CN113946043A

CN113946043A - Excitation light source and fluorescence microscope comprising same

Info

Publication number: CN113946043A
Application number: CN202110801595.0A
Authority: CN
Inventors: 刘伟杰; 林文宇; 陈振星; 黄锦圣
Original assignee: Hong Kong University of Science and Technology HKUST
Current assignee: Hong Kong University of Science and Technology HKUST
Priority date: 2020-07-16
Filing date: 2021-07-15
Publication date: 2022-01-18

Abstract

An excitation light source for a fluorescence microscope and a method of controlling the excitation light source, the excitation light source comprising: the base plate and arrange in proper order emitting diode, light filter and ball lens above the base plate, wherein: one side of the light-emitting diode is installed on the substrate, the optical filter is arranged on the other side of the light-emitting diode, at least the spherical lens is arranged in one lens sleeve, the spherical lens is arranged at one end, far away from the substrate, of the lens sleeve, and light emitted by the light-emitting diode is emitted from the lens sleeve through the optical filter and the spherical lens. The invention also provides a fluorescence microscope comprising the excitation light source. The fluorescence microscope can replace a large and expensive light splitting component of the existing fluorescence microscope, reduces cost, improves portability, and can meet the requirements of various application scenes by using light emitting diodes with different wavelengths.

Description

Excitation light source and fluorescence microscope comprising same

Technical Field

The present invention relates to the field of fluorescence microscopes, and more particularly, to an excitation light source for a fluorescence microscope, a fluorescence microscope including the excitation light source, and a method of controlling an excitation light source for a fluorescence microscope.

Background

In recent years, large-scale infectious diseases such as novel coronavirus (COVD-19), Ebola virus, African swine influenza, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and Zika virus have spread worldwide, and have had a serious influence on human society. In addition, we are also faced with increasing food hygiene and safety issues. The key of infectious disease prevention and control lies in early discovery to inhibit the spread of virus and health hidden trouble. In the prevention of infectious diseases, the most important is the rapid diagnosis.

Fluorescence microscopes can accurately image some features at the submicron scale and are therefore widely used in biological and nano-material research. The key to fluorescence microscopy imaging is the separation of the excitation light from the emission light of the fluorescent sample, and therefore conventional microscopes typically include a light splitting assembly consisting of an excitation filter (fig. 1, b), an emission filter (fig. 1, c), and a dichroic mirror (fig. 1, e), and are therefore costly. In addition, because other optical components in the optical path of the microscope are highly sensitive to position information, the optical splitting component is typically bulky in design and not portable. Moreover, the conventional spectral assembly shown in fig. 1 also lacks versatility since the filters and dichroic mirrors are designed only for specific wavelengths.

Therefore, there is a need for an improved approach to the excitation light source of fluorescence microscopes.

Disclosure of Invention

To address at least one of the above problems, the present invention provides an excitation light source for a fluorescence microscope, a fluorescence microscope including the excitation light source, and a method of controlling an excitation light source for a fluorescence microscope.

The invention provides at least the following technical scheme:

according to an aspect of the present invention, there is provided an excitation light source for a fluorescence microscope, the excitation light source including a substrate, and a light emitting diode, a filter, and a ball lens arranged above the substrate in this order, wherein:

one side of the light-emitting diode is installed on the substrate, the optical filter is arranged on the other side of the light-emitting diode, at least the spherical lens is arranged in one lens sleeve, the spherical lens is arranged at one end, far away from the substrate, of the lens sleeve, and light emitted by the light-emitting diode is emitted from the lens sleeve through the optical filter and the spherical lens.

In one embodiment, the other end of the lens sleeve opposite to the end far away from the substrate is connected with the substrate and the light emitting diode and the optical filter are embedded into the lens sleeve; alternatively, the first and second electrodes may be,

the lens sleeve is not connected with the substrate, the spherical lens is arranged in the lens sleeve, and the light emitting diode and the optical filter are arranged in the corresponding accommodating parts respectively.

In one embodiment, the excitation light source further comprises a fresnel lens arranged near the end of the lens sleeve remote from the substrate and arranged such that light emerging from the lens sleeve is focused through the fresnel lens onto a sample to be illuminated.

