CN216771467U

CN216771467U - Confocal small dish

Info

Publication number: CN216771467U
Application number: CN202120924373.3U
Authority: CN
Inventors: 吴晶
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-06-17
Anticipated expiration: 2031-04-30

Abstract

The invention aims to provide a confocal small dish which can be effectively matched with an upright laser confocal/two-photon microscope for use. The invention relates to a confocal small dish, wherein a hollow cylinder (2) is fixedly arranged at the bottom of the confocal small dish (1). The confocal small dish has the advantages that: (1) water locking can be ensured; (2) the fixation of the sample can be ensured; (3) the manufacturing cost is low, and the printing time is short; (4) the size is flexible; (5) can be repeatedly cleaned and used for many times.

Description

Confocal small dish

Technical Field

The invention belongs to the field of medical instruments, and relates to a confocal capsule.

Background

Confocal laser microscopy is an emerging technology that has been developed in recent years and is widely used in the biological and medical fields. Two-photon excitation has many natural advantages over single-photon excitation: (1) two photons use longer excitation wavelengths and thus have greater penetration depth in biological tissue; (2) two-photon excitation belongs to a nonlinear process and needs higher photon density, so that the excitation has space constraint, the out-of-focus excitation is greatly reduced, and the optical slicing capability is good; (3) less out-of-focus excitation and lower photon energy further reduce photobleaching and photodamage, improve the survival time of tissues and particularly facilitate the imaging of biological samples; (4) the two-photon excitation wavelength and the fluorescence wavelength are far apart in the spectrum, so that the separation of the excitation light and the fluorescence signal is easier to realize.

In two-photon microscopic imaging, as most of the observation objects are biological tissues with certain depth, a water mirror with longer working distance is generally selected for observation. The intermediate between the objective lens and the tissue needs to be flooded with water during the observation to achieve a higher resolution deep in the tissue. Compared with an inverted two-photon microscope, the upright two-photon microscope is more suitable for craniotomy observation of living brain tissues, and is more suitable for ventral shooting by the inverted microscope like organs (intestines, liver and the like) of the abdominal cavity. If the inverted two-photon microscope is adopted for detection, the water locking problem can be easily solved by using a confocal dish, and when the upright two-photon microscope is adopted, the surface of a biological tissue is usually not a plane and has a certain radian, if water is directly dripped on the tissue, the water locking is difficult, and the technical difficulty is brought to the observation by using a water mirror. Therefore, when in vitro large tissues are observed, more inverted two-photon microscopes are selected to directly place the tissues in a small dish for detection. However, when a certain position on the tissue needs to be observed, such as the condition of cells near a pinhole for injecting virus/medicine, on an inverted microscope, because the part to be observed needs a downward objective lens, the target position is difficult to find, and the time for finding the shooting part is greatly increased. And if the upright two-photon microscope is adopted, the target position is clear at a glance, but the biggest problem is water locking.

There are two common solutions: (1) by means of an auxiliary sucking disc water locking system (the device is high in manufacturing cost and complicated in use), an observed tissue is adsorbed, and water is locked by using a concave part of a sucking disc, but the biggest defect of the adsorption water locking is that the visual field cannot be changed, and the observable area is very limited; (2) if the traditional confocal dish is adopted, water directly passes through the tissue block, and when the objective lens presses the tissue from the upper part to the lower part, the tissue can float around and cannot be fixed and imaged well.

Currently, commercially available confocal cuvettes are generally only suitable for inverted confocal microscopes, and not for upright confocal microscopes. If the sample is placed in a common confocal dish, when the upright confocal microscope is used for detection, the objective lens is pressed down from the upper part, so that the sample can drift everywhere due to the pressure, and can not be fixed.

Therefore, the structure of the existing confocal small dish is improved, a brand new confocal small dish is obtained, and the technical problem is effectively solved.

Disclosure of Invention

The invention aims to provide a confocal small dish which can be effectively matched with an upright laser confocal/two-photon microscope for use.

The invention relates to a confocal small dish, which is characterized in that a hollow cylinder (2) is fixedly arranged at the bottom of the confocal small dish (1).

The horizontal height of the hollow cylinder (2) is lower than that of the confocal capsule (1).

The materials of the confocal capsule include but are not limited to: PLA material, resin, glass and the like, preferably PLA material.

PLA (polylactic acid) is a novel biodegradable material, is prepared from starch raw materials provided by renewable plant resources (such as corn), has good mechanical properties and physical properties, and also has good strength, extensibility and degradability, so that the PLA material is the most preferred PLA material in the aspects of economic cost, safety, environmental protection and the like. Particularly, for the invention, the water locking effect of the water-retaining agent is obviously better than that of other materials.

