TW200837385A - Modulation differential confocal microscopy - Google Patents

Abstract

By employing the modulation technique (with phase-lock loop detection) and the differential spatial displacement sensitivity of confocal imaging, we are claiming a scanning imaging technique with spatial response much better than conventional confocal microscopy and a capability to determine the kinetic mechanical properties of objects. The axial response curve of confocal microscopy is approximately a sinc2 curve while the lateral response curve is the square of the Airy disc function. The corresponding spatial resolution is the full width at half maximum of these curves. In addition to the very high resolution claimed with differential confocal microscopy, our technique also detects the phase difference between the modulation and the response of the measured object that gives extra information to determine the mechanical properties of the object (such as Young's modulus). Combined with scanning mechanisms, this technique can be used to determine, with great accuracy, the specimen's kinetic behavior in addition to its 3D profiles.

Description

200837385

P060072-TW IX. INSTRUCTIONS: [Technical Field] The present invention provides a new optical microscopy, the working principle of which utilizes the mobile modulation and cataloging system and the common three-degree spatial analysis of f and surface techniques. Degree 'to obtain extremely high lateral and longitudinal displacement resolution (ie, displacement sensitivity 1 'dlsplacement sensitivity), and can estimate the mechanical properties of the object by the phase difference between the measured object's response and the modulation and change frequency. In recent years, a total of (4) micro-surgery has combined laser spectroscopy techniques, such as laser-excited fluorescent photon emission, Raman scattering, harmonic generation, photocurrent (_ b_Xunru (5) current), etc., is an extremely useful tool in the fields of biological tissue research, observation of materials and semiconductor component characteristics. [Prior Art] Confocal ambition was proposed by Marvin Minsky in 1957, and was awarded the national patent in 1961. However, limited to the high-power spatial c〇herem light source at the time, it was 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 and the spatial filter in front of the photodetector are blocked. Signal light outside the focus area is not detected and thus has a longitudinal depth resolution 6 200837385

P060072-TW capability. The typical signal longitudinal response curve of traditional confocal microscopy is about a dnc square function, and the direction-finding response curve is the square of the Airy disc function, and the spatial resolution is the full width at half maximum of these reaction curve functions, which is about the total of the focused beam. The conf〇cal parameters are equal. The difference between the present invention and the traditional co-(4) micro-surgery is that it is in the linear oblique line region of the transverse-level response curve, and the displacement of the target causes the confocal lateral or longitudinal signal sensitive island change, and the _ energy region improves the lateral or Longitudinal displacement resolution. The lateral or longitudinal displacement resolution of the technique (4) determines the limit of the noise, and can measure the mechanical properties of the object (such as the Young's modulus) when the sample is moved and the phase of the modulated signal is moved. The displacement resolution is different from the spatial resolution of the limit of the diffraction limit. The spatiai method can be used to adjust the displacement sensitivity. m rate. Ji Ya does not specialize in the promotion of spatial resolution [invention] In view of the above-mentioned traditional confocal display, the difference between the difference and the Lai-lock-inversion +, the unprecedented spatial displacement resolution of the original. Hunting to achieve the linear slope area of the curve, the signal focus microscopy space reversal to detect the tiny height of the surface of the object; Second: the object position sensitive change The invention can obtain extremely high transverse and wide displacement changes. The principle of utilization is as follows: 冋 "with the resolution of longitudinal displacement. It works in traditional confocal microscopy, such as the optical signal intensity of the piece 7 200837385

The relationship between P060072-TW and the longitudinal displacement of the target is about the -sine square function curve (Fig.), and the resolution is the square of the Airy disc function (Fig. = The height corresponding to the maximum value of the square function curve is the focus of the focus component for the probe. Although the maximum signal strength is obtained at the focus, the slope of the intensity of the target is zero, indicating that the confocal >^ is not sensitive to the target displacement. Especially when the signal light comes from the surface of the object or the interface inside the object, the target is placed

