CN115877554A - Corneal confocal microscope system with high resolution and rapid imaging - Google Patents

Abstract

The invention discloses a corneal confocal microscope system for high-resolution rapid imaging, wherein a light beam from a laser light source in the system passes through a light filter and a polarization encoder, then reaches a first lens through a beam expander and is focused on a slit; the light beam passing through the slit reaches the scanning mirror through the second lens, the scanning mirror changes the propagation direction, then the light beam passes through the scanning lens, is bent by the beam splitter, enters the microobjective through the sleeve lens and is focused on a cornea to be imaged; the cornea is illuminated by laser to generate a scattered light signal, the scattered light signal returns to the detection light path through the beam splitter, and the scattered light signal passes through the polarization selector and the optical filter and is exposed by the imaging part in a rolling shutter mode to form an image; and the displacement table is axially moved to realize the three-dimensional imaging of the cornea. The laser light source is used for line scanning, and the laser imaging system has the characteristics of high resolution, quick imaging, simple and stable system and strong penetrating power. The rolling shutter is used for blocking out-of-focus light rays, so that the focal depth can be conveniently adjusted, and the cornea can adapt to different tissue structures.

Description

Corneal confocal microscope system with high resolution and rapid imaging

Technical Field

The invention relates to the field of optical microscopic imaging and biomedical imaging, in particular to a corneal confocal microscope system for high-resolution rapid imaging.

Background

The concept of confocal Microscopy was first proposed by Marvin Minsky in 1957 for observing neural networks in living brain tissue (see Microcopy Apparatus, U.S. Pat. No. 5,3013467A). The confocal microscope system is a scanning imaging system and is characterized in that conjugate pinholes are arranged behind a light source and in front of a detection plane to shield stray light beams around non-imaging points, so that the imaging resolution of the system is greatly improved, and the capability of independently imaging parts with certain depth of an imaging object is realized.

Confocal corneal microscopy is a technique for imaging the structure of a living corneal cell using a confocal microscope to assist in the study of changes in the physiological and pathological structures of keratopathy at the level of the living cell, and to assist in disease diagnosis and observation of therapeutic responses, etc. Maurice first applied confocal systems to corneal microimaging in 1974, and successfully obtained microscopic images of the corneal endothelium (see David Maurice, A scanning Slit Optical Microscope, invest, optimholl. Vis. Sci.13, 1033-1037 (1974)). With a series of improvements, the confocal system used by Maurice has been used more widely in clinic.

There are two main types of confocal corneal microscopes currently available on the market: the first type is slit-light confocal microscope, the system usually uses a halogen lamp for filtering out infrared and ultraviolet parts, a pair of conjugate slits (slit) is used for replacing a pinhole, and a linear light beam scans and images in a one-dimensional direction, so that the system has the characteristics of simple structure and quick imaging. However, because the penetration depth of the incoherent light source is shallow, the slit-light confocal system is difficult to image the turbid cornea, the imaging resolution is limited, and the image definition is limited. The second type is a laser point scanning cornea confocal microscope, the microscope system uses laser as a light source, two scanning devices are used for respectively realizing scanning in two-dimensional directions, and the system structure is more complex than that of the former type. In addition, the laser point scanning corneal confocal microscope system has a slow imaging speed, the image is easily affected by the movement of the eyeball to generate distortion, and the patient is difficult to fit due to long-time operation and feels uncomfortable to eyes. The common characteristics of the two confocal microscope systems are that the size of the used slit and pinhole is not convenient to adjust, and the imaging focal depth cannot be adjusted to adapt to the structures of different tissues of the cornea.

Disclosure of Invention

Aiming at the defects of the conventional confocal corneal microscope technology, the invention provides a corneal confocal microscope system with high resolution and rapid imaging, and clear and rapid corneal confocal microscopic imaging is realized.

