NON-FLUORESCENT IMAGING OPTICAL SECTIONING METHOD AND DEVICE BASED ON ANNULAR OFF-AXIS ILLUMINATION FOCAL PLANE CONJUGATION

• US 20190137747A1
• Filed: 12/31/2018
• Published: 05/09/2019
• Est. Priority Date: 05/10/2017
• Status: Active Grant

• Current Assignee: Ji'Nan University
• Original Assignee: Ji'Nan University
• Inventors: ZHONG, Jingang, MA, Xiao
• Granted Patent: US 10,642,016 B2
• Time in Patent Office: Days
• CPC Class Codes: G02B 21/02 Objectives
• CPC Class Codes: G02B 21/084 having annular illumination around the objective
• CPC Class Codes: G02B 21/10 affording dark-field illumination G02B21/14 and G02B21/125 take precedence
• CPC Class Codes: G02B 21/12 affording bright-field illumination G02B21/14 takes precedence
• CPC Class Codes: G02B 21/26 Stages; Adjusting means therefor
• CPC Class Codes: G02B 21/33 Immersion oils , or microscope systems or objectives for use with immersion fluids
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Abstract

The invention discloses a non-fluorescent imaging optical sectioning method and device based on annular off-axis illumination focal plane conjugation. In an infinity-corrected optical system constituted by an objective lens with a large numerical aperture and a tube lens, off-axis light beams are used for bright-field microscopic imaging of a non-fluorescent sample; light beams emitted by M sub-sources which form an annularly distributed light source illuminate the sample off-axis at a large inclination angle; all sub-sources are lit to illuminate the sample at the same time, forming a superposed image of the sample illuminated by the M sub-sources individually on an image focal plane of the tube lens, and the camera shoots the image to obtain an optical-section image of a layer in the sample; and under the control of a translation mechanism, a sample stage allows different layers in the sample to coincide with an object focal plane of the objective lens to obtain optical-section images of multiple layers in the sample.

Claims

1. A non-fluorescent imaging optical sectioning method based on annular off-axis illumination focal plane conjugation, characterized in that the method comprises the following steps:

an objective lens with a large numerical aperture and a tube lensconstitute an infinity-corrected optical system, an object focal plane of the objective lens and an image focal plane of the tube lens form a pair of object-image conjugate planes, a layer in the non-fluorescent sample coincides with the object focal plane of the objective lens, light beams emitted by an annularly distributed light source illuminate the sample off an optical axis of the objective lens to form a bright field image at the image focal plane of the tube lens, and the image at the image focal plane of the tube lens is shot by a digital camera;

the annularly distributed light source consists of M sub-sources, and light beams emitted by each of the sub-sources illuminate the sample at an inclination angle θ

off the optical axis of the objective lens;

each of the sub-sources illuminates the sample to form an image on the image focal plane of the tube lens, all sub-sources are lit to illuminate the sample simultaneously, forming a superposed image I(x, y) of the sample illuminated by the M sub-sources individually on the image focal plane of the tube lens;

I(x, y)=I₁(x, y)+I₂(x, y)+ . . . +I_M(x, y), and the camera shoots the image to obtain an optical-section image of a layer in the sample;

under the control of a translation mechanism, a sample stage drives the sample to move in a direction of the optical axis of the objective lens, so that different layers in the sample coincide with the object focal plane of the objective lens, and optical-section images of multiple layers in the sample are obtained;

where M is an integer, M≥

4;

the inclination angle θ

is an included angle between the beam illuminating the sample and the optical axis of the objective lens, ranging from 30°

≤

θ

<

90°

;

I₁(x, y), I₂(x, y), . . . , I_M(x, y) are images formed on the image focal plane of the tube lens when each of the sub-sources illuminates the sample individually, and (x, y) is pixel point coordinates of a photosensitive surface of the camera.

