CN114460735A

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

Info

Publication number: CN114460735A
Application number: CN202210050450.6A
Authority: CN
Inventors: 曾爱军; 乔适; 胡敬佩; 董延更; 黄惠杰
Original assignee: Shanghai Institute of Optics and Fine Mechanics of CAS
Current assignee: Shanghai Institute of Optics and Fine Mechanics of CAS
Priority date: 2022-01-17
Filing date: 2022-01-17
Publication date: 2022-05-10

Abstract

A microscopic imaging device and method based on wavefront correction of a spatial light modulator can be used for realizing correction imaging of the aberration of a microscopic objective caused by a cover glass with any specification and simultaneously correcting the aberration of the microscopic objective and other system aberrations. The device utilizes a wavefront sensor to detect the backward wavefront of an objective lens and simultaneously utilizes a spatial light modulator to compensate. The device omits the manufacture of compensation test pieces of cover glass with different thicknesses, has good correction effect on the aberration of the microscope objective and other systems, and has great application value in the field of optical microscopic imaging.

Description

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

Technical Field

The invention relates to the field of optical imaging, in particular to a wavefront correction imaging device based on detection of a wavefront sensor, which can be used for realizing correction imaging of microscope objective aberration caused by cover glass of any specification and simultaneously correcting objective aberration and other system aberrations.

Background

The microscope can be divided into two lens groups of an objective lens and an ocular lens, the objective lens and the ocular lens of the traditional microscope are relatively closed through the lens cone connecting structure and cannot be used for continuously compensating aberration, and only simple observation needs to be realized, so that the requirement on imaging is not high. However, with the continuous development of scientific demands, the objective lens and the ocular lens are relatively separated, so that the possibility of subsequent aberration compensation is provided. However, the ocular lens and the human eye are replaced by the high-resolution lens and the CCD, and the ocular lens and the CCD are generally used together, so that the aberration optimization of the ocular lens cannot be continuously realized. Therefore, the objective lens is continuously optimized into a feasible scheme for improving the imaging quality, and the direct action of the objective lens and the object light can simultaneously optimize the influence of the object on the imaging and the aberration of the objective lens.

Parallel light is focused at a focus when passing through an ideal microscope objective, when other refractive index media exist near the focus, light beams with different aperture angles pass through an interface and then focus positions can deviate from the focus and are distributed along the axial direction, and if the light beams are reversely used for imaging, the phenomenon can greatly reduce the imaging quality. However, in microscope imaging, the object to be observed is usually fixed under a cover glass and then observed through a microscope. A cover slip of a certain thickness is taken into account in the design of the microscope objective. However, commercially available microscope objectives either do not consider the effect of the cover glass on imaging, or only use cover glass with a single thickness or a small thickness range, and if the cover glass beyond the specification of the applicable range is used, clear imaging cannot be achieved, and especially the aberration introduced by the cover glass needs to be accurately corrected in the occasion of high-resolution imaging. In addition, any real objective lens cannot achieve 0 aberration, and a commercially available microscope objective lens always has certain aberration which cannot be corrected due to the design requirements and the manufacturing difficulty. When the cover glass aberration and the objective lens aberration act simultaneously, aberration correction is made complicated.

Calibration for microscope imaging through coverslips based on the above features must look at two aspects. Firstly, the aberration caused by the cover glass, and because the cover glass is a transparent glass sheet with uniform thickness and flat surface, the convergent light beam passes through the cover glass under the condition of using an ideal objective lens, and the behavior of the light beam and the wave front at any position can be accurately obtained through ray tracing calculation. However, the information available for the microscope objective is very small, and neither the original design file nor the aberration information can be obtained through simple testing, which greatly increases the difficulty of correcting the aberration of the microscope objective.

Disclosure of Invention

The invention provides a microscopic imaging device and a microscopic imaging method based on wavefront correction of a spatial light modulator, and aims to solve the technical problems.

The embodiment of the invention comprises the following steps:

and the laser is used for providing a light source for the correction imaging device.

And the beam expanding and collimating system is used for expanding and collimating the laser beam into parallel light with a proper size to be incident into a subsequent system.

And the spatial light modulator is used for carrying out phase modulation on the incident parallel light to generate light containing a pre-distorted phase wavefront.

And the beam-shrinking system is used for solving the problem that the emergent light spot of the spatial light modulator is not matched with the pupil of the microscope objective.

And the microscope objective is used for focusing the light transmitted through the cover glass.

And the wave front sensor is used for detecting the wave front of the emergent surface of the microscope.

And the computer is used for receiving and processing the signals detected by the wavefront sensor, and generating modulation signals according to the processing result and transmitting the modulation signals to the spatial light modulator for modulation.

The technical principle of the invention is as follows:

light emitted from a laser is collimated and expanded by an expanding system, then enters a spatial light modulator, enters a pupil of a microscope objective through a beam reducing system, and finally focuses on air, oil and the like in an object space medium specified by the microscope objective, but if other materials with refractive indexes exist near an object space focus, the working refractive index and the actual refractive index of the object space of the microscope are not matched, the original focus can be changed into a focal line distributed along an optical axis, and the imaging quality is poor.