The purpose of the invention is realized by the following technical scheme: a corneal confocal microscope system for high-resolution rapid imaging comprises an illumination light path and a detection light path;

the illumination light path comprises a laser light source, an optical filter, a beam expander, a polarization encoder, a first lens, a slit, a second lens, a scanning mirror, a scanning lens, a beam splitter, a sleeve lens and a micro objective fixed on a displacement table;

after passing through the optical filter, light beams from the laser light source pass through the beam expander and are subjected to polarization encoding by the polarization encoder, and then reach the first lens and are focused on the slit; the light beam passing through the slit reaches the scanning mirror through the second lens, and the propagation direction of the light beam is changed by the scanning mirror; the light beam emitted from the scanning mirror is reflected by the beam splitter after passing through the scanning lens, enters the microobjective through the sleeve lens and is focused on a cornea to be imaged;

the detection light path comprises a polarization selector, an optical filter and an imaging piece; the cornea generates a scattered light signal through laser illumination, the scattered light signal returns to the detection light path through the beam splitter, firstly, the polarization selector selects the scattered light with a specific polarization state, then the stray light signal is filtered through the optical filter, finally, the imaging part exposes in a rolling shutter mode to form an image, and the number of pixel lines exposed at the same time is adjusted by controlling the rolling shutter, so that the exposure condition of defocused scattered light is changed, and the focal depth is adjusted; and axially moving the displacement table to realize the three-dimensional imaging of the cornea. Furthermore, the first lens is a cylindrical lens, the cylindrical surface is a light beam incidence surface, and the cylindrical axis direction is parallel to the slit direction and the rotating shaft direction of the scanning mirror.

Further, the polarization encoder is composed of a linear polarizer, a quarter wave plate and a half wave plate.

Further, the slit is positioned on the back focal plane of the first lens and positioned on the front focal plane of the second lens.

Further, the scanning mirror scans the linear light beam on the focal plane of the microscope objective lens in a one-dimensional direction inside the cornea; the scanning mirror is optically conjugate with the entrance pupil of the microscope objective.

Further, the scanning lens is a lens group that corrects aberrations, which can produce a flat image plane when the light beam is scanned, and the center of the scanning lens entrance pupil coincides with the pivot point of the scanning mirror.

Furthermore, the beam splitter reflects the illumination light and transmits the scattered light from the cornea, and forms an included angle of 45 degrees with the illumination light path and the detection light path.

Further, the back focal plane of the sleeve lens is superposed with the entrance pupil plane of the microscope objective lens; the sleeve lens and the scanning lens constitute a 4f optical system.

Further, the back focal plane of the first lens, the back focal plane of the scanning lens, the focal plane of the microscope objective and the photosensitive surface of the imaging piece have a conjugate relation.

Further, the microscope objective is fixed on a displacement stage having a capability of moving in the optical axis direction.

Further, the polarization selector is composed of a phase retarder and a linear polarizer.

Further, the imaging element is a CMOS camera with a rolling shutter imaging mode, and the scanning mirror and the rolling shutter synchronously move, so that cornea defocusing scattered light is not exposed and does not participate in image formation.

Further, the working wavelength of the laser light source and the optical filter can be adjusted between 600nm and 840 nm.

Further, a polarization encoding and polarization selecting device is contained in the optical path to carry out polarization encoding imaging.

Compared with the prior art, the invention has the following beneficial effects:

the laser light source is used for line scanning, and the system has the characteristics of high resolution, quick imaging, simple and stable system and strong penetrating power. The shutter is used for blocking the defocusing light rays instead of a solid pinhole, so that the focal depth can be conveniently adjusted to adapt to different tissue structures of the cornea.

Drawings

Fig. 1 is a schematic structural diagram of a corneal confocal microscope system for high-resolution and fast imaging according to an embodiment of the present invention.

