An illumination device for solving the concave liquid surface effect in orifice plate imaging

Abstract

This invention discloses an illumination device for solving the concave liquid surface effect in well plate imaging, belonging to the field of microscope light source technology. The device includes an inverted microscope illumination source and several annular light sources distributed in a dome-like pattern around it. The inverted microscope illumination source is composed of a central light source, a collimating lens, and a condenser lens in sequence. The annular light sources contain multiple LED beads, and the size of the rings matches the diameter of the microwells in a common cell culture plate, with adjustable angles. Through the synergistic effect of traditional vertical illumination and dome-shaped oblique illumination, this invention effectively improves the imaging quality of well plates, adapts to microwell plates of different diameters, is easy to operate, and highly practical, providing strong support for cell observation and other related work.

Description

Technical Field

[0001] This utility model relates to the field of microscope light source technology, and more specifically to an illumination device for solving the concave liquid surface effect in orifice plate imaging. Background Technology

[0002] In cutting-edge research fields such as life sciences, drug screening, and cell therapy, long-term dynamic microscopic imaging of live cells has become a core experimental method for obtaining cell behavior data, evaluating drug efficacy, and validating mechanisms. Traditional methods typically involve seeding live cells into standard cell culture plates (e.g., 6, 12, 24, 48, 96, 384 wells) and then using an inverted microscope for transmission or fluorescence imaging from the bottom. However, the culture medium in microplates contains high concentrations of serum, protein, and nutrients, resulting in significant surface tension and a tendency to form concave liquid surfaces with large curvature near the well walls (crescent effect).

[0003] This concave crescent effect can severely negatively impact imaging. Firstly, inverted microscopes typically use a condenser to generate a conical beam with a specific numerical aperture. When this beam passes through the concave surface, the curvature of the liquid-gas interface causes the incident angle to change continuously with radial position. According to Snell's law, the angle of refraction also increases non-linearly; near the well wall, the effective illumination flux decreases sharply, and sometimes no light enters the culture medium due to the obstruction of the well wall, resulting in "dark rings" or "shadows." Secondly, the Fresnel equation derived from Maxwell's equations shows that as the incident angle increases, the proportion of reflected light energy rises while the proportion of refracted light energy decreases. The high incident angle at the edge of the concave surface causes a large amount of light energy to be reflected back into the air, further reducing the light intensity reaching the cell level, leading to a deterioration in the signal-to-noise ratio and loss of detail in the edge region. Thirdly, the wall thickness of microplates is typically 0.3–1 mm, and they have a certain height. At the junction of the concave meniscus and the well wall, some light is physically blocked by the opaque or translucent wall material, forming geometric shadows. Combined with the aforementioned optical effects, this drastically reduces the quality of edge imaging. The coupled effect of these three mechanisms makes it difficult to clearly image cells within approximately 0.5–1 mm of the well wall under a conventional inverted microscope, severely hindering whole-well statistics, edge cell dynamics tracking, and image-based high-throughput phenotypic analysis. Statistics show that in 96-well plates, the invalid imaging area affected by shadows can account for 10–15% of the single-well area, and in 384-well plates, it can even exceed 20%, directly reducing the integrity and statistical power of experimental data.

[0004] To overcome the shadowing problem of concave liquid surfaces, existing technologies mainly propose two approaches:

[0005] (1) Liquid surface smoothing: For example, CN108707551A uses additional liquid injection to change the concave liquid surface into a flat liquid surface. Although this method can eliminate the black ring, the mechanical disturbance and changes in liquid composition introduced may alter the cell microenvironment, leading to fluctuations in parameters such as osmotic pressure, pH, and shear force, inducing cell stress or phenotypic drift, and affecting the reproducibility of the experiment.

[0006] (2) Aperture zone illumination: For example, CN111708109A uses a switchable multi-aperture aperture to illuminate different radial areas sequentially and then splice them together. This method requires a large number of through-hole combinations to be preset, and the operation process is cumbersome; moreover, the aperture size is fixed and cannot adapt to various orifice plate specifications with diameters ranging from 4mm to 60mm, thus limiting its versatility.

[0007] Therefore, how to provide a novel lighting device that does not require changes to the culture medium composition, does not require complex mechanical switching, is compatible with all types of microplates, and can fundamentally compensate for the optical path distortion of the concave liquid surface is a problem that urgently needs to be solved by those skilled in the art. Utility Model Content

[0008] In view of this, the present invention provides an illumination device for solving the concave liquid surface effect in orifice plate imaging. Through the synergistic effect of traditional vertical illumination and dome oblique illumination, the imaging shadow problem caused by the concave liquid surface effect of micro-orifice plate in traditional inverted microscope imaging experiments is solved.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides an illumination device for solving the concave liquid surface effect in orifice plate imaging, comprising:

[0011] An inverted microscopic illumination source and a plurality of annular light sources arranged around the inverted microscopic illumination source, wherein the plurality of annular light sources are distributed in a dome shape.

[0012] Preferably, the inverted microscope illumination source includes an intermediate light source, a collimating lens, and a condenser lens arranged sequentially.

[0013] Preferably, the ring light source includes multiple light-emitting units.

[0014] Preferably, the light-emitting unit is an LED lamp bead.

[0015] Preferably, the size of each of the plurality of ring light sources is matched with the diameter of the micropores in the cell culture plate.

[0016] Preferably, the angle of the ring light source is adjustable.

