characterized in that by obtained general view image (OV) capture as general view image Data acquisition system.

Wherein, in step G, the copy part of the general view sets of image data is asserted copy image data acquisition system, it is described The contrast for the contrast reduction that part has relative to the obtained general view image is copied, and

The image data (9) of the copy image data acquisition system offsets corresponding image of the general view sets of image data Data (9), so that obtaining with the calculated general view relative to described obtained general view image (OV) increased contrast Image (OVcomp).

3. method according to claim 1 or 2, which is characterized in that be transformed to the general view image (OV) to be orthogonal to institute State the picture plane of axis of reference (B) extension.

Technical field

The present invention relates to the method for general view image is provided, synoptic chart is especially provided in the microscopy methods using mating plate The method of picture.

Background technique

Such as general view image is provided before, during or after the image data of captures object, to provide a user capture The method and apparatus of the image data of quick overview with object.General view image for example can be used for selecting the region to be imaged (interested region, ROI) and/or the quality for assessing captured image data.

Maximum intensity projection (MIP) and minimum are known as from the possible method of offer general view image known in the art Intensity projection.Text below will discuss maximum intensity projection, also be referred to as MIP.

High-level schematic illustrates the principle (standard MIP) of MIP in Fig. 1.In the volume of object 1 (such as biological sample) In, simultaneously or sequentially capture image data 9.Described image data 9 for example indicate the marker 2 of varying strength.Marker 2 It such as can be the structure or molecule equipped with luminescent dye, and excited by exciting radiation to emit such as fluorescence spoke It penetrates.The transmitting position of the detection radiation of capture transmitting and corresponding intensity.Term " marker " packet in the meaning of this specification The molecule for including dyestuff and with the structure of dye marker or marking in this way.

If the axis beam being parallel to each other essentially passes through object 1 and places (as example as shown in arrow), can incite somebody to action Picture point with highest (maximum) intensity projects in the projection plane 3 extended along each of axis.For simplicity, Marker 2 and picture point are equivalent herein, unless this specification clearly refers to different meanings.If having low intensive marker 2 Be located on one in axis herein with the marker 2 with high intensity, then in several cases, the label with higher-strength Object 2 is projected, and is covered and be not shown by the region 2 with higher-strength with more low intensive marker 2.Fig. 1 is shown There is more low intensive marker as the marker 2 of the higher-strength of filled circles and as open circles in several cases 2。

MIP allows quickly to establish general view image with relatively low operation cost.With the two-dimensional position of corresponding marker 2 Relevant information keeps complete, and information relevant to the spatial orientation of marker 2 is lost.If carried out from different directions multiple MIP, then it seems possible for generating the synoptic chart that can be rotated (if necessary, on different direction of observations).

10 2,005 029 225 A1 of DE only is referred to as example herein, and it discloses manufacture maximum intensity projection/minimums The system and method for intensity projection.

Purposes of the MIP in the field of imaging of medical is for example described in Prokop, M. et al. (1997: maximum intensity is thrown Penetrate the purposes in CT angiography: basis comment；Irradiation image 1997,17:433-451) in.

Summary of the invention

The present invention is based on purposes a possibility that specifying the improvement for generating general view image in mating plate microscopy.

The purpose is realized by the method according to claim 1.Advantageous embodiment is the theme of dependent claims.

Present aspect is for providing the general view image for the object (such as biological sample) arranged in sample plane.Method includes connecting The continuous following steps A to F carried out.In the other configurations of method, other steps and/or intermediate steps can be carried out.

In step, mating plate is generated, wherein mating plate generates along primary optic axis and at least partly in sample plane On.In stepb, the light (detection light) from sample plane is captured along the second optical axis, wherein primary optic axis and the second optical axis Intersect in sample plane and surrounds right angle together.In addition, primary optic axis and the second optical axis and the ginseng for being orthogonal to sample plane It examines axis and respectively surrounds the angle being not zero.

In step C, the light of capture is imaged in the form of the image data of at least one picture captured detector In detection plane, wherein the picture captured is inclined as extending in plane relative to sample plane.Inclination corresponds to the first light Inclination of the axis relative to sample plane.In addition, in step D, in the relative movement of mating plate and object, by sample plane Middle mobile mating plate carrys out multiple pictures at least one region of captures object.Relative movement can be progressively or continuously or The sequence combination of two kinds of possibilities.The inclination storehouse of picture is formed relative to the picture of the inclined capture of sample plane.As plane is opposite In parallel to each other.