Detailed Description

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

As shown in fig. 1, the present invention provides a confocal corneal microscope system with high resolution and fast imaging, the confocal corneal microscope system of the present embodiment includes: the device comprises a laser light source 1, a filter 2, a beam expander 3, a polarization encoder 4, a first lens 5, a slit 6, a second lens 7, a scanning mirror 8, a scanning lens 9, a beam splitter 10, a sleeve lens 11, a microscope objective lens 12, a displacement table 13, a polarization selector 15, a filter 16 and an imaging element 17. All optical elements and the cornea 14 to be imaged are located in a coaxial optical path.

In this embodiment, a light beam from a laser light source 1 passes through a filter 2, then passes through a beam expander 3 and a polarization encoder 4, reaches a first lens 5, and is focused on a slit 6. The light beam passing through the slit 6 reaches the scanning mirror 8 through the second lens 7, and the traveling direction is changed by the scanning mirror 8. The light beam emitted from the scanning mirror 8 passes through the scanning lens 9, is then deflected by the beam splitter 10, passes through the sleeve lens 11, enters the microscope objective 12, and is focused on the cornea 14 to be imaged. The scattered light from the cornea 14 is collected back by the microscope objective 12, and then passes through the beam splitter 10, the polarization selector 15, the optical filter 16, and reaches the imaging element 17, and an image is formed. And the displacement table 13 is axially moved to realize three-dimensional imaging of the cornea. The optical path contains a polarization encoder 4 and a polarization selector 15 for polarization encoded imaging.

In this embodiment, the light source 1 provides a laser beam to the microscope system. The laser is a white light laser, the wavelength range is 470nm to 2400nm, and the power is controlled in a certain range, so that the power of the light beam entering the cornea 14 is not more than 2.5mW, and the eye is prevented from being damaged.

The optical filter 2 functions to select a desired light source wavelength and output collimated light, and the optical filter of the present example is a tunable linewidth output filter system for selecting a desired laser wavelength and outputting a collimated light beam, and can output laser light having a linewidth of 10nm or more in the range of 600nm to 840 nm. Preferably, the center wavelength of the output laser is 690nm to obtain a strong scattered light signal.

The laser source 1 and the filter 2 may be replaced by a monochromatic laser source with a wavelength in the visible, near infrared range, preferably between 680nm and 800 nm.

The beam expander 3 is used to modify the diameter of the collimated beam output by the filter 2, preferably to a diameter greater than the length of the slit 6. The length of the slit 6 in this embodiment is 3mm, and the preferred output beam diameter is 8mm.

The polarization encoder 4 is used to encode the polarization of the light beam. In a preferred embodiment, the polarization encoder 4 consists of a linear polarizer, a quarter-wave plate and a half-wave plate.

The light beam passing through the polarization encoder 4 is focused by the first lens 5. The first lens 5 is a cylindrical lens, the cylindrical axis direction of which is parallel to the direction of the slit 6 and the rotating shaft direction of the scanning mirror 8, and has the function of shaping the incident collimated light beam into a linear light spot at the focal point. In a preferred embodiment, the first lens 5 is a plano-convex cylindrical lens with a focal length of 25mm, and the cylindrical surface is a light beam incident surface.

The slit 6 can be regarded as an approximately rectangular diaphragm having a long side much longer than a wide side, and the long side direction coincides with the cylindrical axis direction of the first lens 5 and is placed at the focal point of the first lens 5. The slits 6 serve to form a linear light source with good spatial coherence and in a preferred embodiment are 3mm long and 5 μm wide.

The light beam passing through the slit 6 passes through the second lens 7 and is incident on the scanning mirror 8. Preferably, the slit 6 and the scanning mirror 8 are centered on two focal planes of the second lens 7, respectively, and the light beam forms a linear focal spot at the scanning mirror 8.

The scanning mirror 8 is used to change the deflection angle of the light beam at the entrance pupil of the microscope objective 12, so that the linear light beam at the focal plane of the microscope objective 12 is scanned one-dimensionally in the cornea 14. In the preferred embodiment, the scanning mirror 8 is a galvanometer mirror, with the axis of rotation parallel to the direction of the slit 6.