2. The non-fluorescent imaging optical sectioning method based on annular off-axis illumination focal plane conjugation according to claim 1, characterized in that,

the M sub-sources of the annularly distributed light source are divided into N groups;

one group of sub-sources are lit each time to illuminate the sample, and the camera sequentially shoots the images formed each time the sample is illuminated, obtaining N images;

I₁(x, y), I₂(x, y), . . . , I_N(x, y);

the N images are used to remove defocused images to obtain an optical-section image I(x, y) in one layer of the sample;

an algorithm for removing defocused images is;

linear superposition algorithm for N images I(x, y)=I₁(x, y)+I₂(x, y)+ . . . +I_N(x, y), or an algorithm for extracting the maximum value of pixel points in the same coordinates of the N images I(x, y)=max [I₁(x, y), I₂(x, y), . . . , I_N(x, y)], or a threshold operation algorithm for filtering out smaller values of three-dimensional gradient of optical-section images of multiple layers in the sample

3. The non-fluorescent imaging optical sectioning method based on annular off-axis illumination focal plane conjugation according to claim 1, characterized in that,

the annularly distributed light source is formed by rotating the sub-sources around the optical axis of the objective lens 4 under the control of a rotating mechanism, the camera synchronously shoots an image every time the sub-sources are rotated by an azimuth angle Δ

ϕ

, and the images shot within 360 degrees of rotation are linearly superposed or the maximum value of the images is taken to obtain an optical-section image of one layer in the sample; and

the range of the azimuth angle Δ

ϕ

is 0<

Δ

ϕ

≤

90°

.

4. The non-fluorescent imaging optical sectioning method based on annular off-axis illumination focal plane conjugation according to claim 2, characterized in that,

the annularly distributed light source is formed by rotating the sub-sources around the optical axis of the objective lens 4 under the control of a rotating mechanism, the camera synchronously shoots an image every time the sub-sources are rotated by an azimuth angle Δ

ϕ

, and the images shot within 360 degrees of rotation are linearly superposed or the maximum value of the images is taken to obtain an optical-section image of one layer in the sample; and

the range of the azimuth angle Δ

ϕ

is 0<

Δ

ϕ

90°

.

5. A non-fluorescent imaging optical sectioning device based on annular off-axis illumination focal plane conjugation, characterized in that the device comprises sequentially an annularly distributed light source, an objective lens, a tube lens and a camera to form an infinity-corrected optical system, wherein a sample is placed on a sample stage, and the sample stage is translatable along an optical axis of the objective lens under the control of a translation mechanism;

the objective lens and the tube lens have the same optical axis which passes through the center of the annularly distributed light source;

light beams emitted by the annularly distributed light source illuminate the sample at an inclination angle θ

off the optical axis of the objective lens, and a photosensitive surface of the camera coincides with an image focal plane of the tube lens; and

a computer controls the movement of the translation mechanism and the image shooting of the camera.

6. The non-fluorescent imaging optical sectioning device based on annular off-axis illumination focal plane conjugation according to claim 5, characterized in that,

the annularly distributed light source consists of M sub-sources and is uniformly distributed to form an annular shape, and a light beam emitted by each of the sub-sources illuminates the sample at an inclination angle θ

off the optical axis of the objective lens.

7. The non-fluorescent imaging optical sectioning device based on annular off-axis illumination focal plane conjugation according to claim 5, characterized in that,

the numerical aperture NA of the objective lens should be large enough so that when the light source illuminates the sample off the optical axis of the objective lens, a bright field image is formed at the image focal plane of the tube lens, and the NA is in the range of 0.75≤

NA<

2.0.

8. The non-fluorescent imaging optical sectioning device based on annular off-axis illumination focal plane conjugation according to claim 5, characterized in that,

a refractive index matching lens, which is a hemispherical plano-convex lens or a conical lens, is arranged between the annularly distributed light source and the sample, and a bottom surface of the lens is bonded with a slide glass encapsulating the sample.

9. The non-fluorescent imaging optical sectioning device based on annular off-axis illumination focal plane conjugation according to claim 5, characterized in that,

the annularly distributed light source consists of two sub-sources arranged on a semi-circular ring which is arranged on a rotating platform, wherein a rotating axis of the rotating platform coincides with the optical axis of the objective lens, and the two sub-sources are symmetrically distributed on both sides of the optical axis of the objective lens and rotate around the optical axis of the objective lens under the control of the rotating platform, thus form an annular illumination of the sample off the optical axis of the objective lens;

and

a computer simultaneously controls the movement of the translation mechanism, the rotation of the rotating platform and the synchronous shooting of the camera.