In step E, inclined stack virtual is transformed to normalization Z storehouse, wherein by relative to axis of reference Correct orientation distributes to the image data of the picture of capture.The process is also known as " going to tilt ".Preferably along axis of reference or with Its parallel axis come realize to especially normalization Z storehouse layer and/or region schematic diagram observation.

It is characterized in that according to the method for the present invention, in step F, carries out maximum intensity projection in normalization Z storehouse, In all picture points along optical axis or along in parallel one, axis be located at another behind, along the optical axis of objective lens (that is along the second optical axis) assesses all picture points about their corresponding intensity, and selects along each axis Picture point with maximum intensity.During selecting picture point, optionally it can be lower than the threshold value with the lower threshold of fixing intensity Without selection.

In step F, by by each of picture point selected as virtual projection imaging to being parallel to detector As plane projection plane in, the general view image that generates.

For simplicity, sample carries the surface of part (such as glass or sample stage) in the description with sample plane etc. Together.

Here, picture point is also understood to mean that picture element (pixel).For example, detector can have specified resolution Rate (pixel of every unit area).The intensity of picture element or the intensity of pixel are understood to the intensity of picture point.

Mating plate especially in the microscopy field of high-resolution microscopy and/or fluorescence microscopy be understood to plate or The space of the shape illumination of piece.The spacial alignment of mating plate is preferably limited by the direction of propagation for being formed as the lighting radiation of mating plate It is fixed.

Mating plate is to provide the region in primary beam path for illumination sample volume, the lighting radiation in the region of sample volume Spatial dimension is no more than 10 μm measured for example in detection direction or on the direction of the optical axis of objective lens, and wherein therefore It is suitable for that sample volume is detected or measured according to the principle of mating plate microscopy.

Have been found that secondary maximum (also known as " secondary lobe ") can be in the generation intermediate frequency of mating plate actually in mating plate microscopy Occur numerously.These secondary maximum are substantially parallel to practical mating plate and extend, and individual mating plate is indicated in terms of their effect simultaneously And there is radiation intensity more lower than practical mating plate.Although lower radiation intensity, secondary maximum can for example cause to mark transmitting Remember the excitation of object or the reflection to lighting radiation.Because these unexpected excitations and transmitting or reflection, detector capture Additional image data, obtain picture (the general view image to obtain in this case) in cause defocus be imaged region or Structure, and/or phantom structure.Phantom structure is to indicate the image data for the structure of the object to be imaged being not present, or cause The image data of imaging of the structure at errors present.

Carrying out MIP in normalization Z storehouse along the optical axis of objective lens leads to following advantageous effect: at least partly not The region or structure correctly captured is covered by the practical structures with higher-strength.The result is that improved general view image.Pass through root It is also known as LSFM-MIP herein according to the picture that method of the invention obtains.

In the advantageous configuration of method, general view image (more particularly its image data) is captured as general view image Data acquisition system.In step G, the copy part of general view sets of image data is asserted copy image data acquisition system, the copy part Contrast with the contrast reduction relative to obtained general view image.The image data of the copy image data acquisition system is with picture The mode of elementization offsets corresponding image data of general view sets of image data, so that obtaining with relative to obtaining The calculated general view image of the increased contrast of general view image.The process is known as unsharp in the field of image procossing Masking ".

Obtained general view image or calculated general view image can be transformed to be orthogonal to axis of reference extension picture it is flat Face.Due to such transformation, corresponding general view image is as user is along axis of reference observation general view image.Therefore, it uses Person intuitively uses and explanation becomes easier to general view image.