The light beam emitted from the scanning mirror 8 passes through the scanning lens 9, is reflected by the beam splitter 10, passes through the sleeve lens 11, enters the entrance pupil of the microscope objective lens 12, and is focused inside the cornea 14.

The scanning lens 9 is a special lens group with corrected curvature of field, and functions to generate a flat image plane when the direction of the outgoing beam of the scanning mirror 8 changes. The entrance pupil plane of the scanning lens 9 coincides with the center of the scanning mirror 8, and the back focal plane coincides with the front focal plane of the sleeve lens 11. The line light source at the slit 6 forms a conjugate image at the focal plane of the scanning lens 9.

Preferably, the back focal plane of the telescopic lens 11 coincides with the entrance pupil plane of the microscope objective 12 such that the center of the scanning mirror 8 has a conjugate relationship with the entrance pupil plane of the microscope objective 12.

Preferably, in order to fill the entrance pupil of the microscope objective lens 12 with the light beam, the ratio of the effective focal length of the telescopic lens 11 to the effective focal length of the scanning lens 9 is equal to the ratio of the size of the entrance pupil of the microscope objective lens 12 to the size of the light spot of the scanning mirror 8, so that the light beam fills the entrance pupil of the microscope objective lens 12. The sleeve lens 11 and the scanning lens 9 constitute a 4f optical system.

The microscope objective 12 is used to focus the beam of light inside the cornea 14 for illumination and to collect back the scattered light from the cornea 14. Preferably, a flat field achromatic immersion microscope objective with a numerical aperture of greater than 1.05, a working distance of greater than 1mm, and a horizontal immersion liquid is selected to ensure imaging resolution and avoid direct contact with the corneal surface.

The displacement table 13 has a one-dimensional moving capability, and has the function of driving the microscope objective 12 to move back and forth along the axial direction, so that the image plane is positioned at different depths of the cornea 14, and the three-dimensional imaging of the cornea is realized. In a preferred embodiment, the axial movement range of the displacement table 13 is 800 μm and the minimum movement step is 5nm.

The backscattered light from the inside of the cornea 14 is collected back by the microscope objective 12, passes through the sleeve lens 11, the beam splitter 10, the polarization selector 15, and the optical filter 16, and enters the imaging element 17. The beam splitter 10 is used to connect the illumination light path and the detection light path, reflect the illumination light, project the scattered light from the cornea 14, and form an angle of 45 degrees with both light paths, and the preferred splitting ratio is 8. The polarization selector 15 is composed of a phase retarder and a linear polarizer, and functions to selectively transmit the scattered light of a specific polarization state. The filter 16 filters stray light signals that do not belong to the detection spectrum, and transmits the stray light signals.

The imaging element 17 is a CMOS camera whose light-sensitive surface is in conjugate relation with the focal plane (sample plane) of the microscope objective 12, the back focal plane of the first lens 5 and the back focal plane of the scanning lens 9. When the light beam is incident on the photosensitive surface of the imaging member 17, the imaging member 17 controls exposure using a rolling shutter. Rolling shutters are one way of operating CMOS cameras, among others, for exposing and processing data line by line for pixel elements on a sensor array. In the preferred embodiment, the sensor array of the imaging member 17 and the scanning mirror 8 are software controlled to ensure that the rows being exposed are in conjugate relationship with the line beam focused at the objective focal plane so that the defocused scattered light is not exposed.

Wherein, adjusting the exposure line number of the imaging part 17 exposed at the same time can adjust the exposure condition of the scattered light out of focus, and change the imaging focal depth. Generally, the width of the simultaneously exposed pixels is comparable to the width of the line beam on the photosensitive surface of the imaging member 17. In order to adapt to a complex cornea structure and improve the image quality, when the cornea tissue with a large characteristic structure and weak scattering capability is imaged, the number of lines exposed at the same moment is increased, and the focal depth is increased; when the cornea tissue with small characteristic structure and strong scattering ability is imaged, the number of exposed lines at the same time is reduced.

