CN111338070B - Structured light generating device and structured light illuminating microscope - Google Patents
Structured light generating device and structured light illuminating microscope Download PDFInfo
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Abstract
The invention provides a structured light generating device and a structured light illuminating microscope, wherein the structured light generating device can quickly generate high-quality structured light in different directions and different phase states, effectively eliminates the time of phase shift and direction switching in the process of generating the structured light, and ensures that the image acquisition speed is not limited by the generation speed of the structured light. Moreover, the structured light generating device has universality, and can be used for the structured light generation of N directions (such as two directions forming 90 degrees with each other, or three directions forming 120 degrees with each other) and M-step phase shift (such as three-step phase shift or five-step phase shift).
Description
Technical Field
The invention relates to the technical field of microscopy, in particular to a structured light generating device and a structured light illuminating microscope.
Background
The structured light microscope is a common microscopic imaging technology which breaks through diffraction limit, and high-time-space resolution observation of subcellular organelles in living cells can be realized by utilizing the technology.
The structured light illumination microscope becomes a super-resolution microscopic imaging technology which is most suitable for observing living cells by the performances of quick data acquisition rate, low excitation light intensity, high space-time resolution, low phototoxicity to cells and the like.
When the technology is used for synthesizing a super-resolution image, a plurality of structured light illumination modes need to be generated, so that the imaging speed of the system is limited by the generation speed of the structured light. Accurately capturing fast moving structured light is crucial for studying the dynamic processes of living cells, slow acquisition speeds can lead to motion-induced artifacts and can miss fast dynamic processes, leading to erroneous biomedical research conclusions.
In a conventional structured light microscope, a projection grating generates a coherent light beam and generates a structured illumination cluster, a rotating stage rotates the grating to generate structured light in different directions, and a piezoelectric displacement stage translates the grating to generate phase shift of the structured light.
The spatial light modulator and the digital micro-array as the photoelectric information processing device can realize the rapid modulation of the light field distribution in an electric control way, and the speed of generating the structured light is improved to a certain extent. However, these devices cannot acquire images when switching the structured light mode, and devices such as a spatial light modulator and a digital microarray have a very low light utilization rate, and an image of the same quality can be obtained by extending the exposure time of the system.
Therefore, how to provide a technology capable of rapidly generating structured light is a technical problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, to solve the above problems, the present invention provides a structured light generating apparatus and a structured light illuminating microscope, and the technical solution is as follows:
a structured light generating device, comprising: the angle between the N groups of structured light generating units is 360/N degrees, so that the structured light stripes generated by the N groups of structured light generating units mutually form 360/N degrees on the surface of a sample to be detected, N is more than or equal to 2 and is a positive integer;
wherein the structured light generating unit includes: the light source, the light field modulation device and the light path coupling device;
when one group of the structured light generation units is in an illumination state, the rest groups of the structured light generation units are in a non-illumination state, and the light field modulation devices in the rest groups of the structured light generation units perform phase shifting.
Preferably, in the above structured light generating apparatus, the light field modulation devices in the N groups of structured light generating units have the same phase start position and are all in the first phase state.
Preferably, in the structured light generating apparatus described above, when image capturing is performed on a first phase state for a first group of structured light generating units, the light sources in the first group of structured light generating units are turned on;
and the light sources in the rest groups of the structured light generating units are in an off state.
Preferably, in the structured light generation apparatus described above, when image acquisition in the first phase state is performed on the second group of structured light generation units, the light sources in the second group of structured light generation units are turned on;
the light sources in the remaining groups of structured light generation units are all in an off state, and the light field modulation devices in the first group of structured light generation units move to a second or other phase state.
Preferably, in the above structured light generating apparatus, when the image capturing of N sets of the structured light generating units in the first phase state is completed,
turning on light sources in the first group of structured light generation units, and carrying out image acquisition on the first group of structured light generation units in the second or other phase states;
and the light sources in the rest groups of the structured light generation units are all in a closed state, and the light field modulation devices in the rest groups of the structured light generation units perform phase shift until the light field modulation devices are in the second or other phase states.
Preferably, in the above structured light generating apparatus, when the image capturing of N sets of the structured light generating units in the p-1 th phase state is completed,
turning on light sources in the first group of structured light generation units, and carrying out image acquisition on the p-th or other phase states of the first group of structured light generation units;
the light sources in the other groups of the structured light generation units are all in a closed state, and the light field modulation devices in the other groups of the structured light generation units perform phase shift until the light field modulation devices are in the p-th or other phase states;
and continuing to collect the images until the image collection of the N groups of the structured light generating units on the p-th or other phase states is completed.
Preferably, in the structured light generating device described above, the structured light generating device further includes: n power switches;
and each power switch correspondingly controls one power supply.
Preferably, in the structured light generating apparatus described above, the light source is an LED light source.
Preferably, in the structured light generating apparatus described above, the displacement stage is a piezoelectric displacement stage.