Detailed description of the invention

The present invention is explained in greater detail below based on attached drawing and exemplary embodiment.In attached drawing:

Fig. 1 shows the schematic diagram of the principle of maximum intensity projection according to prior art,

Fig. 2 shows the schematic diagrames of the arrangement of mating plate microscopy, and for the institute of image data capture and image procossing The schematic diagram of the method and step of choosing,

Fig. 3 shows the schematic diagram of the selected method and step of the generation of image procossing and general view image,

Fig. 4 show the arrangement of mating plate microscopy schematic diagram and it is according to the present invention for image data capture and The schematic diagram of the selected method and step of image procossing,

Fig. 5 shows the selected method and step of the generation for image procossing and obtained general view image of the invention Schematic diagram,

Fig. 6 shows the comparable schematic diagram of object detail and obtained general view image below: a) using according to existing Have the MIP of technology, b) according to the method for the present invention first configuration MIP and c) second according to the method for the present invention is matched The calculated general view image set and d) using using deconvolution step and the general view image of MIP according to prior art.

Specific embodiment

Fig. 2 shows the arrangement of mating plate microscopy in a simplified manner.The object 1 to be imaged is arranged in sample and carries part 4 On (have as sample plane 5 towards upper surface).Illumination objective lens 6 pass through sample with its primary optic axis A1 and carry the direction sample of part 4 Product plane 5 and object 1.Illumination objective lens 6 are configured, so that passing through the illumination objective lens 6 and such as its of cylindrical lens or scanner Its possible element (being not shown here), mating plate 8 can generate and perpendicular to figure plane along primary optic axis A1.Therefore Show in cross section mating plate 8.

The objective lens 7 equally occurred carry part 4 across sample with its optical axis (referred to as the second optical axis A2) and are similarly directed toward Sample plane 5 and object 1.Primary optic axis A1 and the second optical axis A2 intersect in sample plane 5, and in 90 ° relative to each other Angle.For simplicity, the space that the top towards upper surface of part 4 is carried in sample is similarly known as sample plane 5.Detectable substance The focal position of mirror 7 or focal plane are overlapped with mating plate 8 so that be located at mating plate 8 in region by objective lens 7 by clearly at In picture to the picture plane and region of the image data 9s of referred to as blur-free imaging.Make the image data 9s of blur-free imaging by heavy line Area visualization (seeing below).

Fig. 2 shows two coordinate systems.Cartesian coordinate system with axis x, y and z especially with sample carry part 4, Sample plane 5 is related with axis of reference B.Sample plane 5 is prolonged in the x/y plane as defined by axis x and y (x-axis and/or y-axis) It stretches, and axis of reference B is upwardly extended in the side of axis z (z-axis).The coordinate system is also known as the (ginseng of coordinate system 12 for normalizing Z storehouse See Fig. 3).

With axis X ', the second coordinate system of Y ' and Z ' be especially it is related with primary optic axis A1 and the second optical axis A2.First Optical axis A1 is parallel to as two axis Xs ' and Y ' defined by X '-Y ' plane.Second optical axis A2 is on the direction of axis Z '.The seat Also known as detection coordinate system 11 of mark system (referring to Fig. 3).

Primary optic axis A1 and the second optical axis A2, which is respectively surrounded relative to the axis of reference B perpendicular to sample plane 5, to be not zero Angle, the angle is respectively known as α 1 and α 2 herein.

For example, showing the object area of four almost sphericals on located horizontally from the emitter in object 1.These objects Region is the albumen for example with fluorochrome label, and for simplicity therefore hereinafter referred to as marker 2.1 to 2.4.Fluorescent dye is can be excited by the lighting radiation of illumination objective lens 6 and mating plate 8, and in the state of excitation, emit Fluorescent radiation is as detection radiation.Passing through objective lens 7 for the detection radiative capture along the second optical axis A2 is image data 9. The transmitting of excitation and the exciting radiation of fluorescent dye only just occurs when corresponding marker 2.1 to 2.4 is located in mating plate 8.

Object 1 is moved relative to mating plate 8 (by arrow mark) along sample plane 5.Image data is respectively in different time points It is captured during t1 to t5, and is respectively stored as captured image data 9.

Fig. 2, which has been shown as example, captures image data 9 in time point t3.In the time point t1 that the past occurs, marker 2.1 are located in mating plate 8.The detection radiation emitted by the marker 2.1 is captured with together with as the two-dimensional position in plane And it is assigned to time point t1.It is corresponding as plane is shown as example by being parallel to the fine line of X '-Y ' plane, and for example In the detection surface of similarly unshowned detector.The space to corresponding marker 2.1 to 2.4 is respectively marked by filled circles Differentiate capture.For simplicity, Fig. 2 and 4 diagrammatically illustrates the picture plane for being assigned to independent time point t1 to t5.Used Detector allows the origin by determining the detection radiation of capture come to 2 two-dimensional localization of region.This can be used as example and passes through tool There is the detector of the position sensing of the array (matrix) of detecting element to realize.