Firstly, when the microscope objective focuses under the designed refractive index, the wave surface is spherical wave; however, when other refractive index materials are present, such as a cover glass, focusing can still be achieved inside the cover glass, spherical waves are formed inside the cover glass, and the wave surface in the designed refractive index medium is no longer spherical waves. When focusing in the cover glass, the wave front image of the light at any vertical optical axis plane behind the microscope objective can be obtained through theoretical calculation, and the wave front image is uniquely determined when the position is determined. And selecting the emergent surface of the microscope as a reference wavefront image, wherein the transmission characteristics of the light beam behind the microscope objective lens are the same as long as the actual wavefront image is the same as the reference wavefront image at the same position.

In the calibrated device, the wavefront detected by the wavefront sensor is co-located with the reference wavefront map. The light emitted by the laser passes through the correction device to obtain an actual wavefront image on the wavefront sensor, the actual wavefront image is differenced with the reference wavefront image to obtain a difference value between the actual wavefront and the wavefront at ideal focusing, and a new modulation phase image is generated according to the difference value information and is transmitted to the spatial light modulator for new modulation; and continuously acquiring the modulated wavefront map on the wavefront sensor and repeating the operation until the difference is optimized to be below a target value, wherein the optimization purpose is considered to be achieved.

Besides the optimization of the aberration caused by the cover glass, the optimization process also comprises the global optimization of the aberration of the microscope objective and the whole system because the emergent wavefront of the microscope objective is directly optimized.

The invention has the technical effects that:

(1) the invention can realize aberration compensation of cover glass with any specification, but does not need to carry out actual test on cover glass with different specifications.

(2) The invention can simultaneously correct the aberration of the microscope objective caused by design, manufacture, assembly and the like on the basis of the technical effect (1), thereby further improving the imaging quality.

(3) The invention has simple steps and stable structure when correcting the aberration of the cover glass and the aberration of the objective lens, and can adjust the correction precision according to different use occasions.

Drawings

Fig. 1 is a schematic diagram of a spatial light modulator wavefront correction-based microscopic imaging device according to the present invention.

Fig. 2 is a flow chart of the operation of the microscopic imaging method based on the wavefront correction of the spatial light modulator.

Fig. 3 is a schematic view of an ideal microscope objective when focused in a cover glass (this figure is merely illustrative of the method of obtaining the reference wavefront and does not represent actual dimensions and scale).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention easier to understand, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limitations of the present invention, and that equivalent substitutions for a element or elements of the described embodiments of the present invention are within the scope of the present invention.

An ideal objective lens is a point when focused at a prescribed object-side refractive index. When the cover glass 9 exists near the focal point, the light beam originally focused at one point due to the difference between the refractive index of the cover glass and the refractive index at the time of focusing becomes a focal line distributed along the optical axis, and when imaging continues to be performed through the cover glass 9, the imaging effect is limited.

As shown in fig. 3, when the cover glass 9 is placed at the focal point and focused to a point inside the cover glass 9. Spherical wave transmission is performed from the focal point to the ideal objective lens emergent surface when no cover glass is used for focusing, and spherical wave transmission is performed from the cover glass 9A surface to the focal point in the structure shown in figure 3, but aspheric wave transmission is performed from the cover glass 9A surface to the ideal objective lens emergent surface. The wavefront from the 9A surface to the exit surface of the ideal objective lens is a pre-distorted wavefront which introduces aberration correction to the cover glass 9, and the wavefront and the thickness of the cover glass 9 and the refractive index form a unique determined relationship, so as long as the exit wavefront of the microscope objective lens 5 is the exit wavefront corresponding to the ideal objective lens 8 when focusing in the corresponding cover glass 9 in fig. 3, a good focusing effect can be obtained inside the cover glass 9 with the corresponding thickness, and a good imaging effect can also be obtained when the wavefront is reversely used for imaging.

Based on the principle described above, the device and method for microscopic imaging based on wavefront correction of spatial light modulator of the present invention are proposed, fig. 1 is a schematic diagram of the invented device, fig. 2 is a flow chart of the method of the present invention, and the device and method of the present invention are further described in detail with reference to fig. 1 and fig. 2.

A microscopic imaging device based on wavefront correction of a spatial light modulator, which is specifically shown in fig. 1.

The laser 1 provides a light source for the entire device. Since the light emitted from the laser 1 is a gaussian beam having a small divergence angle, the light can be considered approximately 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 (SLM), so that the beam expanding collimating lens group 2A is subsequently used to expand the laser beam. Wherein, the middle confocal plane of the beam expanding collimator set 2A is added with a small-hole filter 2B to filter the stray light of the system.