A structured light illuminated microscope comprising the structured light generating device of any of the above.
Compared with the prior art, the invention has the following beneficial effects:
the present invention provides a structured light generating device comprising: the angle between the N groups of structured light generating units is 360/N degrees, so that the structured light stripes generated by the N groups of structured light generating units mutually form 360/N degrees on the surface of a sample to be detected, N is more than or equal to 2 and is a positive integer; wherein the structured light generating unit includes: the light source, the light field modulation device and the light path coupling device; when one group of the structured light generation units is in an operating state, the rest groups of the structured light generation units are in a non-operating state, and the light field modulation devices in the rest groups of the structured light generation units perform phase shifting.
That is to say, the structured light generating device can rapidly generate high-quality structured light in different directions and different phase states, effectively eliminates the time of phase shift and direction switching in the process of generating the structured light, and enables the image acquisition speed not to be limited by the generation speed of the structured light.
Moreover, the structured light generating device has universality, and can be used for the structured light generation of N directions (such as two directions forming 90 degrees with each other, or three directions forming 120 degrees with each other) and M-step phase shift (such as three-step phase shift or five-step phase shift).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a structured light generating device according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Referring to fig. 1, fig. 1 is a schematic structural diagram of a structured light generating device according to an embodiment of the present invention.
The structured light generating device includes: the angle between the N groups of structured light generating units is 360/N degrees, so that the structured light stripes generated by the N groups of structured light generating units mutually form 360/N degrees on the surface of a sample to be detected, N is more than or equal to 2 and is a positive integer;
wherein the structured light generating unit includes: the light source, the light field modulation device and the light path coupling device;
when one group of the structured light generation units is in an illumination state, the rest groups of the structured light generation units are in a non-illumination state, and the light field modulation devices in the rest groups of the structured light generation units perform phase shifting.
It should be noted that, in the embodiment of the present invention, the angles between the N groups of structured light generating units may also be other self-defined angles, so that the structured light stripes generated by the N groups of structured light generating units form corresponding angles with each other on the surface of the sample to be measured.
In addition, the optical path coupling device shown in fig. 1 is described by taking a reflective optical path coupling device as an example, and may also be a transmissive optical path coupling device.
In the embodiment, the structured light generating device can quickly generate high-quality structured light in different directions and different phase states, effectively eliminates the time of phase shift and direction switching in the process of generating the structured light, and ensures that the image acquisition speed is not limited by the generation speed of the structured light.
Moreover, the structured light generating device has universality, and can be used for the structured light generation of N directions (such as two directions forming 90 degrees with each other, or three directions forming 120 degrees with each other) and M-step phase shift (such as three-step phase shift or five-step phase shift).
Specifically, for a super-resolution image acquired by a structured light microscope, the structured light needs to be divided into N directions within 360 degrees, and an included angle of 360/N degrees is formed between each two directions; the structured light fringes in each direction need to move p phases within one optical field modulation device (grating) period, so that N × p images need to be collected for synthesis.
Then, each time the structured light illumination microscope takes a super-resolution image of a biological sample, the structured light fringes in the N fringe directions need to be shifted p times, and N × p structured light illumination modes are total.
In the present application, light emitted from the light source passes through a structured light generating device (herein, a grating is taken as an example) clamped on the displacement table, enters the illumination light path through a light path coupler (such as a dichroic mirror) and finally forms structured light illumination on the sample plane.
The structured light generating units on the N channels are arranged according to the directions forming 360/N degrees with each other, so that structured light bright stripes forming 360/N degrees with each other can be generated on the surface of the sample.
By controlling the on-off of the light source entering the light path, the conversion of the structured light stripes in different directions can be realized, thereby avoiding the time consumed by mechanical rotation in the prior art. The switching speed of the structured light stripes in different directions is only the speed of the light source switch, and the speed of the light source switch can be extremely fast (for example, microsecond magnitude), so that the fast switching of the structured light directions is realized.
In each channel, the displacement platform drives the grating to displace 1/p grating phases, so that phase displacement of the structured light stripes in each direction can be realized, when one channel is subjected to structured light modal acquisition, the displacement platforms of other p-1 channels perform grating phase displacement in advance, and thus when the channel is switched to other channels, the displacement platforms are ready to be displaced, so that the time consumed by grating phase displacement is avoided.
Further, it can be seen that the structured light generating apparatus can eliminate the time for phase shift and direction switching in the structured light generating process, thereby achieving high-quality and rapid structured light generation.
Further, based on the above embodiments of the present invention, the phase starting positions of the light field modulation devices in the N groups of structured light generation units are the same and are all the first phase states.
In this embodiment, at the initial position of the installation fixture, the initial positions of the light field modulation devices in the N groups of structured light generation units may be the same, and all of the initial positions are in the first phase state, so as to facilitate directly performing image acquisition in the first phase state.
