TWI329207B - Modulation differential confocal microscopy - Google Patents

Description

1329207

P060072-TW IX. INSTRUCTIONS: [Technical Field] The present invention provides a new optical microscopy whose working principle utilizes a three-dimensional spatial analysis of motion modulation, phase lock detection and confocal microscopy Degree, in order to obtain extremely high lateral and longitudinal displacement resolution (ie displacement sensitivity), and can estimate the mechanical properties of the object by the phase difference between the reaction of the object to be measured and the modulation frequency. In recent years, confocal microscopy has combined laser spectroscopy techniques such as laser excitation fluorescence, multiphoton excitation, Raman scattering, harmonic generation, and optical beam induced. Current) is an extremely useful tool in the fields of biological tissue research, observation of materials and semiconductor device characteristics. [Prior Art] Confocal microscopy was proposed by Marvin Minsky in 1957 and obtained the US national patent in 1961. However, limited spatial c〇herent light sources without high power at the time were not immediately available for widespread attention and application. Confocal microscopy did not begin to develop until after the invention of the laser. The signal light of confocal microscopy is from the laser beam that is focused by a numerical aperture of a numerical aperture, the fluorescent light, reflected light or scattered light emitted by the object at the focus; the light outside the focus area is The spatial filter in front of the light detector is blocked. Signal light outside the focus area is not detected and thus has a longitudinal depth analysis 6

P060072-TW month b force. The typical signal longitudinal response curve of the traditional confocal display is about a sine square function. The lateral response curve is the square of the Airy disc function, and the spatial resolution is the full width and width of the function of these reaction curves. The confocal parameters are equal. The difference between this and traditional confocal microscopy is that it works in the linear oblique line of the lateral or longitudinal response curve, and the displacement of the target will result in a sensitive differential change of the confocal lateral or longitudinal signal', thus greatly improving the lateral or vertical direction. Displacement resolution. This technique _ lateral or longitudinal displacement resolution (four), the limit of the noise control determines 'and can be used to measure the mechanical characteristics of the object (such as Young's coefficient) by measuring the phase difference between the movement of the miscellaneous (4) and the coordinate (4). The displacement resolution is different from the spatial resolution (spatial res〇lmi〇n) limited by the diffraction limit of the light wave. The modulation sensitivity can be greatly improved by using modulation and differential methods (d1Splacement sensi lying y), but this is not equivalent to increasing the space ratio. SUMMARY OF THE INVENTION In view of the conventional confocal microscopy and signal processing described above, the space displacement rate is not continued. In the linear slope region of the curve, the signal is strong:: the height of the burst = ?! · 〗, the height and lateral displacement changes. Use the principle 2 横向 lateral and longitudinal displacement resolution. Its work in the traditional total money micro-surgery, _ (four) obtained optical signal strength

The relationship between P060072-TW and the longitudinal displacement of the target is about a sine square function curve (Fig. 1). The horizontal resolution is the square of the Airy disc function (Fig. 2). The height corresponding to the maximum value of the sine square function curve is the focus of the focusing element for detection. Although the maximum signal strength is obtained at the focus, the slope of the intensity of the "is intensity on the target is love, and the confocal signal is not sensitive to the displacement of the target at this position, especially when the signal light comes from When the surface of an object or the interface inside the object is placed at the focus, the highest longitudinal resolution is not obtained. If the height of the target is first adjusted to the linear slope of the sine square function curve (the part marked by the box in Figure 1), the longitudinal displacement of the target will cause the difference of the intensity of the optical core known by the detector. The change in signal size caused by the change 'is extremely sensitive to small differences in height' and thus can greatly increase the vertical resolution. The same principle can also be used to greatly increase the sensitivity of the lateral displacement (as indicated by the box in Figure 2). The effect is equivalent to laterally differentiating the original 2 confocal image to highlight the high spatial frequency components of the image. The present invention is a modulated differential confocal microscopy system, as shown in Figure 3. This system uses a light source 1 with spatial coherence (Μ, focusing the detection light to the surface of the object 1〇6 with a large value hole, and then detecting the reflected light on the surface of the object 106 with a photodetector. ^ 0g , 里光检测益1 1 1 一 一 工 工 滤波器 滤波器 121 121 121 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 121 以 121 以 121 以 121 以 以 。 。 Zone, then carry out the two-dimensional material / W the confocal longitudinal detector 111 signal to the surface height of the differential: We record the light detection 'can be extremely high 1329207

P060072-TW [Simple description of the diagram] Figure 1: Relationship between signal intensity and longitudinal displacement of the target in confocal microscopy. The box marked part is a linear slope area. Figure 2: Relationship between signal intensity and lateral displacement of the target in confocal microscopy. The box marked part is a linear slope area. Figure 3: Prototype system architecture for modulated differential confocal microscopy. Figure 4: When a microscope objective with a numerical aperture of 0.85 is used to detect the focusing element, the magnitude of the signal measured by the photodetector varies with the height of the target. The solid dot is the measured signal value, and the solid line is the linear fitting curve of the measured value. [Main component symbol description] 101 laser light source 102 beam expander 103 beam splitter 104 optical scanner 105, 109 focusing element 106 object 107 high frequency position adjuster 108 displacement platform 110 pinhole 111 light detector 112 phase lock Loop 113 computer 121 spatial filter 14