The highest vertical resolution is not available at the focus. If the target height is first trimmed to the linear slope region of the sinc square function curve (the part indicated by the box in Figure 1), then the longitudinal displacement of the target will cause a differential change in the intensity of the light signal detected by the crying. The resulting signal size is extremely sensitive to small differences in height, which 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 the lateral differential processing of the original confocal image to highlight the high spatial frequency of the image. . The invention is a modulated differential confocal microscopy system, as shown in FIG. This system uses a light source 1 具有 having a spatial coherence, focusing the detecting light to the surface of the object 1 〇 6 with a large numerical aperture focusing element 105, and measuring the amount of reflected light on the surface of the object 106 by an optical debt detector. The optical debt detector is placed in a spatial response from the longitudinal response of the confocal imaging. The southness of the object to be measured 106 is finely adjusted to the linear slope region of the aforementioned confocal longitudinal reaction, and then subjected to two-dimensional scanning. We recorded the differential change of the signal height of the photodetector Π1 to the surface height, which can be extremely high. 8 200837385

P060072-TW Accuracy is obtained on the surface of the object, and the height of the space is changed by three degrees under the motion modulation. In the part of the adjustment, the sample can be displaced by the sound wave or piezoelectric actuator, and the relative position between the no-head (or its expansion) function can be changed. In addition, the liquid crystal can be placed in the optical path, and the relative position of the focus point and the sample is periodically changed. In this way, the signal change caused by the shift modulation can be detected by the lock-in amplification circuit. Due to the use of the phase-locked loop, the signal-to-noise ratio is greatly improved, so that the displacement degree exceeds the simple confocal or differential microscopy. In addition, the phase difference loop of the phase-locked loop (the difference between the reference signal of the periodic modulation and the signal to be measured) can be used to interpret the mechanical dynamic response of the object to be measured, and obtain the critical mechanical constant. Such as the Young's Coefficient (Ymmg, s Modulus), etc., is a unique advantage of the present invention. In the detailed description of the actual system described below, the principles applied to the invention, the objects used and their advantages will be more clearly shown. The principle of modulation-phase-lock detection is as follows: Basically, the modulation signal can be expressed as: d(t)^d0+d} cos(^) + 〇(2ω) (1) Only 4 items are detected by the phase-locked loop The high harmonics are negligible. The system signal is the convolution of the modulation signal and the sample reaction (conv〇luti〇n): ping) = c/lC〇S (a>0®i? (0, if the reaction of the sample under the displacement modulation is assumed = indicates (or expands), then the system signal D(1)=1 f cos〇' -multi), (2) da + ω 9 200837385

P060072-TW Multiple = W〇 is the phase shift and contains the reaction time constant (or characteristic mechanical response) of the sample. The time constant, r, can be expressed as r = I = ItanW (3) and the phase can be detected by the phase-locked loop, that is, two orthogonal (phase difference is 9 〇. degrees) output frequency y; /^rc〇s_) and is the phase • offset (offset). • [Embodiment] FIG. 3 is a schematic system architecture of a modulated differential confocal microscopy system of the present invention. The light beam emitted by the laser light source 101 is first enlarged by a beam expander 1 〇 2 and then focused by the focusing element 105 onto the object 1 〇 6 . The multiple of the beam expansion 疋 allows the enlarged beam radius and the focal length of the focusing element 1 to be as close as possible to the numerical aperture of the focusing element 105. This can reduce the full width at half maximum of the longitudinal response function to increase the resolution of the longitudinal space, while reducing the size of the spot at the focus after focusing the beam to achieve a higher lateral resolution in spring. The object to be tested 106 is placed on a 3D high frequency displacement actuator 107, and the 3D high frequency position adjuster 107 is placed on another 3D displacement platform controlled by the computer 113 (3D translation) Stage) 108. First, the distance between the object 106 and the focusing element 105 is adjusted to be close to the focal length of the focusing element 1〇5, and then adjusted to the linear slope region of the axial response curve of the confocal microscopy by the 3D high-frequency position adjuster 107, that is, the adjustment Work area for variable confocal microscopy. The computer controlled 3D displacement platform 1 〇 8 is used to cause the object 106 to be scanned in three dimensions on a plane perpendicular to the ray. 10 200837385