Further, according to the above embodiment of the present invention, when the first group of structured light generation units is subjected to image acquisition in the first phase state, the light sources in the first group of structured light generation units are turned on;
and the light sources in the rest groups of the structured light generating units are in an off state.
When the second group of structured light generation units are subjected to image acquisition in a first phase state, turning on light sources in the second group of structured light generation units;
the light sources in the remaining groups of structured light generation units are all in an off state, and the light field modulation devices in the first group of structured light generation units move to a second or other phase state.
In this embodiment, when the first group of structured light generation units is subjected to image acquisition in the first phase state, only the light sources in the first group of structured light generation units need to be turned on, and the light sources in the remaining groups of structured light generation units are all in the off state.
In the next process, when the second group of structured light generation units are subjected to image acquisition in the first phase state, only the light sources in the second group of structured light generation units need to be turned on, the light sources in the other groups of structured light generation units are all in the off state, and the light field modulation device in the first group of structured light generation units moves to the second or other phase states.
Similar to the above-mentioned process, in the nth process, only the light source in the nth group of structured light generation units may be turned on, the light field modulation devices in the other groups of structured light generation units may all move to the second or other phase states, and the light sources corresponding to the light field modulation devices are all in the off state.
Further, according to the above-mentioned embodiment of the present invention, when the image capturing of the N sets of the structured light generation units in the first phase state is completed,
turning on a light source in the first group of structured light generation units, and acquiring an image of the first group of structured light generation units in the second phase state;
and the light sources in the rest groups of the structured light generation units are all in a closed state, and the light field modulation devices in the rest groups of the structured light generation units perform phase shift until the light field modulation devices are in the second or other phase states.
In this embodiment, when the image acquisition of the structured light in all directions on the first phase state has been completed, the image acquisition of the next phase state (second phase state) is started.
In the acquisition process of the first phase state, the light field modulation device in the first group of structured light generation units is moved to the second phase state, so that the image acquisition of the first group of structured light generation units on the second phase state can be performed only by turning on the light source in the first group of structured light generation units.
When the light field modulation device in the second group of structured light generation units moves to the second phase state, only the light source in the second group of structured light generation units is turned on, and image acquisition of the second group of structured light generation units in the second phase state is performed.
Similar to the above-mentioned process, in the nth process, when the light field modulation devices in the nth group of structured light generation units are moved to the second phase state, only the light sources in the nth group of structured light generation units are turned on, the light field modulation devices in the other groups of structured light generation units are all moved to the next phase state, and the light sources corresponding to the light field modulation devices are all in the off state.
Further, according to the above-mentioned embodiment of the present invention, when the image capturing of the N sets of the structured light generating units in the p-1 th phase state is completed,
turning on a light source in the first group of structured light generation units, and acquiring an image of the first group of structured light generation units in the p-th phase state;
the light sources in the other groups of the structured light generation units are all in a closed state, and the light field modulation devices in the other groups of the structured light generation units perform phase shift until the light field modulation devices are in the p-th or other phase states;
and continuing to collect the images until the image collection of the N groups of the structured light generating units on the p-th or other phase states is completed.
In this embodiment, after the acquisition of the structured light in all directions in the second phase state is completed, the image acquisition is performed until the p-th phase, similar to the above-described procedure.
Because the light field modulation device in the first group of structured light generation units moves until the p-th phase state in the acquisition process of the p-1 phase state, the light source in the first group of structured light generation units is only required to be turned on at the moment to acquire the image of the first group of structured light generation units in the p-th phase state.
In the process of image acquisition of the first group of structured light generation units in the p-th phase state, the light field modulation devices in the other groups of structured light generation units perform phase shift until the first group of structured light generation units are in the p-th phase state, and image acquisition is continuously performed until the image acquisition of the N groups of structured light generation units in the p-th phase state is completed.
After all states of a complete structured light direction and phase are acquired, and in the image acquisition process of the p-th phase state, the light field modulation devices in the 1 st group to the N-1 st group of structured light generation units all start to move to the next first phase state.
Further, based on the above embodiments of the present invention, the structured light generating apparatus further includes: n power switches;
and each power switch correspondingly controls one power supply.
The light source is an LED light source.
In this embodiment, the light source may optionally include, but is not limited to, an LED light source, which may be on the order of microseconds in switching speed.
Further, based on the above embodiment of the present invention, the displacement stage is a piezoelectric displacement stage.
In this embodiment, the displacement stage includes, but is not limited to, a piezoelectric displacement stage, whose open loop response time may reach sub-millisecond order.
Further, based on all the above embodiments of the present invention, in another embodiment of the present invention, there is provided a structured light illumination microscope, which includes the above structured light generation device.
The structured light generating device and the structured light illuminating microscope provided by the invention are described in detail above, and the principle and the implementation mode of the invention are explained in the text by applying specific examples, and the description of the above examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
It should be noted that, in the present specification, the embodiments are all described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments may be referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
It is further noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include or include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The previous description of the disclosed embodiments is provided to enable any person 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 generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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.
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