Any wave band spot scanning imaging device without using dichroscope

Abstract

The utility model provides an arbitrary wave band spot scanning imaging device without using a dichroscope, comprising a laser module used as a light source to generate laser; the non-polarization beam splitter is located at the rear end of the laser module; exciting light emitted by the laser module reaches the gathering assembly under the reflection effect of the non-polarization beam splitter, the exciting light is focused on a sample to excite fluorescence after being deflected by the gathering assembly, and the sample excites the fluorescence to reach the non-polarization beam splitter from the gathering assembly. The fluorescence reaches the detector under the transmission effect of the non-polarization beam splitter, and a fluorescence signal is obtained through the detector. According to the utility model, an angle difference mode is adopted to prevent exciting light from returning to the gathering assembly along with fluorescence, no matter what kind of laser light is adopted by the laser module as a light source, a large amount of calibration and adjustment are not needed after a dichroscope is replaced, the debugging time is shortened, and the switching requirement of different laser imaging in a short time is met.

Description

Technical Field

[0001] The utility model belongs to the technical field of laser imaging, and in particular relates to an arbitrary waveband point scanning imaging device without using a dichroic mirror. Background Art

[0002] Currently, point scanning imaging technologies include confocal imaging, two-photon imaging / multi-photon imaging, STED super-resolution imaging and fluorescence lifetime imaging. Dichroic mirrors play an irreplaceable role in point scanning imaging technology, because dichroic mirrors are needed to separate the excitation light from the fluorescence generated by the sample so that the fluorescence can reach the detector part for imaging.

[0003] However, the dichroic mirror has great limitations and can only distinguish between excitation light and fluorescence in one or several wavelength bands. If you want to change the excitation light wavelength or different samples during use, it is beyond the applicable scope of the dichroic mirror and you can only replace the dichroic mirror. However, since the dichroic mirror requires extremely high mechanical precision in the optical path, if the direction of the excitation light beam changes only a small angle, the excitation light spot that excites the sample is likely to move or even deviate from the original optical path, causing the returned fluorescence beam to no longer focus on the pinhole or no fluorescence beam to return. This problem can only be solved by recalibrating the deflection angle of the dichroic mirror, and this process takes a long time, and it is impossible to achieve switching between different laser imaging in a short time. Utility Model Content

[0004] The utility model provides an arbitrary-band point scanning imaging device without using a dichroic mirror, aiming to solve the problem that it takes a long time to recalibrate the dichroic mirror when changing the excitation light wavelength or different samples, and it is impossible to switch between different laser imaging in a short time.

[0005] The utility model is implemented as follows: an arbitrary band point scanning imaging device without using a dichroic mirror comprises:

[0006] A laser module, the laser module serving as a light source to generate laser light;

[0007] A non-polarizing beam splitter, which is located at the rear end of the laser module;

[0008] A focusing component, the focusing component includes an objective lens, a tube lens and a reflector, the excitation light output by the non-polarizing beam splitter passes through the tube lens to reach the reflector, and the reflector focuses the excitation light to the objective lens and projects it onto the sample surface;

[0009] A sample stage and a signal collection unit, wherein the sample is placed on the sample stage, and the sample stage is arranged tilted relative to the focal plane of the objective lens. Excitation light is focused on the sample in a non-vertical state via a focusing component to excite a fluorescence signal. Under the effect of the tilted arrangement of the sample stage, the excitation light is reflected to other areas while the fluorescence enters the focusing component and returns to the non-polarizing beam splitter. The fluorescence reaches the signal collection unit under the transmission effect of the non-polarizing beam splitter, and the fluorescence signal is collected and analyzed by the signal collection unit.

[0010] Preferably, an optical path adjustment component is provided between the non-polarizing beam splitter and the focusing component.

[0011] Preferably, the optical path adjustment component includes a scanning device and a scanning lens, and the excitation light reflected from the non-polarizing beam splitter is reflected by the scanning device and then focused by the scanning lens to reach the objective lens of the focusing component.

[0012] Preferably, the scanning device is a galvanometer, and the scanning lens is a lens.

[0013] Preferably, the scanning lens focuses the excitation light to an upper focal plane conjugate with the focal plane of the focusing component objective lens.

[0014] Preferably, the laser module comprises a first laser, which generates pulsed laser as excitation light.

[0015] Preferably, the laser module includes a first laser and a second laser.

[0016] Preferably, the signal collection unit comprises a filter assembly and a detector, and the filter assembly is arranged between the detector and the filter assembly.

[0017] The filter assembly includes a lens, a filter and a pinhole, which are arranged at the front end of the detector from far to near. The fluorescence transmitted from the non-polarizing beam splitter passes through the lens, the filter and the pinhole in sequence and finally reaches the detector.