It should be noted that the beam expanding

collimator lens groups

2A and 2B are used to ensure that the phase modulation capability of the spatial light modulator 3 is fully exerted and the utilization rate of the light sensing unit is increased, so any structure with equivalent functions is within the protection scope of the present invention.

The beam-expanded and filtered laser beam is incident on the spatial light modulator 3 with an appropriate beam size. Wherein the spatial light modulator 3 does not change the beam propagation characteristics or wavefront characteristics in the device when not loaded with any pre-distorted phase pattern.

In general, the size of the light-sensing surface of the spatial light modulator 3 does not match the size of the pupil of the microscope objective, so the addition of the beam-reducing lens group 4 ensures that the light from the spatial light modulator 3 enters the microscope objective 5 completely.

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

The light passing through the reduction lens group 4 is incident on the microscope objective 5 with a suitable beam size. The microscope objective 5 is placed in the opposite direction to that when the microscope objective is used for imaging, namely, the original object is in the focus position when the original object is focused.

A microscopic imaging method based on wavefront correction of a spatial light modulator, as shown in fig. 2 specifically, includes the following steps:

step 1: calculating a wave front diagram which is close to the exit surface of the ideal objective lens 8 when ideally focused by a cover glass and is recorded as a reference wave front diagram WP0 as shown in FIG. 3; the reference wavefront map is stored in the computer 7.

Step 2: a computer 7 is connected to the spatial light modulator 3 for displaying the phase map P_iTransmitted to the spatial light modulator 3 and producing a phase map P modulated and initially loaded onto the spatial light modulator₀Is a 0 phase diagram.

And step 3: the wavefront sensor 5 records a wavefront map at the same position as the reference wavefront WP0 in the step 1, and records the wavefront as a measured wavefront map WP 1; the wavefront sensor 5 is connected to a computer 7, and the wavefront map is transmitted to the computer 7.

And 4, step 4: the difference between the reference wave front map WP0 and the measured wave front map WP1 is obtained in the computer 7, and the PV value of the difference map is extracted.

And 5: comparing the PV value with the judgment value, if the PV value is greater than the judgment value, not meeting the correction requirement, adjusting the phase diagram according to the difference value of each pixel point relative to the corresponding pixel point of the reference wavefront diagram, returning to the step 2, and continuing to execute the step 2 to the step 5; and if the PV value is smaller than the judgment value, the correction requirement is met, and a corrected device is obtained.

The ideal objective 8 in fig. 3 described above is an ideal objective having the same optical characteristics as the microscope objective 5 in fig. 2 but without aberrations. The judgment value can be adjusted according to an actual use scene, the PV value judgment value can be reduced when high-resolution imaging is needed, 0.25 lambda can be selected when the imaging requirement is not high, and lambda is the working wavelength of the system.

Claims

1. A spatial light modulator wavefront correction based microscopic imaging apparatus, comprising:

a laser for providing a light source;

the beam expanding and collimating system is used for expanding and collimating the laser beam into parallel light;

the spatial light modulator is used for carrying out phase modulation on incident parallel light to generate light containing a pre-distorted phase wavefront;

the beam-shrinking system is used for solving the problem that the emergent light spot of the spatial light modulator is not matched with the pupil of the microscope objective;

the microscope objective is used for focusing the light modulated by the spatial light modulator;

2. The wavefront-correction imaging device of the microscope objective based on the wavefront sensor detection as claimed in claim 1, wherein the wavelength of the output laser light of the laser (1) is within the working wavelength range of the spatial light modulator (3) and the microscope objective (5).

3. A wavefront sensor detection based microscope objective wavefront correction imaging device according to claim 1, characterized in that the wavefront sensor (6) is placed close to the exit surface of the microscope objective (5) to detect the wavefront close to the exit plane of the microscope objective (5).

4. The wavefront-sensor-detection-based microobjective wavefront correction imaging apparatus according to claim 1, wherein the beam expanding collimator set comprises a beam expanding collimator set (2A) and a pinhole filter (2B) for filtering out stray light of the system.

5. A microscopic imaging method based on wavefront correction of a spatial light modulator is characterized by comprising the following steps:

step 1: calculating a wavefront map on the exit surface of the microscope containing the ideal focus of the coverslip, i.e., reference wavefront WP 0;

step 2: loading a phase diagram to a spatial light modulator through a computer, and setting the initially loaded phase diagram as a 0 phase diagram;

and step 3: the wavefront sensor detects a wavefront map at the same position as the reference wavefront in the step 2, namely a measured wavefront map WP 1;

and 4, step 4: the reference wave front map WP0 is differenced with the measured wave front map WP1, and the PV value of the difference is recorded;

and 5: and (4) comparing the PV value obtained in the step (4) with a judgment value:

if the PV value is larger than the judgment value, modifying the phase diagram loaded to the spatial light modulator according to the difference value of each pixel point of the actually measured wavefront relative to the reference wavefront and the same pixel point, and repeating the step 2 to the step 5;

and if the PV value is smaller than the judgment value, acquiring the correction system under the current cover glass.