In time point t2, no one of marker 2.1 to 2.4 is located in mating plate 8, and therefore corresponding without capturing Image data 9 and as plane is only illustrated as line, the region of the image data 9s with blur-free imaging.In time point t3, second Marker 2.2 is moved in mating plate 8 and emits detection radiation, is captured as image data 9 and saved.In marker 2.3 In the case where 2.4, same process occurs in time point t4 and t5.

In order to provide intuitive intelligible general view image OV (referring to Fig. 3 to the user of the arrangement of mating plate microscopy With 6), captured image data 9 are transferred to normalization Z storehouse 10.For this purpose, being used by being known as tilting the method and step of DS Captured image data 9 are transformed to virtual normalization Z storehouse 10 by the method for calculating.For example, if user is in each feelings The a part or (preferably horizontal) layer of normalization Z storehouse 10 to be occurred are arranged under condition, then they will be given along reference Axis B observation digitizes the impression of object 1 now.The image data 9 of marker 2.1 to 2.4 is with laterally correctly square with positioning Formula is shown.

Fig. 3 shows captured image data 9 from detection coordinate system 11 to the transformation of the coordinate system 12 of normalization Z storehouse 10 Method and step.The detection captured objective lens 7 radiates in a manner of spatial discrimination that (such as Fig. 2 schematically shows to capture Out), so that the location information of marker 2.1 to 2.4 exists in detection coordinate system 11 in the form of image data 9.This by The solid line of interruption is schematically shown, and has corresponding with inclination (referring to fig. 2 and the Fig. 4) of the second optical axis A2 inclination.For For the sake of concise, the not exclusively overlapping each other of coordinate system 11 and coordinate system 12 is detected.

The coordinate system 12 of normalization Z storehouse 10 is limited, and the coordinate of image data 9 is (special from the detection transfer of coordinate system 11 It is not conversion) arrive coordinate system 12.If appropriate, the picture number of missing is supplemented by interpolation method or other known method According to 9.

As described in the principle hereinbefore with reference to Fig. 1, then MIP can be carried out along Z axis.Respectively along axis (by arrow Labeling head) the corresponding maximum intensity of image data 9 be located is projected onto projection plane 3, and obtains general view image OV.

Notable of the present invention be can the imaging aberration as caused by the unexpected secondary maximum 8a of mating plate 8.Illustrate in Fig. 4 Property shows the secondary maximum 8a of also known as secondary lobe.

If as being already mentioned above about Fig. 2, then object 1 and mating plate 8 are displaced relative to each other, and time t1 extremely T5 captures image data 9, then additional image data 9 mistakenly occurs.Fig. 4 again illustrates time point t3 as example.The Two markers 2.2 are located in mating plate 8.Detection radiation is transmitted by least one fluorescent dye that the lighting radiation of mating plate 8 excites, It is captured under the form of image data 9 in a manner of room and time resolution objective lens 7.However, at time point T3, third marker 2.3 are illuminated and are excited via one lighting radiation in secondary maximum 8a to emit detection radiation. It means that, in addition to the image data 9 of the second marker 2.2, also capturing third marker 2.3 now in time point t3 Image data 9, wherein the relatively low-intensity and third marker 2.3 due to the lighting radiation of secondary maximum 8a are not at detectable substance The fact in the focal plane of mirror 7, thus the detection of third marker 2.3 is radiated with the image data 9 compared with low-intensity and acutance Form is captured.The detection radiation of position outside the focal plane as caused by one in secondary maximum 8a effect is expressed as image The picture of data 9u, defocus is imaged and is marked by hollow ellipse.

Different from Fig. 2, in each of the time point t1 to t5 as example selection, the image data 9s of blur-free imaging Region be expressed as the form of heavy line, and the region of the image data 9u of the imaging of defocus can be schematically shown.In Time point t4, second and the 4th marker 2 even in secondary maximum 8a, therefore in addition to the figure of the blur-free imaging from focal plane As data 9s region other than, capture defocus imaging image data 9u two regions.