[0018] Preferably, the signal collection unit comprises a filter assembly and a detector, and the filter assembly is arranged between the detector and the filter assembly;

[0019] The filter assembly includes a lens and a filter, which are arranged at the front end of the detector from far to near. The fluorescence transmitted from the non-polarizing beam splitter passes through the lens and the filter in sequence and finally reaches the detector.

[0020] Preferably, a beam spot adjustment device is provided between the laser module and the non-polarizing beam splitter.

[0021] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0022] The arbitrary-band point scanning imaging device without the need for a dichroic mirror provided by the utility model adopts an inclined arrangement of the sample stage, so that the excitation light that fails to generate fluorescence on the sample surface will also be reflected to other areas due to the reflection effect, and cannot return to the objective lens of the focusing component along with the fluorescence, while a small amount of excitation light mixed in the fluorescence is subjected to corresponding filtering processing by the filter component; an angle difference method is adopted to prevent the excitation light from returning to the focusing component along with the fluorescence, no matter what kind of laser light is used as the light source of the laser module, there is no need to spend a lot of time on calibration and adjustment after replacing the dichroic mirror, thus shortening the debugging time and meeting the switching needs of different laser imaging in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The utility model provides a confocal imaging structure schematic diagram of an arbitrary waveband point scanning imaging device without using a dichroic mirror.

[0024] Figure 2 The utility model provides a schematic diagram of the structure of a focusing assembly and a sample stage optical path of an arbitrary waveband point scanning imaging device without using a dichroic mirror.

[0025] Figure 3 The utility model provides an arbitrary-band point scanning imaging device without using a dichroic mirror, which uses 473nm excitation light to perform confocal microscopic imaging on lily of the valley rhizome cells.

[0026] Figure 4 The utility model provides an arbitrary-band point scanning imaging device without using a dichroic mirror, which uses 577nm excitation light to perform confocal microscopic imaging on lily of the valley rhizome cells.

[0027] Description of reference numerals:

[0028] 1. Laser module; 2. Light spot adjustment device; 3. Non-polarizing beam splitter; 4. Scanning device; 5. Scanning lens; 6. Focusing component; 7. Lens; 8. Filter; 9. Pinhole; 10. Detector. DETAILED DESCRIPTION

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0030] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0031] The present invention provides an arbitrary band point scanning imaging device without using a dichroic mirror, such as Figure 1-Figure 4 As shown, the arbitrary wavelength point scanning imaging device without using a dichroic mirror comprises:

[0032] A laser module 1, wherein the laser module 1 is used as a light source to generate laser light;

[0033] A non-polarizing beam splitter 3, wherein the non-polarizing beam splitter 3 is located at the rear end of the laser module 1;

[0034] The focusing component 6 and the detector 10, the excitation light emitted by the laser module 1 reaches the focusing component 6 under the reflection of the non-polarizing beam splitter 3, the excitation light is deflected by the focusing component 6 and focused on the sample to excite fluorescence, and the sample excitation fluorescence will reach the non-polarizing beam splitter 3 from the focusing component 6, and the fluorescence reaches the detector 10 under the transmission of the non-polarizing beam splitter 3, and the fluorescence signal is obtained through the detector 10;

[0035] The focusing assembly 6 includes an objective lens, a tube lens and a reflector. The excitation light output by the non-polarizing beam splitter 3 passes through the tube lens to reach the reflector. The reflector focuses the excitation light to reach the upper focal plane conjugate with the focal plane of the objective lens of the focusing assembly 6. The sample is placed on a sample stage, and the sample stage is arranged at a focal plane inclined to the focal plane of the objective lens.

[0036] An optical path adjustment component is provided between the non-polarizing beam splitter 3 and the focusing component 6, and the optical path adjustment component includes a scanning device 4 and a scanning lens 5. The excitation light reflected from the non-polarizing beam splitter 3 is reflected by the scanning device 4 and then focused by the scanning lens 5 to reach the upper focal plane conjugate with the focal plane of the objective lens of the focusing component 6;

[0037] The fluorescence collected by the focusing component 6 through the objective lens will pass through the optical path adjustment component in reverse direction to reach the non-polarizing beam splitter 3, and the non-polarizing beam splitter 3 transmits the fluorescence so that it passes through the non-polarizing beam splitter 3 to reach the detector 10. A filter component is provided between the non-polarizing beam splitter 3 and the detector 10, and the filter component filters the excitation light mixed in the fluorescence to improve the quality of the fluorescence received by the detector 10;

[0038] The sample stage is a stage in the prior art, and the plane of the sample stage is arranged at a certain inclination angle with the focal plane of the objective lens; since the sample stage is placed obliquely to the focal plane of the objective lens, the excitation light will reach the sample surface in a non-vertical state, so the excitation light that fails to generate fluorescence on the sample surface will also be reflected to other areas due to reflection, and cannot return to the objective lens of the focusing component 6 along with the fluorescence, and a small amount of excitation light mixed in the fluorescence is filtered accordingly by the filter component; Figure 3 and Figure 4 As shown, confocal microscopy imaging of lily of the valley rhizomes using different lasers can obtain clear and stable images.