In one embodiment, half of the ball lenses are nestingly fixed inside the end of the lens sleeve remote from the base plate, and the other half of the ball lenses are located outside the end of the lens sleeve remote from the base plate.

In one embodiment, one or more sets of light emitting diodes are disposed on the substrate, each set of light emitting diodes including at least three light emitting diodes, each set of light emitting diodes emitting light of the same wavelength, different sets of light emitting diodes emitting light of different wavelengths.

In one embodiment, the individual light emitting diodes of each group of light emitting diodes are substantially evenly spaced on the substrate, and the one or more groups of light emitting diodes collectively form a substantially symmetrical shape.

In one embodiment, the lens sleeve is removably mounted to enable replacement of lens sleeves having different lengths; or the length of the lens sleeve itself can be adjusted.

According to another aspect of the invention, there is provided a fluorescence microscope comprising an objective lens and an excitation light source according to any of the above, a lens sleeve of the excitation light source being arranged around the objective lens.

In one embodiment, the fluorescence microscope further comprises a microcontroller coupled thereto, the microcontroller configured to perform at least one of:

reading an image captured by the fluorescence microscope;

controlling movement of a sample stage of the fluorescence microscope;

controlling movement of the excitation light source and other optical components of the fluorescence microscope; and/or

Sending the read image captured by the fluorescence microscope and/or the image processed data to a user.

According to another aspect of the present invention, there is provided a method of controlling an excitation light source, wherein the excitation light source includes a substrate, and a light emitting diode, a filter, and a ball lens, which are sequentially disposed above the substrate, at least the ball lens being disposed in a lens sleeve, the method including:

filtering light emitted by the light emitting diode by using the optical filter; and

causing the filtered light to exit the lens sleeve through the ball lens.

In one embodiment, the excitation light source further comprises a fresnel lens arranged near an end of the lens sleeve remote from the substrate and arranged such that light emerging from the lens sleeve is focused through the fresnel lens onto a sample to be illuminated.

In one embodiment, one or more sets of light emitting diodes are disposed on the substrate, each set of light emitting diodes including at least three light emitting diodes, the at least three light emitting diodes in each set of light emitting diodes being substantially evenly spaced on the substrate, the method further comprising: the method includes the steps of causing each group of light emitting diodes to emit light of the same wavelength, causing different groups of light emitting diodes to emit light of different wavelengths, and selecting any one of the one or more groups of light emitting diodes to operate.

In one embodiment, the method further comprises:

controlling the divergence degree of the light emitted from the lens sleeve by adjusting the length of the lens sleeve or adjusting the distance between the optical filter and the spherical lens;

controlling the power density of light reaching the sample to be irradiated by adjusting the distance between the spherical lens and the Fresnel lens; and/or

And controlling the light passing through the Fresnel lens to be focused on the sample to be irradiated by adjusting the distance between the Fresnel lens and the sample to be irradiated.

The excitation light source for the fluorescence microscope and the method for controlling the excitation light source for the fluorescence microscope provided by the invention replace a large and expensive light splitting component of the fluorescence microscope in the current market, reduce the cost and improve the portability of the excitation light source and the fluorescence microscope. In addition, under the condition that one or more groups of light-emitting diodes are arranged on the substrate, any one group of light-emitting diodes in the one or more groups of light-emitting diodes can be selected to work, so that the requirements of the fluorescence microscope on the light-emitting diodes with different wavelengths in different application scenes can be met, and the universality and the versatility of the excitation light source are improved.

Drawings

Non-limiting and non-exhaustive embodiments of the present invention are described by way of example with reference to the following drawings, in which:

FIG. 1 shows a schematic diagram of a prior art fluorescence microscope;

FIG. 2 is a schematic diagram illustrating one embodiment of an excitation light source for a fluorescence microscope according to the present invention;

FIG. 3 is a schematic diagram of an arrangement of LEDs on a circular ring-shaped substrate of an excitation light source according to an embodiment of the invention;

FIG. 4a shows a 3D rendering of an arrangement of LEDs on a U-shaped ring-like substrate of an excitation light source according to another embodiment of the invention;

FIG. 4b shows a perspective view of the LED arrangement on a U-shaped ring-like substrate according to the embodiment of FIG. 4 a;