The confocal capsule is integrally formed and can be prepared by a 3D printing technology.

The sizes of the confocal small dish (1) and the hollow cylinder (2) can be adjusted according to actual needs. Preferably, the radius of the confocal cuvette (1) is: 17.5-20mm, more preferably 21-25 mm. The wall thickness of the confocal small dish (1) is as follows: 0.5 to 1mm, more preferably 0.2 to 0.4 mm. The height of the confocal small dish (1) is as follows: 8-10 mm.

Similarly, the radius of the hollow cylinder (2) is: 5-7.5mm, more preferably 10 mm. The wall thickness of the hollow cylinder (2) is: 0.5 to 1mm, more preferably 0.1 to 0.4 mm. The height of the hollow cylinder (2) is: 3-5mm, more preferably 2 mm.

The confocal small dish has the advantages that: (1) water locking can be ensured; (2) the fixation of the sample can be ensured; (3) the manufacturing cost is low, and the printing time is short; (4) the size is flexible; (5) can be repeatedly cleaned and used for many times.

The present invention first attempts to apply 3D printing technology to an upright confocal laser/two-photon microscope. The 3D printing technology is flexible and convenient, so that design parameters are adjusted according to different experiments, series of small dishes with different inner diameter sizes are designed, and good fixing effect on biological tissues with different sizes can be achieved. By utilizing the small dishes, the blood vessels and the medicine distribution of the appointed parts of tissues with different sizes such as isolated brain tissues, tumor tissues and the like and the imaging of transparent cerebral neurons and the like are completed through the personalized orthostatic two-photon microscope, and the new progress of rapidly realizing two-photon detection of the target position of the large tissue is obtained.

Drawings

FIG. 1 is a diagram of a confocal dish

FIG. 2 is a diagram showing the dimension of a confocal dish

FIG. 3 is a diagram showing the dimension of a confocal cuvette

FIG. 4 is a diagram of a confocal cuvette prepared by 3D printing technology

FIG. 5, confocal dish picture (10mm inner diameter)

A is a plan view and B is a sectional view.

FIG. 6, confocal small dish picture (15mm inner diameter)

A is a plan view and B is a sectional view.

FIG. 7 is a diagram showing the state of use of the confocal cuvette

FIG. 8 is a diagram showing the state of use of the confocal measurement capsule

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to be limiting.

Example 1 confocal dish

The bottom of the small confocal dish (1) is fixedly provided with a hollow cylinder (2), and the horizontal height of the hollow cylinder (2) is lower than that of the small confocal dish (1).

The confocal small dish is made of PLA material.

Example 2, 3D printing confocal dish

And loading the 3D drawing into 3D printing control software Cura, and storing the 3D drawing into a format which can be identified by an aurora 3D printer. According to the size and weight of a required carrier, a PLA material is selected, and a plurality of 3D printing parameters (layer height, shell thickness, whether grid is filled or not, filling density, printing speed, temperature and the like) are adjusted and searched, so that the confocal capsule is obtained.

Example 3 use of confocal dish

As shown in figures 7 and 8, when we observed isolated tissues, the tissue can be well fixed by using the capsule of the invention, and the observation is carried out by using an upright confocal/two-photon microscope. Meanwhile, when the small dish is filled with water, the tissue cannot float around, and the special objective lens with a thick diameter and a long working distance can be used for well imaging from the upper part. Particularly when the tissues have pinholes and other specified positions to be observed, the invention can quickly find the target area for observation by adopting the upright microscope, and is convenient and quick to use.

Claims

1. A confocal small dish is characterized in that a hollow cylinder is fixedly arranged at the bottom of the confocal small dish (1), the horizontal height of the hollow cylinder (2) is lower than that of the confocal small dish (1),

wherein, the material of the confocal small dish is selected from: PLA materials, resins, glass;

wherein, the radius of the confocal small dish (1) is as follows: 17.5-20mm, and the wall thickness of the confocal small dish (1) is as follows: 0.5-1mm, and the height of the confocal small dish (1) is as follows: 8-10 mm;

wherein, the radius of the hollow cylinder (2) is: 5-7.5mm, the wall thickness of the hollow cylinder (2) is: 0.5-1mm, the height of the hollow cylinder (2) is: 3-5 mm.

2. The confocal capsule of claim 1 wherein the confocal capsule is integrally formed.