[0039] The main removal method in the present application is to use an inclined sample stage to avoid the excitation light returning to the focusing component 6 along with the fluorescence. No matter what kind of laser light is used as the light source of the laser module 1, there is no need to spend a lot of time on calibration and adjustment like replacing the dichroic mirror, which shortens the debugging time and meets the switching needs of different laser imaging in a short time. The optical path system can be applied to a variety of technical means in point scanning imaging, such as confocal imaging, two-photon imaging / multi-photon imaging, STED super-resolution imaging technology and fluorescence lifetime imaging; when using different technical routes of confocal imaging, two-photon imaging / multi-photon imaging, STED super-resolution imaging technology and fluorescence lifetime imaging, the laser module 1, the filter component and the detector 10 will be adapted and replaced according to different technical methods; wherein, the detector 10 adopts existing technical equipment, and in the fluorescence lifetime imaging technology, a time-correlated single-photon counter will be loaded on the detector 10 to test the fluorescence lifetime;

[0040] As a preferred implementation in this embodiment, the laser module 1 includes a first laser, which generates a pulsed laser as an excitation light. The first laser generates excitation light as a single light source, and the single light source is mainly used for confocal imaging, two-photon imaging / multi-photon imaging and fluorescence lifetime imaging;

[0041] In the STED super-resolution imaging technology, the laser module 1 includes a first laser and a second laser, and both the first laser and the second laser generate pulsed lasers;

[0042] As a preferred implementation in this embodiment, the scanning lens 5 adopts a common lens, the scanning device 4 is a galvanometer, and the collimated laser beam reflected by the non-polarizing beam splitter 3 is deflected by the galvanometer and directed to the scanning lens 5;

[0043] In this embodiment, the scanning lens 5 is equivalent to the eyepiece of the traditional focusing assembly. The scanning lens 5 focuses the laser to an upper focal plane conjugate with the focal plane of the objective lens of the focusing assembly in the sample. The fluorescence returned from the focusing assembly 6 first passes through the scanning lens 5 to reach the scanning device 4, and then is deflected to reach the non-polarizing beam splitter 3.

[0044] In a further preferred embodiment of the utility model, the filter assembly adopts two groups of different structures. In the two-photon imaging / multi-photon imaging technology, the filter assembly includes a lens 7 and a filter 8, and the lens 7 and the filter 8 are arranged at the front end of the detector 10 in a manner from far to near; the fluorescence transmitted from the non-polarizing beam splitter 3 passes through the lens 7 and the filter 8 in sequence and finally reaches the detector 10;

[0045] In the confocal imaging, STED super-resolution imaging and fluorescence lifetime imaging techniques, the filter assembly includes a lens 7, a filter 8 and a pinhole 9, and the lens 7, the filter 8 and the pinhole 9 are arranged at the front end of the detector 10 in a manner from far to near; the fluorescence transmitted from the non-polarizing beam splitter 3 passes through the lens 7, the filter 8 and the pinhole 9 in sequence and finally reaches the detector 10;

[0046] In this embodiment, the filter 8 is used to filter light other than the required fluorescence. The filter 8 is a bandpass filter. Under normal circumstances, the filter 8 does not need to be replaced when the fluorescent material is not replaced. Here, the excitation light that enters the focusing component 6 synchronously with the fluorescence during the diffuse reflection process is mainly filtered;

[0047] In a further preferred embodiment of the utility model, the ratio of transmittance to reflectivity of the non-polarizing beam splitter 3 can be 10:90, 30:70, 50:50, 70:30, 90:10 or other ratios, and the energy lost by the excitation light passing through the non-polarizing beam splitter 3 can be compensated by increasing the power of the laser module;

[0048] In a further preferred embodiment of the utility model, a beam spot adjustment device 2 is provided between the laser module 1 and the non-polarizing beam splitter 3, and the beam spot adjustment device 2 adjusts the size of the spot emitted by the excitation light;

[0049] The working principle of the utility model is as follows:

[0050] The excitation light starts from the laser module 1, passes through the beam spot adjustment device 2 to adjust the spot size, and is reflected by the non-polarizing beam splitter 3. The reflected power is determined by the ratio of the transmittance to the reflectance of the non-polarizing reflector. It passes through the galvanometer 4 and the scanning lens 5 to reach the focusing component 6. After the excitation light passes through the focusing component 6, it irradiates the sample stage in a non-vertical state. The excitation light deflected by several arc minutes is focused on the sample and excites fluorescence on the sample surface. Since the optical path is not perpendicular to the optical platform, most of the laser light will not be reflected back on the sample and the sample stage. Only a very small amount of laser light will return to the focusing component 6 due to diffuse reflection accompanied by fluorescence. After passing through the objective lens, reflector and tube lens in the focusing component 6, it passes through the scanning lens 5 and the galvanometer 4 in turn, and finally reaches the non-polarizing beam splitter 3. According to the ratio of the transmittance to the reflectance of the selected non-polarizing beam splitter, part of the fluorescence is transmitted through the filter component to enter the detector 10 to obtain a fluorescence signal, and a very small amount of laser light generated in the diffuse reflection process will be filtered by the filter 8.

[0051] It should be noted that, for the above-mentioned embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the utility model is not limited by the described action sequence, because according to the utility model, some steps may be performed in other sequences or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the utility model.

[0052] The above embodiments are only used to illustrate the technical solutions of the utility model, rather than to limit the protection scope of the utility model. Obviously, the described embodiments are only some embodiments of the utility model, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the utility model. Although the utility model has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the utility model according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the utility model in essence, and these technical solutions also belong to the scope of protection of the utility model.

Claims

1. An arbitrary wavelength point scanning imaging device without using a dichroic mirror, characterized in that: include: A laser module (1), the laser module (1) serving as a light source to generate laser light; A non-polarizing beam splitter (3), wherein the non-polarizing beam splitter (3) is located at the rear end of the laser module (1); A focusing component (6), the focusing component (6) comprising an objective lens, a tube lens and a reflector, the excitation light output by the non-polarizing beam splitter (3) passes through the tube lens to reach the reflector, and the reflector focuses the excitation light to the objective lens and projects it onto the sample surface; A sample stage and a signal collection unit, wherein the sample is placed on the sample stage, and the sample stage is arranged at an angle to the focal plane of the objective lens, and excitation light is focused on the sample in a non-vertical state via a focusing component (6) to excite a fluorescence signal, and under the effect of the inclined arrangement of the sample stage, the excitation light is reflected to other areas while the fluorescence enters the focusing component (6) and returns to the non-polarizing beam splitter (3), and the fluorescence reaches the signal collection unit under the transmission effect of the non-polarizing beam splitter (3), and the fluorescence signal is collected and analyzed by the signal collection unit.

2. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 1, characterized in that: An optical path adjustment component is provided between the non-polarizing beam splitter (3) and the focusing component (6).

3. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 2, characterized in that: The optical path adjustment component comprises a scanning device (4) and a scanning lens (5); the excitation light reflected by the non-polarizing beam splitter (3) is reflected by the scanning device (4) and then gathered by the scanning lens (5) to reach the objective lens of the gathering component (6).

4. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 3, characterized in that: The scanning device (4) is a galvanometer, and the scanning lens (5) is a lens.

5. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 4, characterized in that: The scanning lens (5) focuses the excitation light to reach the upper focal plane conjugate with the focal plane of the objective lens of the focusing component (6).

6. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 5, characterized in that: The laser module (1) comprises a first laser, which generates pulsed laser as excitation light.

7. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 5, characterized in that: The laser module (1) comprises a first laser and a second laser.

8. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 6 or 7, characterized in that: The signal collection unit comprises a filter component and a detector (10), wherein the filter component is arranged between the detector (10) and the filter component; The filter assembly comprises a lens (7), a filter (8) and a pinhole (9), wherein the lens (7), the filter (8) and the pinhole (9) are arranged at the front end of the detector (10) in a manner from far to near, and the fluorescence transmitted from the non-polarizing beam splitter (3) passes through the lens (7), the filter (8) and the pinhole (9) in sequence and finally reaches the detector (10).

9. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 6, characterized in that: The signal collection unit comprises a filter component and a detector (10), wherein the filter component is arranged between the detector (10) and the filter component; The filter assembly comprises a lens (7) and a filter (8), wherein the lens (7) and the filter (8) are arranged at the front end of the detector (10) in a manner from far to near, and the fluorescence transmitted from the non-polarizing beam splitter (3) passes through the lens (7) and the filter (8) in sequence and finally reaches the detector (10).

10. The arbitrary wavelength point scanning imaging device without using a dichroic mirror as claimed in claim 1, characterized in that: A light beam spot adjustment device (2) is provided between the laser module (1) and the non-polarizing beam splitter (3).

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