[0017] As can be seen from the above technical solution, compared with the prior art, this utility model discloses an illumination device for solving the concave liquid surface effect in well plate imaging. The inverted microscopic illumination source can provide stable and uniform illumination for the central area of ​​the microwells, ensuring clear cell imaging in the central area. The ring light source, distributed in a dome shape and with the ring size matching the diameter of the microwells in the cell culture plate, can accurately illuminate the edge area of ​​the microwells. Combined with brightness adjustment, it can effectively eliminate edge shadows, making the cell imaging of the entire microwell area clearer and more realistic. In the device of this utility model, the inverted microscopic illumination source is composed of a central light source, a collimating lens, and a condenser lens arranged in sequence, which can ensure stable and concentrated illumination of the central area. The ring light source uses multiple LED beads as light-emitting units. LED beads have the characteristics of high brightness, low energy consumption, and long life, which can provide stable illumination. Moreover, the angle of the ring light source is adjustable, and the illumination angle can be flexibly adjusted according to actual imaging needs to further optimize the illumination effect. The ring size of several ring light sources is matched with the diameter of the microwells in the cell culture plate, making the device applicable to imaging of microwells of different diameters, thus expanding the application range of the device. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 A front view of the lighting device provided by this utility model.

[0020] Figure 2 A top view of the lighting device provided by this utility model.

[0021] Figure 3 This is a diagram showing the light emitted by a traditional inverted microscope light source onto a micropore.

[0022] Figure 4 This is a diagram showing the light illuminating the micro-holes through the lighting device of this utility model.

[0023] Figure 5 This is an image of the edge of a micro-hole in a traditional inverted microscope.

[0024] Figure 6 An edge view of a micro-hole under the lighting device provided by this utility model. Detailed Implementation

[0025] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0026] This utility model discloses an illumination device for solving the concave liquid surface effect in orifice plate imaging, such as... Figure 1-2 As shown, it includes:

[0027] An inverted microscopic illumination source and a plurality of annular light sources arranged around the inverted microscopic illumination source, wherein the plurality of annular light sources are distributed in a dome shape.

[0028] Furthermore, the inverted microscope illumination source includes an intermediate light source, a collimating lens, and a condenser lens arranged sequentially.

[0029] In another embodiment, the ring light source includes multiple light-emitting units.

[0030] In another embodiment, the light-emitting unit is an LED lamp bead.

[0031] In another embodiment, the size of the rings of the plurality of annular light sources is matched with the diameter of the micropores in the cell culture plate.

[0032] In another embodiment, the angle of the ring light source is adjustable.

[0033] Because the micropores contain cell culture medium, the liquid exhibits surface tension, resulting in a concave liquid surface (crescent effect). Figure 3 The optical principle of a traditional inverted microscope illuminating microwells is quite complex. Due to the presence of a condenser lens, the light from the traditional inverted microscope enters the cell culture medium at a certain angle. The behavior of these rays at the concave surface is complex, represented by ①-④, which depict four main optical reflection and refraction phenomena. According to Snell's law, the angle of refraction of light entering the cell culture medium (optically denser medium) from air (optically less dense medium) increases sequentially from ④ to ① due to the increasing angle of incidence. This results in less light entering the well edges, and at ①, the well wall may even block all light from entering the culture medium. Simultaneously, according to Maxwell's equations of electromagnetic waves, the energy of refracted light entering the culture medium decreases with increasing incident light, while the energy of reflected light increases with increasing incident light. In other words, the energy (light intensity) of refracted light entering the cell medium decreases from ④ to ①. Furthermore, at ①, the well wall may block all light from entering the culture medium. Considering all these factors, the final result is that there is little or no light at the edges of the microplate, hence the well edges appear black in the photographed images, making it impossible to observe cells. Figure 4 The light diagram of the illumination device provided by this utility model shows that at the edge of the micropores, the oblique dome light plays a major role. ⑤-⑧ represent the four main optical reflection and refraction phenomena. Since oblique rays ⑤, ⑦, and ⑧ are incident into the culture medium at small angles, the refraction angle is even smaller. According to the light path diagram, the light is more easily concentrated. The energy and light intensity of the light entering the edge of the micropores are higher than those of the reflected light. Meanwhile, ⑥ is incident perpendicularly, with almost no reflection, and all the energy (light intensity) enters the cell culture medium. Thus, the light rays ⑤, ⑥, ⑦, and ⑧ will all illuminate the edge of the micropores and reach the imaging device through the cells at the bottom and the objective lens. Therefore, the overall brightness of the well plate edge and the brightness of the center will not be significantly different. If there is a difference, the brightness of the conventional illumination in the center and the oblique dome illumination can be adjusted to correct it. Figure 5 and Figure 6 The images show the edge of a microwell in a 96-well cell culture plate under conventional inverted microscope illumination and the dome illumination source of this invention, respectively.

[0034] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0035] The above description of the disclosed embodiments enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An illumination device for solving the concave liquid surface effect in orifice plate imaging, characterized in that, include: An inverted microscopic illumination source and a plurality of annular light sources arranged around the inverted microscopic illumination source, wherein the plurality of annular light sources are distributed in a dome shape.

2. The illumination device for solving the concave liquid surface effect in orifice plate imaging according to claim 1, characterized in that, The inverted microscope illumination source includes an intermediate light source, a collimating lens, and a condenser lens arranged in sequence.

3. The illumination device for solving the concave liquid surface effect in orifice plate imaging according to claim 1, characterized in that, The ring light source includes multiple light-emitting units.

4. The illumination device for solving the concave liquid surface effect in orifice plate imaging according to claim 3, characterized in that, The light-emitting unit is an LED lamp bead.

5. The illumination device for solving the concave liquid surface effect in orifice plate imaging according to claim 1, characterized in that, The size of each of the several ring-shaped light sources is matched to the diameter of the micropores in the cell culture plate.

6. The illumination device for solving the concave liquid surface effect in orifice plate imaging according to claim 1, characterized in that, The angle of the ring light source is adjustable.

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