FIG. 4c shows a schematic electrical diagram of the arrangement of light emitting diodes on a U-shaped ring-like substrate according to the embodiment of FIG. 4 a;

FIG. 5 is a schematic diagram of a portion of a U-shaped ring excitation light source with corresponding receptacles;

FIG. 6 shows a schematic view of a fluorescence microscope according to an embodiment of the invention;

FIG. 7 is a view of a fluorescence microscope with a portion of the U-shaped ring excitation light source with corresponding receptacles as shown in FIG. 5 from different perspectives;

FIG. 8a shows a schematic diagram of an experimental setup for measuring the relationship between the distance between a ball lens and a light emitting diode and the power density;

FIG. 8b shows a graph of distance between a ball lens and a light emitting diode versus power density;

FIG. 9a shows a calibration plate target image obtained from testing an excitation light source fabricated according to one embodiment of the present invention using a 4-fold objective lens with a numerical aperture of 0.1;

FIG. 9b shows calibration plate target images obtained from a fluorescence microscope (10-fold objective with a numerical aperture of 0.25), an iolight fluorescence microscope in one comparative example, and a Nikon E200 microscope in another comparative example, respectively, using the excitation light source of the present invention;

fig. 10 shows an image of celery obtained using a 4-fold objective lens to test an excitation light source made in accordance with an embodiment of the invention;

fig. 11 shows another image of celery using a 4 x objective lens to test an excitation light source made according to one embodiment of the invention.

Detailed Description

In order to make the above and other features and advantages of the present invention more apparent, the present invention is further described below with reference to the accompanying drawings. It is understood that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the specific details need not be employed to practice the present invention. In other instances, well-known steps or operations are not described in detail to avoid obscuring the invention.

In accordance with an aspect of the present invention, referring to fig. 2-4, an excitation light source for a fluorescence microscope is provided. The excitation light source is suitable for various types of fluorescence microscopes.

The excitation light source comprises a substrate, and a light emitting diode, a light filter and a spherical lens which are sequentially arranged above the substrate, wherein:

one side of the light-emitting diode is installed on the substrate, the optical filter is arranged on the other side of the light-emitting diode, at least the spherical lens is arranged in one lens sleeve, the spherical lens is arranged at one end, far away from the substrate, of the lens sleeve, and light emitted by the light-emitting diode is emitted from the lens sleeve through the optical filter and the spherical lens. In one embodiment as shown in fig. 2, the other end of the lens sleeve D opposite to the end far from the substrate a is connected with the substrate a and the light emitting diode B and the filter C are nested inside the lens sleeve. Specifically, the light emitting diode B is disposed inside the upper end of the lens sleeve D. For example, the light emitting diode B may be a surface mount diode (SMD LED), a low power surface mount diode (SMD LED) has a smaller size and generates less heat than a high power light emitting diode, may be integrated in a small module, and a low power light emitting diode has a higher price advantage than a high power light emitting diode, and may effectively reduce manufacturing costs. The lower surface of the surface mount diode (SMD LED) is closely attached to the upper surface of a filter C located at the lower portion thereof, which may be an excitation filter, for passing only light of a desired wavelength, i.e., excitation light, from the light emitting diode B. A filter C is typically used over one light emitting diode B.

The excitation light source further includes a fresnel lens disposed near the other end of the lens sleeve opposite the one end remote from the substrate. The excitation light emitted from the light emitting diode may be focused using a combination of a ball lens E, a lens sleeve D and a fresnel lens F, wherein the lens sleeve D may control the direction and divergence of the light focused on the ball lens E and the fresnel lens F, and one lens sleeve D and one ball lens E may be generally used for one light emitting diode B. The excitation light travels a certain distance L2 in the lens sleeve D and reaches the ball lens E. Half of the spherical lens E is fixed inside the end of the lens sleeve D far away from the base plate in a nested manner, and the other half of the spherical lens E is positioned outside the end of the lens sleeve D far away from the base plate. The excitation light is collimated into a substantially parallel light beam after passing through the spherical lens E, the light beam continues to travel a certain distance downwards to the textured side of the fresnel lens F placed below the lens sleeve D, and the light beam is then redirected and focused through the fresnel lens F onto the sample to be illuminated.