The above-described embodiments are intended to illustrate rather than to limit the invention, and any modifications and variations of the present invention are within the spirit of the invention and the scope of the appended claims.

Claims (12)

1. A corneal confocal microscope system for high-resolution rapid imaging is characterized by comprising an illumination light path and a detection light path;

the illumination light path comprises a laser light source, a filter, a beam expander, a polarization encoder, a first lens, a slit, a second lens, a scanning mirror, a scanning lens, a beam splitter, a sleeve lens and a microscope objective fixed on a displacement table;

the light beam emitted by the laser source sequentially passes through the optical filter, the beam expander and the polarization encoder and then reaches the first lens, and is focused on the slit; the light beam passing through the slit reaches the scanning mirror through the second lens, and the propagation direction of the light beam is changed by the scanning mirror; the light beam emitted from the scanning mirror is reflected by the beam splitter after passing through the scanning lens, enters the microobjective through the sleeve lens and is focused on a cornea to be imaged;

the detection light path comprises a polarization selector, an optical filter and an imaging piece; the cornea generates a scattered light signal through laser illumination, the scattered light signal returns to the detection light path through the beam splitter, the specific polarization state is selected through the polarization selector, the stray light signal is filtered through the optical filter, finally, the imaging part exposes in a rolling shutter mode to form an image, the number of pixel lines which are exposed at the same time is adjusted by controlling the rolling shutter, the exposure condition of defocused scattered light is changed, and the focal depth is adjusted; and the displacement table is axially moved to realize the three-dimensional imaging of the cornea.

2. The confocal corneal microscope system with high resolution and fast imaging according to claim 1, wherein the first lens is a cylindrical lens, the cylindrical surface is a light beam incident surface, and the cylindrical axis direction is parallel to the slit direction and the scanning mirror rotation axis direction.

3. The confocal high resolution fast imaging corneal microscope system of claim 1, wherein the slit is located on a back focal plane of the first lens and on a front focal plane of the second lens.

4. The confocal corneal microscope system for high resolution and fast imaging according to claim 1, wherein the scanning mirror scans the linear light beam on the focal plane of the microscope objective lens in one dimension inside the cornea; the scanning mirror is optically conjugate with the entrance pupil of the microobjective.

5. The confocal corneal microscope system for high resolution fast imaging according to claim 1, wherein the scanning lens is a lens group for correcting aberration, capable of generating a flat image plane when the light beam is scanned, and the center of the entrance pupil of the scanning lens coincides with the pivot point of the scanning mirror.

6. The confocal corneal microscope system for high resolution and fast imaging according to claim 1, wherein the beam splitter reflects the illumination light and transmits the scattered light from the cornea at 45 ° angles to both the illumination light path and the detection light path.

7. The confocal corneal high resolution fast imaging microscope system according to claim 5, wherein the sleeve lens back focal plane coincides with the microscope objective entrance pupil plane; the sleeve lens and the scanning lens constitute a 4f optical system.

8. The high resolution fast imaging confocal corneal microscope system according to claim 1, wherein the first lens back focal plane, the scanning lens back focal plane, the microscope objective focal plane, and the imaging element photosensitive plane have a conjugate relationship.

9. The confocal corneal high resolution rapid imaging microscope system according to claim 1, wherein the microscope objective is fixed on a displacement stage capable of moving in the direction of the optical axis.

10. The confocal corneal microscope system with high resolution and fast imaging according to claim 1, wherein the imaging element is a CMOS camera with rolling shutter imaging, and the scanning mirror and the rolling shutter move synchronously, so that the defocused corneal scattered light is not exposed and does not participate in image formation.

11. The confocal corneal microscope system for high resolution and rapid imaging according to claim 1, wherein the operating wavelength of the laser source and the optical filter can be adjusted between 600nm and 840 nm.

12. The confocal corneal microscope system with high resolution and rapid imaging according to claim 1, wherein the optical path comprises a polarization encoding and polarization selecting device for polarization encoded imaging.

Images