CN109884051A - Harmonic confocal microscopy measurement method based on image scanning - Google Patents
Harmonic confocal microscopy measurement method based on image scanning Download PDFInfo
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- 238000000691 measurement method Methods 0.000 title claims abstract description 16
- 238000004624 confocal microscopy Methods 0.000 title abstract 2
- 238000003384 imaging method Methods 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 230000005284 excitation Effects 0.000 claims abstract description 9
- 230000003287 optical effect Effects 0.000 claims abstract description 7
- 238000007493 shaping process Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 6
- KPHWPUGNDIVLNH-UHFFFAOYSA-M diclofenac sodium Chemical compound [Na+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KPHWPUGNDIVLNH-UHFFFAOYSA-M 0.000 claims description 3
- 238000005259 measurement Methods 0.000 abstract description 5
- 238000000386 microscopy Methods 0.000 abstract 3
- 238000004621 scanning probe microscopy Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 7
- 238000005286 illumination Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000007274 generation of a signal involved in cell-cell signaling Effects 0.000 description 1
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Abstract
基于图像扫描的谐波共焦显微测量方法属于非线性光学测量领域；在谐波显微测量中，用位置敏感CCD探测代替传统共焦扫描显微中的点探测，可将谐波显微测量分辨率提升两倍。飞秒激光脉冲经扫描振镜反射后进入4f光学系统进行光束整形，接着由显微物镜会聚在样品内部形成谐波信号发生所需的激发聚焦光斑。样品激发出的谐波信号，被显微物镜收集后再一次进入4f系统。信号经二向分色镜分离后，基频信号进入光束诊断相机；谐波信号依次经过共焦针孔和窄带滤光片后被位置敏感CCD接收。对于每一个扫描位置，整个图像均被位置敏感CCD记录，通过算法合成所有扫描位置记录的图像，可以提升谐波显微成像分辨率。
The harmonic confocal microscopy measurement method based on image scanning belongs to the field of nonlinear optical measurement; in the harmonic microscopy measurement, the position-sensitive CCD detection is used to replace the point detection in the traditional confocal scanning microscopy, which can resolve the harmonic microscopy measurement. rate doubled. The femtosecond laser pulses are reflected by the scanning galvanometer and then enter the 4f optical system for beam shaping, and then converged by the microscope objective lens inside the sample to form the excitation focusing spot required for the generation of harmonic signals. The harmonic signal excited by the sample is collected by the microscope objective and then enters the 4f system again. After the signal is separated by a dichroic mirror, the fundamental frequency signal enters the beam diagnostic camera; the harmonic signal passes through the confocal pinhole and narrow-band filter in turn and is received by the position-sensitive CCD. For each scanning position, the entire image is recorded by the position-sensitive CCD, and the images recorded at all scanning positions are synthesized by an algorithm, which can improve the resolution of harmonic microscopy imaging.
Description
Technical field
The invention belongs to optical microphotograph fields of measurement, relate generally to a kind of for three-dimensional micro- in nano-device and biological sample
The ultraprecise non-contact measurement method of fine texture measurement.
Background technique
It is generated using the nonlinear optical effect of sample itself, such as second harmonic, triple-frequency harmonics generates, and can carry out biology
The micro-imaging of sample unstressed configuration label, the micro-structure detection of nano-device, the diagnosis etc. of pathogenic mechanism.But harmonic wave micro-imaging
The resolution ratio of method is still limited to the optical transfer function and diffraction limit of microscopic system.Many super-resolution fluorescences were aobvious in recent years
Microtechnology, which promotes imaging resolution to diffraction limit, even breaks through diffraction limit.Wherein Structured Illumination is micro- for one of which
Important technology.In Structured Illumination is micro-, sample is illuminated and is inspired by the excitation intensity distribution Jing Guo spatial modulation
Signal be collected by traditional wide field imaging device.By different location and direction be mobile and rotation excitation intensity mode,
And each wide-field image is recorded, the image after synthesis can be nearly twice by increase resolution.But structure light is needed to illuminating bundle
It is modulated and imaging device is relative complex.
Micro imaging method of the another kind based on Structured Illumination thought is image scanning micro imaging method.Image scanning
Micro imaging method, which focuses the laser under diffraction limit, regards a kind of special Structured Illumination as, carries objective lens numerical hole
The permitted all possible spectrum mode of diameter.A detection is changed to by this method on the basis of conventional confocal scans microscopic method
Position sensing CCD detection, for each scan position, whole image is recorded.The image that all records are synthesized by algorithm, can
To promote micro-imaging resolution ratio.Image scanning micro imaging method is integrated as further mentioning with harmonic wave micro-imaging technique
It rises harmonic wave micro-imaging resolution ratio and provides a new technological approaches.
Summary of the invention
The present invention devises a kind of harmonic wave confocal micro-measurement method based on image scanning, with position sensing CCD mostly as
The photomultiplier tube point that element detection replaces conventional harmonic to scan micro- middle use detects, to realize harmonic wave micro-imaging indirectly
The Structured Illumination of technology, while keeping imaging system simpler compared to traditional structure lighting system and easy to operate.It will be every
The entire harmonic signal image that a scan position is detected synthesizes final harmonic image by algorithm, can be humorous by tradition
The micro- imaging resolution of wave is promoted nearly twice.