One or more sets of light emitting diodes may be disposed on the substrate a, wherein each set of light emitting diodes includes, for example, at least three light emitting diodes (e.g., four or more), each set of light emitting diodes emitting light of the same wavelength, and different sets of light emitting diodes emitting light of different wavelengths. The individual leds in each group of leds are substantially evenly spaced on the substrate (with a spacing deviation of around 10%), and the one or more groups of leds together form a substantially symmetrical shape, such as a substantially axisymmetric shape (e.g., a circular or U-like ring as exemplified below). The shape of the substrate on which the light emitting diodes are arranged may be similar to the shape that the one or more groups of light emitting diodes jointly form, for example also an axially symmetric shape (as an annular or U-like ring as exemplified below).

In one embodiment, as shown in fig. 3, multiple sets of leds of different wavelengths may be soldered on a circular Printed Circuit Board (PCB) substrate. The 18 light emitting diodes are radially arranged on a circular ring Printed Circuit (PCB) substrate in a circular ring shape at the same interval distance, wherein the light emitting diodes LED 311, LED 312 and LED 313 are arranged in an equilateral triangle on the circular ring substrate as a first group of light emitting diodes having the same wavelength to improve the uniformity of light irradiated onto the sample. The light emitting diodes LED 321, LED 322, and LED 323 respectively adjacent to the light emitting diodes LED 311, LED 312, and LED 313 in the clockwise direction are arranged in an equilateral triangle on the circular ring substrate as a second group of light emitting diodes having the same wavelength (the wavelength is different from the wavelength of any other group of light emitting diodes). By analogy, the 18 leds shown in fig. 3 can be divided into 6 groups of leds in the manner described above, wherein the leds of each group each have a different wavelength. The light emitting diode sets with different wavelengths make the excitation light source of the present invention adaptable for use in many types of research, testing and analysis, where in one embodiment the 365nm, 465nm, 520nm, 590nm, 625nm, etc. light emitting diode sets installed in the excitation light source have been matched to the absorption spectra of most dyes in the laboratory to be compatible with most dyes on the market.

In another embodiment, as shown in fig. 4a, the substrate a may also be a U-like ring substrate with a fixture. The U-shaped ring substrate can provide a method for quickly and conveniently installing the excitation light source of the fluorescence microscope, wherein the opening part of the U-shaped ring substrate can pass through the objective lens G of the fluorescence microscope without obstruction, and the fixing device on the U-shaped ring substrate can quickly fix the U-shaped ring substrate on the fluorescence microscope, so that the substrate arranged with the light emitting diode groups with different wavelengths can be conveniently disassembled/replaced.

Fig. 4b and 4c show schematic circuit connection diagrams of the LEDs inside the U-shaped ring-like substrate as shown in fig. 4a, wherein the LEDs 411, 412 and 413 are arranged in a substantially symmetrical triangle as a first group of LEDs with the same wavelength, the LEDs of the first group of LEDs are connected to each other through wires and to pin 1 of the circuit board, and pin 1 controls the first group of LEDs to emit uv light. The light emitting diodes LED421, LED 422 and LED 423, which are respectively adjacent to the light emitting diodes LED 411, LED412 and LED 413 in the clockwise direction, are arranged in a substantially symmetrical triangle as a second group of light emitting diodes having the same wavelength and different from the wavelength of any other group of light emitting diodes, the light emitting diodes in the second group of light emitting diodes are connected to each other through a wire and connected to a pin 2 of the circuit board, and the pin 2 controls the second group of light emitting diodes to emit blue light. The light emitting diodes 431, 432 and 433, which are respectively adjacent to the light emitting diodes LED421, 422 and 423 in the clockwise direction, are arranged in a substantially symmetrical triangle as a third group of light emitting diodes having the same wavelength and different from the wavelength of any other group of light emitting diodes, the light emitting diodes in the third group of light emitting diodes are connected with each other and connected to a pin 3 of the circuit board through a wire, and the pin 3 controls the third group of light emitting diodes to emit green light. The light emitting diodes LED 441, LED 442 and LED 443 respectively adjacent to the light emitting diodes LED 431, LED 432 and LED 433 in the clockwise direction are arranged in a substantially symmetrical triangle as a fourth group of light emitting diodes having the same wavelength and different from the wavelength of any other group of light emitting diodes, the light emitting diodes in the fourth group of light emitting diodes are connected to each other through a wire and connected to a pin 4 of the circuit board, and the pin 4 controls the fourth group of light emitting diodes to emit red light. Fig. 4b also shows the position of the cathode/anode in the U-shaped loop-like circuit board, which may be, for example,