It after capture image data 9,9s, 9u, carries out the step of tilting DS, and as described above, generates normalization Z Storehouse 10.For explaining to relevant other according to the present invention, the region of the image data 9s of blur-free imaging hereafter will be only shown The region of the image data 9u of defocus imaging.

For simplicity, Fig. 5 is limited to the coordinate system 12 of normalization Z storehouse 10.If carried out as explained about Fig. 3 MIP, then obtain general view image OV, not only the region of the image data 9s of blur-free imaging contributes to this, but also defocus Three regions of the image data 9u of imaging also contribute.

In contrast, many if carrying out MIP (referring to fig. 2 and Fig. 4) on the direction of the second optical axis A2 --- in mesh Before in the case where it is even all --- the region of the image data 9u of defocus imaging is on projecting direction by the figure of blur-free imaging As the region overlay of data 9s.It used projection plane 3 and is obtained in the method according to the invention by the method General view image OV obliquely shown on upper left side.

General view image OV can be transformed to the picture plane for being orthogonal to axis of reference extension, to make user in Z again The side of axis, which looks up, to be possibly realized.

Therefore, according to the method for the present invention for obtaining with when carrying out MIP along Z axis the case where compared with it is less, The general view image OV that the region of the image data 9u of defocus imaging contributes to it.

Fig. 6 a) actual effect of the invention is shown into 6d).Fig. 6 a) it shows using as about the institute above of Fig. 2 and 3 The general view image OV in the section for the object 1 that the maximum intensity projection (referred to as standard MIP) of description has been created.Such as secondary maximum 8a The region of the image data 9u of defocus imaging is provided, the region is indicated by means of an arrow.

In contrast, Fig. 6 b) in, pass through first such as interpreted about Figure 4 and 5 according to the method for the present invention (LSFM-MIP) is configured to generate general view image OV.In general view image OV, the region of the image data 9u of defocus imaging Ratio substantially reduces.General view image OV presents apparent, has more high contrast, and show imaging structure it is more Details.That be shown as example is the image data 9s of blur-free imaging, is still greatly imaged by defocus in Fig. 6 a) The regional effect of image data 9u.

In other configurations according to the method for the present invention, obtained general view image OV is not demonstrated but is captured as general Look at image collection.

In step G, the copy part of obtained general view image OV is created as copy image data acquisition system, wherein described copy Shellfish part has the contrast of the contrast reduction relative to obtained general view image OV.The respective correspondence of general view sets of image data Image data the image data of copy image data acquisition system is subtracted with the calculating of pixelation so that obtain have relative to The calculated general view image OVcomp of the increased contrast of general view image arrived.Fig. 6 c) in show it is calculated so general Look at image OVcomp.

The general view image OV of LSFM-MIP and calculated general view image OVcomp is on the direction of X ' axis than standard MIP's Situation is longer.If in X ' on the direction of axis by the compression of images of LSFM-MIP, similar mark can be generated in terms of size The image that quasi- MIP is presented.The factor of necessity compression depends on the height and length of the object 1 of imaging herein.In attached drawing 6b) and Such compression is carried out in image 6c).

Fig. 6 d) show with according to the MIP of Fig. 6 a) carry out using deconvolution process (DCV) after object 1 Section.If such MIP is applied to according to prior art Z storehouse (it is previously by deconvolution) obtained, secondary maximum is not It is visible again.Fig. 6 d) although show calculate expense it is lower, it is also possible to by according to the method for the present invention obtain qualitative can compare Compared with general view image.

Appended drawing reference

1 object

2 (excitation) markers

3 projection planes

4 samples carry part

5 sample planes

6 illumination objective lens

7 objective lens

8 mating plates

8a secondary maximum, secondary lobe

9 image datas

The image data of 9s blur-free imaging

The image data of 9u defocus imaging

10 normalization Z storehouses

11 detection coordinate systems

12 (normalization Z storehouse 10) coordinate systems

A1 primary optic axis

The second optical axis of A2

B axis of reference

DS goes inclination (method and step)

OV (obtains) general view image

The calculated general view image of OVcomp

1 angle of α (between primary optic axis A1 and axis of reference B)

2 angle of α (between the second optical axis A2 and axis of reference B)