Claims (1)

1329207 P060072-TW X. Patent Application Range: 4. A confocal imaging system comprising: a laser light source, the light beam emitted by the light source is focused by a beam expanding element and a beam splitting element, and then focused by a focusing element On the measuring object, a feedback light returns to the light splitting component via the original path starting from the object to be measured, and the light splitting component is introduced into a spatial filter to cause a confocal imaging; and a photodetector detects the confocal imaging and connects one a phase-locked loop, the phase-locked loop and a modulation controller exchange signals with each other; the modulation controller controls a modulation module to generate a displacement modulation, and the signal caused by the displacement modulation is detected by the phase-locked loop Changes to improve the signal-to-noise ratio and displacement sensitivity of the confocal imaging system. 2. The confocal imaging system of claim 1, wherein the feedback light is generated by excitation of the object to be measured or by scattering or reflecting incident light. 3. The confocal imaging system of claim 1, wherein the spatial filter comprises a focusing element and a pinhole. 4. The confocal imaging system according to claim 1, further comprising a platform for changing the position of the object to be tested in the space, the platform moving the object to a position, wherein the system has the highest The spatial displacement resolution rate. 15 1329207 P060072-TW 5' The confocal imaging system of claim 4, wherein the method for obtaining the highest spatial displacement resolution is the signal intensity measured using the spatial filter and the surface and focus of the object to be measured The distance relationship of the components is a relationship of a bell-shaped function curve, and an appropriate region is taken in the linear slope region of the function curve so that the differential change caused by the change of the distance is proportional to the height change of the surface of the object to be measured, and the operating system The area. 6. The confocal imaging system of claim 5, wherein the differential change caused by the change in distance is also proportional to the horizontal offset. 7. The confocal imaging system of claim 4, wherein the platform has a one-dimensional or two-dimensional scanning function to achieve measurement of a full surface height and horizontal interface change of the measured object. 8. The confocal imaging system of claim 7, wherein the platform can use a piezoelectric, mechanical, electromagnetic, or sonic displacement platform. 9. The confocal imaging system of claim 7, wherein the platform is a platform device having rapid displacement vibration. 10. The confocal imaging system of claim 1, wherein the 16 1329207 P060072-TW variable module is periodically displaced by a sensitive actuator to change the relative position of the object to be measured. . 11. The confocal imaging system of claim 10, wherein the modulation module uses the phase-locked loop to obtain a phase difference between the two while moving the object to be measured. 12. The confocal imaging system of claim 11, wherein the phase difference is determined by the mechanical properties of the object, and the phase difference signal can be utilized to obtain the mechanical properties of the object to be measured. 13. The confocal imaging system of claim 1, wherein the modulation module periodically changes the relative position of the focus element of the focusing element to the sample, and the phase locked loop detects the modulation. The feedback light. 14. The confocal imaging system of claim 13, wherein the modulation module is disposed in the optical path of the focusing element and the beam splitting element. 15. The confocal imaging system of claim 14, wherein the modulation module can use a liquid crystal panel, a deformable mirror, or other component that changes the phase of the wavefront of the light wave. The confocal imaging system of claim 1, wherein the focusing element is a large numerical aperture focusing element. 17. The confocal imaging system of claim 16, wherein the large numerical aperture focusing element is one of a microscope objective, an optical fiber exit, a Fresnel wave plate or a GRIN mirror. 18. The confocal imaging system of claim 1, wherein the photodetector is an element capable of generating a signal proportional to the amount of received light. 19. The confocal imaging system according to claim 18, wherein the photodetector is an electric diode, a collapsing photodiode, a photomultiplier tube, a charge-and-light component or a fluorescent screen. one. 2. The confocal imaging system of claim 1, wherein the modulation controller is a computer system that receives the k-number of the photodetector and sends a signal to the modulation mode. group. 21. A confocal imaging system, comprising: a laser source, which is a pulsed laser capable of generating a two-photon image, the emitted beam being focused onto a measured object via a focusing element; The partial region is excited by absorbing the frequency of the two-photon, which occurs only in the high-intensity region where the beam is focused, and the effect is equivalent to the excitation of a single photon having the sum of the two photon frequencies and the two-photon The point spread function has a spatial filtering effect of single photon confocal imaging; and the photosensor collects the excitation light emitted by the detected object to form a confocal image. 22. The confocal imaging system of claim 21, further comprising a platform to change the position of the object under test, the platform moving the object to the position - the position has the highest The spatial displacement solution 23, such as ^ 专 · 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 第 共 共 共 共 共 共 共 共 共 共 共= =: Γ 'In the linear slope region of the function curve, the b domain of the field is made such that the differential change caused by the distance of the sound of the object (4) is proportional to the change of the surface of the object, and the operating system is in the region. . 24. The conjugate detector according to claim 21 is a confocal imaging system according to claim 24, wherein the light is 1329207. The P060072-TW detector is one of an electric diode, a collapsed photodiode, a photomultiplier tube, a charge coupled device or a fluorescent screen. 20