5 or 6. The connection manner of the light emitting diodes in the present embodiment can be more intuitively observed from fig. 4 c. In one embodiment, one or more sets of leds with specific wavelengths can be rapidly turned off/on by circuitry in the substrate, wherein multiple beams can be redirected onto the sample via the fresnel lens F to improve power density and spot uniformity.

In addition, in another embodiment of the present invention, since the spherical lens E, the lens sleeve D and the light emitting diode B in the excitation light source are aligned on a straight line and can be glued and fixed to each other, the alignment of the light path is easier than that required by a special mirror and filter arrangement in a conventional system. In this embodiment, the only critical positioning is the distance of the leds B from each other, but the position of the leds B is easily marked on the substrate a and the leds B can be soldered on the substrate a for fixation, so that minor vibrations do not affect the spectroscopic accuracy and additional recalibration of the system is not necessary.

The lens sleeve D is detachably mounted to enable replacement of lens sleeves having different lengths; or the length of the lens sleeve itself can be adjusted. In one embodiment, the distance between the ball lens E and the filter B (which may be an absolute distance from the lower surface of the filter to the upper vertex of the ball lens or a relative distance L2 from the lower surface of the filter to the center of the ball lens) may be changed by replacing a lens sleeve having a different length to adjust the distance from the ball lens E to the fresnel lens F (which may be an absolute distance from the lower vertex of the ball lens to the upper surface of the textured portion of the fresnel lens or a relative distance L3 from the center of the ball lens to the upper surface of the fresnel lens), thereby adjusting the size (L4) and intensity of the light spot irradiated onto the sample stage H. If the light from the spherical lens E is perfectly collimated, the distance between the spherical lens E and the fresnel lens F is negligible, in which case the distance between the spherical lens E and the fresnel lens F should be as close as possible, resulting in a higher intensity of light coupling and a smaller spot. However, if high intensity operating conditions are not required, the light beam exiting the ball lens E can be made to diverge slightly, resulting in lower light intensity and more uniform illumination. The size and intensity of the light spot is dependent on the power of the leds, the size of the ball lens (L1), and the number of leds in each group of leds. In addition, the power density of the excitation light source in the present invention is high, and the power density can be controlled by changing the distance between the ball lens E and the fresnel lens F, as shown in table 1 below, wherein the current and voltage of the light emitting diode are 0.3A and 3.9V, respectively, when the distance between the ball lens E and the fresnel lens F is the smallest.

TABLE 1

Fig. 5 is a schematic diagram of a part of the U-shaped ring excitation light source with corresponding accommodating parts according to another embodiment. Referring to a sectional view shown on the right side of fig. 5, a light emitting diode may be disposed in a light emitting diode groove, one side of which is mounted on a substrate, and a filter groove for arranging a filter and a ball lens groove for arranging a ball lens are also shown. The lens sleeve may be the spherical lens groove itself, or a sleeve placed inside the spherical lens groove. The length of the lens sleeve may be the length of the ball lens groove itself, or the length of the lens sleeve may extend over the ball lens groove, the filter groove, and the light emitting diode groove.

In one embodiment, the lens sleeve is not connected to the substrate, the ball lens is disposed in the lens sleeve, and the light emitting diode and the optical filter are disposed in the respective corresponding receiving portions.

In addition to the shapes of the substrates and the positions of the led groups on the substrates mentioned herein, other reasonable variations that can be considered by those skilled in the art are also intended to be covered by the present invention.

According to another aspect of the invention, and with reference to FIG. 6, there is provided a fluorescence microscope comprising an objective lens G, wherein the objective lens G is not part of the excitation light source described above, but the light distribution should be adjusted according to the specific specifications of the objective lens. A lens sleeve D of the excitation light source is arranged around the objective lens G; wherein the focal lengths of the Fresnel lens F and the objective lens G need to be matched and the numerical aperture of the Fresnel lens F must be higher than that of the objective lens G, and in addition, the shielding of the main body of the objective lens G should be considered, and the Fresnel lens F and the objective lens G should be placed so that they jointly focus the light source on the sample.