P060072-TW The present invention uses a laser or point source as the source here, the focusing element 105 uses the objective lens of the microscope, and the laser source 101 performs the 2D image via the scanning optics for imaging 104. scanning. However, the feedback light causes an interference effect on the laser. Therefore, a beam splitter 103 is used in the optical path. Before the reflected light of the surface of the object is introduced into the photodetector 111 via the optical splitter 103, the focus is passed through the other focusing element 109 and the needle. A spatial filter 121 _ consisting of a pinhole 110 causes confocal imaging and filters out light outside the focal zone. The signal measured by the photodetector 111 is input to the computer 113, and the size of the signal is matched by the computer 113 to the coordinates of the 3D displacement platform 108 to obtain a three-dimensional spatial stereoscopic image. It should be noted that the relationship between the amount of change in the drive current and the height of the object must be calibrated with the same object 1〇6 before measurement. This allows the height of the object to be fine-tuned using the calibrated 3D high-frequency position adjuster 107 while recording the size of the light-detecting _111 to obtain a linear relationship. The measured values in the linear working area can be fitted in a straight line, as shown in Figure 4.夂 • The calibration point spatial position measurement value is the square root error of the system. The (root-mean-square error) value is the longitudinal resolution of the system. According to this set of straight lines, calibration and resolution 4 can be completed. Taking Figure 4 as an example, when a microscope objective with a numerical aperture of 0·85 is used to detect the focusing element, the dynamic range exceeds 0.8 μm, and the longitudinal resolution of the system is 14 m. Modulated differential confocal microscopy can be used as a light source with high spatial coherence. For example, switching to a shorter wavelength source such as a nitrogen mine laser 200837385

P060072-TW (wavelength 325 nm) will give higher longitudinal and lateral resolution. = The money of the system _ is (four) to make _«, the component his value hole: = and = diameter is smaller, the dynamic range is larger; however, the achievable will be reduced. In our prototype system, Du = (9) numerical hole grip is 〇. 4 _ objective lens for detecting focus element: wide range up to 1 〇 micron 'but the longitudinal resolution becomes 8 〇 meters, and the angular resolution is about 1.2 microns.

The longitudinal resolution of the differential collinear microscopy = the instability of the output power of the source, the inherent noise of the optical controller, and the error of the power-to-digital conversion, so that the calibration position of each calibration point is measured. The square root error for the appropriate straight line is not G. Improve the materials used, such as using power stabilized lasers (1). Stablizediaser, low noise photodetectors, and high-resolution analog-to-digital converters (Analog/Digital C〇nverter) can greatly improve the vertical resolution. The modulated differential confocal microscopy disclosed in the present invention can also be used to form a two-photon excited imaging using a passively locked titanium scale; 5 laser (Ti: Sap handsome eLasei>) as a light source. Double-wire excitation means that the excited molecules absorb two photons at the same time, as if they were absorbing a single photon with the sum of the two photon frequencies. Since two photons are required, the transition rate (Tmnsisti〇n Rate) is proportional to the square of the human ray and requires a very high instantaneous power due to the low two-photon absorption cross section. The mode-locked titanium sapphire laser is excited by argon-ion laser excitation, and its adjustable range is from 700nm to 丨〇5〇nm. The pulse width can be shorter than 10fs, and the average power is more than iw. Since the excitation is only generated in the high-intensity region where the beam is focused, there is no need to use pinholes as spatial filtering. 12 200837385

For the P060072-TW, the two-photon point spread function is the same as the single-photon confocal pinhole. Therefore, in the system, imaging does not necessarily pass through the original focusing element, but can be imaged on the other side of the object 106 via the mirror.

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P060072-TW 200837385 [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)

P060072-TW 200837385 X. Patent application scope: 1. A confocal imaging system comprising: a laser light source, the light beam emitted by the light source is focused by a focusing element and a focusing element, and then focused by a focusing element On the measuring object; a feedback light returns to the spectroscopic component through the original path starting from the object to be measured, and the spectroscopic component is introduced into a spatial filter to cause a confocal imaging; and a photodetector detects the confocal imaging and connects a phase-locked loop, the lock phase is returned to the Φ circuit and a modulation controller exchanges signals; the modulation controller controls a modulation module to generate a displacement modulation, and the phase-locked loop detects the displacement modulation The resulting signal changes to improve the signal-to-noise ratio and displacement sensitivity of the confocal imaging system. 2. The confocal imaging system according to claim 1, wherein the feedback light is emitted by the object to be tested, or is generated by scattering or reflecting incident light - 3, as claimed in claim 1 The confocal imaging system of the item, wherein the spatial filter comprises a focusing element and a pinhole. 4. The confocal imaging system of claim 1, further comprising a platform for changing the position of the object under test, the platform moving the object to a position, wherein the system has the highest The spatial displacement resolution rate. A 15 P060072-TW 200837385 5. The confocal imaging system according to claim 4, wherein the method for obtaining the highest spatial displacement resolution is the signal intensity measured by the spatial filter and the surface of the object to be tested. The distance relationship of the focusing elements is a relationship of a bell-shaped function curve, and an appropriate region is taken in the linear slope region of the function curve such that the differential change caused by the change in the distance is proportional to the height change of the surface of the object to be tested, the operating system In 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 modulating 16 200837385 P060072-TW variable module is periodically displaced by a sensitive driver to change the relative position of the object to be measured. . The confocal imaging system according to claim 10, wherein the modulation module obtains a phase difference between the two by using the phase-locked loop under the modulation of moving the object to be tested. 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 according to claim 1, wherein the modulation module detects the relative position of the focus 黠 and the sample by periodically changing the phase-locked loop, and the phase-locked loop detects the modulation. This 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. 17 P060072-TW 200837385 16, 17, m is 19 • 20 The confocal imaging system of claim 1, wherein the focusing element is a large numerical aperture focusing element. A confocal imaging system according to 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. 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. The confocal imaging system according to claim 18, wherein the photodetector is one of an electric diode, a collapsing photodiode, a photomultiplier tube, a charge coupled device or a fluorescent screen. . < The confocal imaging system of claim 1, wherein the modulation controller is a computer system that receives the signal of the photodetector and sends a signal to the modulation module. a confocal imaging system comprising: a laser source, the pulsed laser capable of generating a two-photon image, the emitted beam being focused onto a measured object via a focusing element; Part of the region is excited by the absorption of the two-photon frequency, which occurs only in the high-intensity region where the beam is focused, and the effect is equivalent to a single photon excitation with the sum of the two photon frequencies and the double The point spread function of the photon has a spatial filtering effect of single photon confocal imaging; and a photodetector 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 for changing the position of the object under test, the platform moving the object to be tested to a position, the system Has the highest spatial displacement resolution. 23. The confocal imaging system according to claim 22, wherein the method for obtaining the highest spatial displacement resolution is that the relationship between the signal intensity measured by the spatial filter and the surface of the object to be measured and the focusing element is The relationship of a bell-shaped function curve takes an appropriate region in the linear slope region of the function curve such that the differential change caused by the change in the distance is proportional to the height change of the surface of the object to be measured, and the operating system is in the region. 24. The confocal imaging system of claim 21, wherein the photodetector is an element capable of generating a signal proportional to the amount of received light. 25. The confocal imaging system according to claim 24, wherein the light 19 P060072-TW 200837385 detector is an electric diode, a collapsing photodiode, a photomultiplier tube, and a charge-matching element. Or one of the screens. _ 20