The object of the present invention is achieved like this:
Harmonic wave confocal micro-measurement method based on image scanning, replaces conventional harmonic confocal with position sensing CCD detection
Scan the point detection in micro-.Femto-second laser pulse enters 4f optical system after scanning galvanometer reflects to carry out through beam shaping,
Then excitation focal beam spot needed for sample interior forms harmonic signal generation is focused at by microcobjective.Sample excitation goes out humorous
Wave signal again goes into 4f system after being collected by microcobjective.For signal after two separate to dichronic mirror, fundamental frequency signal enters light
Beam diagnoses camera；Harmonic signal is successively received after confocal pinhole and narrow band filter by position sensing CCD.It is described based on
The harmonic wave confocal micro-measurement method of image scanning it is characterized in that by with structure light thought image scanning microscopic method with
Harmonic wave micro-imaging technique organically combines.
The above-mentioned harmonic wave confocal micro-measurement method based on image scanning, it is characterised in that with position sensing CCD mostly as
The photomultiplier tube point that element detection replaces conventional harmonic to scan micro- middle use detects.
The above-mentioned harmonic wave confocal micro-measurement method based on image scanning, it is characterised in that position is scanned for each
It sets, whole image is recorded by position sensing CCD, and is synthesized the harmonic image that all scan positions are recorded by algorithm
Final high-resolution harmonic image.
The above-mentioned harmonic wave confocal micro-measurement method based on image scanning, it is characterised in that using confocal pinhole to harmonic wave
Signal carries out apodization.
Due to replacing conventional harmonic with the more pixel detections of position sensing CCD in harmonic wave micro imaging method of the invention
The photomultiplier tube point detection of micro- middle use is scanned, so that the Structured Illumination of harmonic wave micro-imaging technique is realized indirectly,
Keep imaging system simpler compared to traditional structure lighting system simultaneously and easy to operate.Each scan position is detected
Entire harmonic signal image final harmonic image is synthesized by algorithm, imaging resolution that can be micro- by conventional harmonic
It is promoted nearly twice.
Detailed description of the invention
Fig. 1 is the harmonic wave confocal micro-measurement method schematic diagram based on image scanning.
Specific embodiment
Embodiment of the invention is described in detail below in conjunction with attached drawing.
The harmonic wave confocal micro-measurement method schematic diagram based on image scanning of the present embodiment is as shown in Figure 1.Femtosecond laser
Pulse enters 4f optical system after scanning galvanometer reflects and carries out then being focused at sample interior by microcobjective through beam shaping
Form the excitation focal beam spot needed for harmonic signal occurs.The harmonic signal that sample excitation goes out, it is another after being collected by microcobjective
It is secondary to enter 4f system.For signal after two separate to dichronic mirror, fundamental frequency signal enters Beam diagnostics camera；Harmonic signal successively passes through
It is received after confocal pinhole and narrow band filter by position sensing CCD.For each scan position, whole image is quick by position
Feel CCD record, and the harmonic image that all scan positions are recorded is synthesized by algorithm by final high-resolution harmonic wave figure
Picture.
Claims (4)
1. the harmonic wave confocal micro-measurement method based on image scanning, replaces conventional harmonic is confocal to sweep with position sensing CCD detection
Retouch the point detection in micro-.Femto-second laser pulse enters 4f optical system after scanning galvanometer reflects connect through beam shaping
By microcobjective be focused at sample interior formed harmonic signal occur needed for excitation focal beam spot.The harmonic wave that sample excitation goes out
Signal again goes into 4f system after being collected by microcobjective.For signal after two separate to dichronic mirror, fundamental frequency signal enters light beam
Diagnose camera；Harmonic signal is successively received after confocal pinhole and narrow band filter by position sensing CCD.It is characterized in that will
Image scanning microscopic method and harmonic wave micro-imaging technique with structure light thought organically combine.
2. the harmonic wave confocal micro-measurement method according to claim 1 based on image scanning, it is characterised in that use position
Set the photomultiplier tube point detection that the sensitive more pixel detections of CCD replace conventional harmonic to scan micro- middle use.
3. the harmonic wave confocal micro-measurement method according to claim 1 based on image scanning, it is characterised in that for
Each scan position, whole image are recorded by position sensing CCD, and by algorithm all scan positions are recorded it is humorous
Wave image synthesizes final high-resolution harmonic image.
4. the harmonic wave confocal micro-measurement method according to claim 1 based on image scanning, it is characterised in that use
Confocal pinhole carries out apodization to harmonic signal.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110638424A (en) * | 2019-09-19 | 2020-01-03 | 哈尔滨工业大学 | Scanning light sheet harmonic wave microscopic imaging method and device |
| CN110664369A (en) * | 2019-09-19 | 2020-01-10 | 哈尔滨工业大学 | An adaptive confocal line scan harmonic microscopy imaging method and device |
| CN111879740A (en) * | 2020-07-14 | 2020-11-03 | 哈尔滨工业大学 | All-optical super-resolution microscopic device based on photon reset technology |
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