According to one embodiment as shown in fig. 6, the excitation light exiting from the spherical lens E reaches the fresnel lens F and is redirected to the surface of the sample on the sample stage H, which is connected to the stepper motor L by a lead screw K, which together control the sample stage H to focus the sample into the objective lens. Because the numerical aperture of the Fresnel lens F is higher than that of the objective lens G, most of the directional excitation light is reflected from the light set received by the objective lens G, the geometric configuration reduces the quantity of the excitation light directly received by the objective lens, and effectively achieves the effect of light separation similar to that achieved by an emission filter; in addition, the spherical lens E and the Fresnel lens F are also cheaper than a dichroic mirror used by a conventional system, and the total cost of the excitation light source using the optical fiber laser can be reduced by more than ten times compared with the conventional system.

As shown in fig. 6, a microcontroller is further connected to the exterior of the eyepiece L and the lead screw K, the microcontroller includes a GPU and a CPU, and the microcontroller can be configured to: (1) reading an image captured by the fluorescence microscope; (2) controlling movement of a sample stage of the fluorescence microscope; (3) controlling movement of the excitation light source and other optical components of the fluorescence microscope; (4) sending the read image captured by the fluorescence microscope and/or the image processed data to a user.

According to an embodiment of the present invention, referring to fig. 7, there is provided a fluorescence microscope using a part of the U-shaped ring excitation light source having the corresponding receiving portion as shown in fig. 5 as a part of the fluorescence microscope. The components are arranged around the fluorescence microscope objective, and the design of the U-ring opening portion facilitates installation of the excitation light source of the present invention and replacement of light emitting diodes and other optical components having different wavelength ranges.

causing the filtered light to exit the lens sleeve through the ball lens.

The excitation light source further comprises a fresnel lens arranged near an end of the lens sleeve remote from the substrate and arranged such that light emerging from the lens sleeve is focused through the fresnel lens onto a sample to be illuminated.

The substrate having one or more sets of light emitting diodes disposed thereon, each set of light emitting diodes including at least three light emitting diodes, the at least three light emitting diodes in each set of light emitting diodes being substantially evenly spaced on the substrate, the method further comprising:

the method includes the steps of causing each group of light emitting diodes to emit light of the same wavelength, causing different groups of light emitting diodes to emit light of different wavelengths, and selecting any one of the one or more groups of light emitting diodes to operate.

The method further comprises the following steps:

controlling the divergence degree of the light emitted from the lens sleeve by adjusting the length of the lens sleeve or adjusting the distance between the spherical lens and the optical filter;

For the fluorescence microscope of the present invention, if a lower power fluorescence microscope system (e.g., 4 x objective) is required, a wide field of view is required, which can be achieved by increasing the length of the sleeve (i.e., increasing the distance between the ball lens E and the led B); if a higher power fluorescence microscope system is required (e.g., a 20 x objective lens), a higher power density is required, which can be achieved by reducing the length of the sleeve (i.e., reducing the distance between the ball lens E and the light emitting diode B). Fig. 8B shows the relationship between the distance between the ball lens E and the light emitting diode B and the power density measured using the experimental setup as shown in fig. 8a, wherein the excitation light reaches the power meter H through a small hole in the middle of the aluminum foil G.

In one embodiment, excitation light sources fabricated according to one embodiment of the present invention were tested using a 4 x objective lens with a numerical aperture of 0.1 from phoenix optics, inc. As shown in FIG. 9a, the maximum resolution of the objective lens is 3um, and the maximum number of 80 scribe lines can be formed on the target of the calibration plate used in the experiment every 1mm, so that the test accuracy is 6.25um at most. As can be seen from fig. 9a, the edges of the test image are very clear, so the maximum resolution of the fluorescence microscope system using the excitation light source can reach above 6.25 um.

