CN114460735A - Microscopic imaging device and method based on spatial light modulator wavefront correction - Google Patents

Abstract

A microscopic imaging device and method based on wavefront correction of a spatial light modulator, which can be used to realize aberration correction imaging of a microscope objective caused by a cover glass of any specification, and simultaneously perform aberration correction on the microscope objective itself and other systems. Correction. The device uses a wavefront sensor to detect the wavefront behind the objective lens and uses a spatial light modulator to compensate. The device eliminates the need to manufacture compensating test pieces of cover glass with different thicknesses, and also has a good correction effect on the aberrations of the microscope objective lens and other systems, and has great application value in the field of optical microscopy imaging.

Description

Microscopic imaging device and method based on spatial light modulator wavefront correction

technical field

The invention relates to the field of optical imaging, in particular to a wavefront correction imaging device based on detection by a wavefront sensor, which can be used to realize the correction imaging of microscope objective image aberration caused by a cover glass of any specification, and simultaneously correct the objective image aberration and other system images. difference is corrected.

Background technique

The microscope can be divided into two lens groups: the objective lens and the eyepiece. The objective lens and eyepiece of the traditional microscope are relatively closed through the lens barrel connection structure and cannot continue to perform aberration compensation, and only need to achieve simple observation, so the imaging requirements are not high. However, with the deepening of scientific research needs, the objective lens and the eyepiece are relatively separated, which provides the possibility for subsequent aberration compensation. However, the eyepiece and the human eye are replaced by a high-resolution lens and CCD, and the combination of the two cannot continue to optimize the aberration of the eyepiece. Therefore, the objective lens continues to be optimized as a practical solution to improve the imaging quality, and the direct interaction of the objective lens with the light on the object side can optimize the influence of the object side on the imaging and the objective image aberration at the same time.

When the parallel light passes through an ideal microscope objective, it is focused at the focal point. When there are other refractive index media near the focal point, the beams with different aperture angles will deviate from the focal point and distribute along the axial direction after passing through the interface. Greatly reduces image quality. However, in microscope imaging, the object to be observed is usually placed under a cover glass to be fixed and then observed under a microscope. Therefore, a cover glass with a certain thickness will be considered when designing the microscope objective. However, commercially available microscope objectives either do not consider the impact of coverslips on imaging, or are only suitable for coverslips with a single thickness or a small thickness range. In particular, where high-resolution imaging is required, aberrations introduced by the coverslip must be precisely corrected. In addition, it is impossible for any real objective lens to achieve 0 aberration, limited by design requirements and manufacturing difficulties, commercially available microscope objectives will always have certain aberrations that cannot be corrected. When the cover glass aberration and the objective image aberration act simultaneously, aberration correction is complicated.

Correction for microscope imaging through coverslips based on the above characteristics must focus on two aspects. The first is the aberration caused by the cover glass. Since the cover glass is a transparent glass sheet with uniform thickness and flat surface, when an ideal objective lens is used to converge the beam and pass through the cover glass, the behavior of the beam and the wavefront at any position can be determined. Accurately obtained by ray tracing calculation. However, there is very little information available for microscope objectives, neither the original design files nor the aberration information can be obtained through simple tests, which greatly increases the difficulty of correcting the microscope objective aberrations.

SUMMARY OF THE INVENTION

The present invention provides a microscopic imaging device and method based on spatial light modulator wavefront correction, aiming at solving the above technical problems.

Embodiments of the present invention include:

A laser is used to provide a light source for the above correction imaging device.

The beam expander collimation system is used to expand and collimate the laser beam into parallel light of suitable size and incident into the subsequent system.

A spatial light modulator for phase-modulating incident parallel light to produce light containing a predistorted phase wavefront.

The beam reduction system is used to solve the problem that the exit spot of the spatial light modulator does not match the pupil of the microscope objective.

Microscope objective for focusing the aforementioned light through the cover glass.

The wavefront sensor is used to detect the wavefront at the exit surface of the microscope.

The computer is used to receive the signal detected by the wavefront sensor and process it, and at the same time generate a modulation signal according to the processing result and transmit it to the spatial light modulator for modulation.

The technical principle of the present invention is:

The light emitted from the laser is collimated and expanded by the beam expander system, then incident on the spatial light modulator, then incident on the pupil of the microscope objective through the beam reduction system, and finally focused in the object-side medium specified by the microscope objective as follows: Air, oil, etc., but if there are materials with other refractive indices near the focal point of the object side, the working refractive index of the microscope object side does not match the actual refractive index, the original focus will become the focal line distributed along the optical axis, and the imaging quality will change. Difference.

First of all, when the microscope objective is focused at the designed refractive index, the wave surface is a spherical wave; but when there are other refractive index materials such as a cover glass, when the focusing can still be achieved inside the cover glass, the inside of the cover glass is spherical. wave, the wavefront in the design index medium is no longer a spherical wave. When focusing in the cover glass, the wavefront diagram of light in any vertical optical axis plane behind the microscope objective can be obtained by theoretical calculation, and the wavefront diagram is uniquely determined when the position is determined. The exit surface of the microscope is selected as the reference wavefront diagram. As long as the actual wavefront diagram and the reference wavefront diagram at the same position are the same, the transmission characteristics of the light beam behind the microscope objective are the same.

In the calibration device, the wavefront detected by the wavefront sensor is in the same position as the above-mentioned reference wavefront diagram. The light emitted by the laser is passed through the above correction device to obtain the actual wavefront image on the wavefront sensor, and the difference between the actual wavefront image and the reference wavefront image can be obtained to obtain the difference between the actual wavefront and the ideal focused wavefront. value information to generate a new modulation phase map and transfer it to the above-mentioned spatial light modulator for new modulation; continue to collect the modulated wavefront map on the wavefront sensor and repeat the above operations until the difference is optimized to be below the target value. achieve optimization purposes.

In addition to the optimization of the aberration caused by the cover glass, the above processing method is to directly optimize the outgoing wavefront of the microscope objective, so the optimization process also includes the global optimization of the aberration of the microscope objective and the entire system.

The technical effect of the present invention is:

(1) The present invention can realize aberration compensation for cover glass of any specification, but does not need to actually test cover glass of different specifications.

(2) On the basis of the technical effect (1), the present invention can simultaneously correct the aberrations of the microscope objective lens due to design, manufacture, assembly, etc., so as to further improve the imaging quality.

(3) The steps used in the present invention to correct the aberration of the cover glass and the aberration of the objective image are simple, the structure is stable, and the correction accuracy can be adjusted according to different application occasions.

Description of drawings

FIG. 1 is a schematic diagram of the microscopic imaging device based on the spatial light modulator wavefront correction of the present invention.

FIG. 2 is a flow chart of the microscopic imaging method based on the wavefront correction of the spatial light modulator of the present invention.

Fig. 3 is a schematic diagram of an ideal microscope objective when it is focused in a cover glass (this figure is only for illustrating the acquisition method of the reference wavefront, and does not represent the actual size and scale).

Detailed ways

In order to make the objects, technical solutions and advantages of the present invention easier to understand, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, not to limit the present invention, and the equivalent replacement of a certain element or a plurality of elements in the embodiments of the present invention is still within the protection scope of the present invention. Inside.

An ideal objective is a point when it focuses at a specified object-side refractive index. When there is a cover glass 9 near the focal point, due to the difference in the refractive index of the cover glass and the refractive index during focusing, the light beam originally focused on one point will become a focal line distributed along the optical axis. When the imaging continues through the cover glass 9, the imaging The effect will be limited.

As shown in FIG. 3 , when the cover glass 9 is placed at the focal point and the inside of the cover glass 9 is focused to a point. When no cover glass is focused, the focus to the exit surface of the ideal objective lens is spherical wave transmission. In the structure shown in Figure 3, the spherical wave transmission is from the cover glass 9A surface to the focal point, but the non-transmission is from the cover glass 9A surface to the ideal objective lens exit surface. Spherical wave transmission. The wavefront from surface 9A to the exit surface of the ideal objective lens is a predistorted wavefront including aberration correction introduced to the cover glass 9, and this wavefront has a unique relationship with the thickness and refractive index of the cover glass 9, so as long as the display The outgoing wavefront of the micro-objective lens 5 corresponds to the outgoing wavefront of the ideal objective lens 8 when focusing in the corresponding cover glass 9 in FIG. You can also get good imaging results when you are imaging.

Based on the principle described above, a microscopic imaging device and method based on spatial light modulator wavefront correction of the present invention is proposed. The apparatus and method of the present invention will be further described in detail with reference to FIG. 1 and FIG. 2 .

A microscopic imaging device based on spatial light modulator wavefront correction, as shown in Figure 1.

Laser 1 provides the light source for the entire device. Since the light emitted from the laser 1 is a Gaussian beam with a small divergence angle, it can be approximated as parallel light. However, the aperture of the laser beam emitted by the laser 1 is too small to reach the photosensitive aperture of the Spatial Light Modulator 3 (English: Spatial Light Modulator, SLM for short), so the beam expander and collimating lens group 2A is used to expand the laser beam. . Among them, a pinhole filter 2B is added on the intermediate confocal surface of the beam expander collimating lens group 2A to filter out the stray light of the system.

It should be noted that the beam expander and collimating lens groups 2A and 2B described above are to ensure that the phase modulation capability of the spatial light modulator 3 is fully utilized and the utilization rate of the photosensitive unit is increased. within the scope of protection of the invention.

The laser beam after beam expansion and filtering is incident on the spatial light modulator 3 with a suitable beam size. Among them, the spatial light modulator 3 will not change the beam propagation characteristics or wavefront characteristics in the device when no predistortion phase map is loaded.

Generally, the size of the photosensitive surface of the spatial light modulator 3 does not match the pupil size of the microscope objective lens, so adding the beam-reducing lens group 4 ensures that all the light from the spatial light modulator 3 enters the microscope objective lens 5 .

It should be noted that, the beam-reducing mirror group 4 described above is to make all the light passing through the spatial light modulator 3 incident into the microscope objective lens 5, and any structure with equivalent functions is within the protection scope of the present invention.

The light passing through the beam-reducing lens group 4 is incident on the microscope objective lens 5 with an appropriate beam size. The microscope objective lens 5 is placed in the opposite direction when used for imaging, that is, the position of the original object is the focus position when focusing.

A microscopic imaging method based on spatial light modulator wavefront correction, as shown in Figure 2, includes the following steps:

Step 1: Calculate the wavefront image that is close to the exit surface of the ideal objective lens 8 when the cover glass is ideally focused as shown in Figure 3, and record this wavefront image as the reference wavefront image WP0; the reference wavefront image is saved in the computer within 7.

Step 2: The computer 7 is connected to the spatial light modulator 3, transmits the phase map P _i to the spatial light modulator 3 and generates modulation, and the phase map P ₀ initially loaded on the spatial light modulator is a 0 phase map.

Step 3: The wavefront sensor 5 records the wavefront image at the same position as the reference wavefront WP0 described in step 1, and records this wavefront as the measured wavefront image WP1; the wavefront sensor 5 is connected to the computer 7, and the above wavefront image is recorded. transfer to computer 7.

Step 4: In the computer 7, the difference between the reference wavefront map WP0 and the measured wavefront map WP1 is obtained to obtain the difference map of the two wavefront maps, and the PV value of the difference map is extracted.

Step 5: Compare the above PV value with the judgment value. If the PV value is greater than the judgment value, it does not meet the correction requirements. Adjust the phase map according to the difference between each pixel point and the corresponding pixel point of the reference wavefront map and return to step 2 to continue to step 2. Go to step 5; if the PV value is less than the judgment value, the calibration requirement is met, and a calibrated device is obtained.

The ideal objective lens 8 in the above-mentioned FIG. 3 is an ideal objective lens with the same optical characteristics as the microscope objective lens 5 in FIG. 2 but no aberration. The above judgment value can be adjusted according to the actual use scene. When high-resolution imaging is required, the PV value judgment value can be reduced. When the imaging demand is not high, it can be selected as 0.25λ, where λ is the working wavelength of the system.

Claims (5)

1. A microscopic imaging device based on spatial light modulator wavefront correction, characterized in that, comprising: Lasers, used to provide light sources; Beam expander collimation system, used to expand and collimate the laser beam into parallel light; a spatial light modulator for phase-modulating incident parallel light to generate light containing a predistorted phase wavefront; The beam reduction system is used to solve the problem that the output spot of the spatial light modulator does not match the pupil of the microscope objective; A microscope objective for focusing light modulated by a spatial light modulator; The wavefront sensor is used to detect the wavefront at the exit surface of the microscope. The computer is used to receive the signal detected by the wavefront sensor and process it, and at the same time generate a modulation signal according to the processing result and transmit it to the spatial light modulator for modulation. 2 . The wavefront correction imaging device for a microscope objective lens based on detection by a wavefront sensor according to claim 1 , wherein the wavelength of the laser output from the laser (1) is determined by the spatial light modulator (3) and the microscope. 3 . within the working wavelength range of the objective lens (5). 3. The wavefront correction imaging device for a microscope objective lens based on detection by a wavefront sensor according to claim 1, characterized in that, the wavefront sensor (6) is placed close to the exit surface of the microscope objective lens (5) to be close to The detection is performed by attaching the wavefront of the exit plane of the microscope objective lens (5). 4 . The wavefront correction imaging device for a microscope objective lens based on detection by a wavefront sensor according to claim 1 , wherein the beam expander collimating lens group comprises a beam expander lens group (2A) and a pinhole filter. 5 . The filter (2B) is used to filter out the stray light of the system. 5. A microscopic imaging method based on spatial light modulator wavefront correction, characterized in that the method comprises the steps: Step 1: Calculate the wavefront image on the exit surface of the microscope when the cover glass is ideally focused, that is, the reference wavefront WP0; Step 2: Load the phase map to the spatial light modulator through the computer, and set the initially loaded phase map to be the 0 phase map; Step 3: The wavefront sensor detects the wavefront image at the same position as the reference wavefront in step 2, that is, the measured wavefront image WP1; Step 4: Make the difference between the reference wavefront map WP0 and the measured wavefront map WP1, and record the PV value of the difference; Step 5: Compare the PV value obtained in step 4 with the judgment value: If the PV value is greater than the judgment value, modify the phase map loaded into the spatial light modulator according to the difference between each pixel point on the measured wavefront relative to the same pixel point on the reference wavefront, and repeat steps 2 to 5; If the PV value is less than the judgment value, obtain the current correction system under the coverslip.

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