In another embodiment, when an excitation light source fabricated according to one embodiment of the present invention is tested using a 10-fold objective lens with a numerical aperture of 0.25 from phoenix optics ltd, as shown in fig. 9b (three pictures from left to right of a fluorescence microscope using an excitation light source of the present invention (a 10-fold objective lens with a numerical aperture of 0.25), an iolight fluorescence microscope in one comparative example, and a Nikon E200 microscope in another comparative example), a fluorescence microscope using an excitation light source of the present invention can develop a line that is 3um from a target plate, and the development quality is very close to a Nikon E200 microscope that is much higher in price, cost, and volume than in the comparative example.

In addition, the fluorescence microscope of the present invention was also tested using celery as a sample, using 450nm ultraviolet light with a long pass ultraviolet excitation filter as a light source, and it can be seen from fig. 10 and 11 that spots and vessels with a specific fluorescent color are clearly shown on the image due to the presence of natural fluorescent dye at the chloroplast and xylem vessels.

The above-mentioned description about the excitation light source described with reference to fig. 2 to 8 can also be extended to the method for controlling the excitation light source for a fluorescence microscope of the present invention, and is not repeated herein to avoid repetition.

The features of the above embodiments may be arbitrarily combined, and for the sake of brevity, all possible combinations of the features in the above embodiments are not described, but should be construed as being within the scope of the present disclosure as long as there is no contradiction between the combinations of the features.

While the present invention has been described in connection with the embodiments, it is to be understood by those skilled in the art that the foregoing description and drawings are merely illustrative and not restrictive of the broad invention, and that this invention not be limited to the disclosed embodiments. Various modifications and variations are possible without departing from the spirit of the invention.

Claims

1. An excitation light source for a fluorescence microscope, comprising a substrate, and a light emitting diode, a filter, and a ball lens sequentially arranged above the substrate, wherein:

2. The excitation light source according to claim 1,

the other end of the lens sleeve opposite to the end far away from the substrate is connected with the substrate, and the light-emitting diode and the optical filter are embedded into the lens sleeve; alternatively, the first and second electrodes may be,

3. An excitation light source according to claim 2, further comprising a fresnel lens arranged near the end of the lens sleeve remote from the substrate and arranged such that light emerging from the lens sleeve is focused through the fresnel lens onto a sample to be illuminated.

4. An excitation light source according to claim 2 or 3, wherein half of the ball lens is fixed inside the end of the lens sleeve remote from the substrate in a nested manner, and the other half of the ball lens is located outside the end of the lens sleeve remote from the substrate.

5. The excitation light source of claim 4, wherein one or more sets of light emitting diodes are disposed on the substrate, each set of light emitting diodes comprising at least three light emitting diodes, each set of light emitting diodes emitting light of a same wavelength, different sets of light emitting diodes emitting light of different wavelengths.

6. An excitation light source according to claim 5 wherein the individual LEDs in each group of LEDs are substantially evenly spaced on the substrate and the one or more groups of LEDs collectively form a substantially symmetrical shape.

7. The excitation light source according to claim 1 or 2,

the lens sleeve being removably mounted to enable replacement of lens sleeves having different lengths; or

The length of the lens sleeve itself is adjustable.

8. A fluorescence microscope comprising an objective and an excitation light source according to any one of claims 1 to 7, a lens sleeve of the excitation light source being arranged around the objective.

9. The fluorescence microscope of claim 8, further comprising a microcontroller coupled thereto, the microcontroller configured to perform at least one of:

reading an image captured by the fluorescence microscope;

controlling movement of a sample stage of the fluorescence microscope;

10. A method of controlling an excitation light source, the excitation light source comprising a substrate and, disposed above the substrate in that order, a light emitting diode, a filter, and a ball lens, at least the ball lens being disposed in a lens sleeve, the method comprising:

causing the filtered light to exit the lens sleeve through the ball lens.

11. A method according to claim 10, wherein the excitation light source further comprises a fresnel lens arranged near an end of the lens sleeve remote from the substrate and arranged such that light emerging from the lens sleeve is focused through the fresnel lens onto the sample to be illuminated.

12. The method of claim 10 or 11, wherein one or more sets of light emitting diodes are disposed on the substrate, each set of light emitting diodes comprising at least three light emitting diodes, the at least three light emitting diodes of each set of light emitting diodes being substantially evenly spaced on the substrate, the method further comprising:

13. The method of claim 11, further comprising: