WO2020207215A1 - 便携式微型荧光显微镜 - Google Patents

便携式微型荧光显微镜 Download PDF

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Publication number
WO2020207215A1
WO2020207215A1 PCT/CN2020/079835 CN2020079835W WO2020207215A1 WO 2020207215 A1 WO2020207215 A1 WO 2020207215A1 CN 2020079835 W CN2020079835 W CN 2020079835W WO 2020207215 A1 WO2020207215 A1 WO 2020207215A1
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Prior art keywords
excitation
objective lens
optical path
image acquisition
fluorescence
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PCT/CN2020/079835
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English (en)
French (fr)
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温云卿
朱之谦
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中国科学院脑科学与智能技术卓越创新中心
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Publication of WO2020207215A1 publication Critical patent/WO2020207215A1/zh

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor

Definitions

  • the invention relates to a microscope imaging device, in particular to a portable miniature fluorescent microscope.
  • the optical path is widely used in the design and manufacture of large fluorescence microscopes.
  • the working principle is that the light emitted by the excitation source passes through the excitation filter to allow the excitation light of a specific wavelength to pass, and then is reflected by the dichroic mirror to irradiate the excitation light on the sample, and the sample illuminated by the excitation light emits emission light, that is, fluorescence .
  • Longer-wavelength fluorescence can pass through a dichroic mirror, and then pass through a fluorescence filter to filter out stray light, allowing specific wavelengths of fluorescence to be illuminated on the detector.
  • the emitted light passes through the objective lens and the projection lens, and then converges into a magnified image on the detector.
  • this kind of optical path design is simple and easy to implement, it has a long optical path and takes up a lot of space. Obviously, it is only suitable for manufacturing large instruments and not suitable for miniature portable devices. Existing miniature devices usually use the optical path design of traditional large-scale fluorescence microscopes, and only have a proportional reduction in size. However, the design of the optical path determines the appearance and structure, and also limits the performance of the instrument.
  • a lens with a very short focal length must be used as the objective lens to shorten the length of the optical path while ensuring sufficient magnification. Therefore, in this optical path, a self-focusing lens must be used as an objective lens.
  • the manufacturing process of the self-focusing lens is complicated, and the uniformity of the density gradient is difficult to control, which results in the small observation range of the device, low imaging resolution, and image distortion.
  • the imaging focusing structure behind the projection lens is very inconvenient. If you want to adjust the focus, you must loosen the screw and rotate the CMOS sensor circuit board base.
  • the wiring harness on the CMOS circuit board is connected from the top of the device to the side of the device to supply power to the LED circuit board.
  • Such a design causes the power supply line to be involved when focusing, and the experimental animal will scratch the power supply line exposed on the side, which may cause a short circuit or an open circuit to damage the equipment. This not only greatly reduces the ease of use of the equipment, but also increases the risk of use.
  • the device uses a simple constant current LED circuit to provide excitation light for imaging.
  • the luminous efficiency of LEDs will decrease as the temperature rises due to heat generation.
  • the change of excitation light intensity directly affects the final imaging brightness, resulting in uncontrollable imaging conditions, and the conditions of each experiment are inconsistent.
  • the purpose of the present invention is to provide a portable miniature fluorescence microscope.
  • the portable micro-fluorescence microscope can effectively overcome the shortcomings of the existing technology, and according to the needs of the current scientific development trend, the optical path is redesigned to make the optical path adaptable.
  • the micro-fluorescence microscope is also small in size and light in weight; and has a large observation field; The imaging resolution is high and the aberration is small.
  • the present invention provides a portable miniature fluorescence microscope.
  • the microscope includes a first optical path and a second optical path; the first optical path is from an excitation source to an objective lens, and the excitation source An excitation element is provided between and the objective lens, the excitation element is configured and arranged to guide the excitation light emitted from the excitation source to the objective lens; the second optical path is from the objective lens to the image acquisition Device, and an emission element is provided between the objective lens and the image acquisition device, the emission element is configured and arranged to project the fluorescence received by the objective lens to the image acquisition device; wherein the objective lens is Aspheric lens; the emitting element includes a reflector, the reflector is arranged in front of the image acquisition device, and is used to change the light receiving position of the image acquisition device.
  • the microscope includes a housing, and the first optical path and the second optical path are built in the housing.
  • the objective lens is arranged between the dichroic mirror and the observed sample.
  • the excitation source, optional converging lens, excitation filter, dichroic mirror and the objective lens are arranged vertically.
  • the optical axis of the first optical path is perpendicular to the sample to be observed.
  • the excitation element includes an excitation filter and a dichroic mirror.
  • the excitation element further includes a condensing lens.
  • the condensing lens is a ball lens.
  • the condensing lens is arranged between the excitation source and the excitation filter.
  • the number of the excitation elements is multiple.
  • the dichroic mirror is a filter that passes short wavelengths and reflects long wavelengths.
  • the emitting element includes the dichroic mirror, the projection lens and the reflector.
  • the number of the emitting elements is multiple.
  • the excitation light emitted by the excitation source first passes through the excitation filter for the first filtering, and then passes through the dichroic mirror for the second filtering, passes through the objective lens, and then irradiates the On the sample being observed.
  • a long-wavelength fluorescence is excited from the sample, and the fluorescence is filtered through a dichroic mirror for the third time, and the fluorescence is reflected through the projection lens and the reflector to finally reach the image Collection device.
  • the fluorescence filtered by the emission filter is condensed after passing through the projection lens.
  • the emission element further includes an emission filter, and the fluorescence is filtered through the emission filter for a fourth time.
  • the emission filter is located at any position on the second optical path before the image acquisition device and after the dichroic mirror.
  • the surface of the reflector is coated with a coating, and the coating is used to filter the fluorescence.
  • the coating film covers the surface of the reflector.
  • the excitation filter is perpendicular to the emission filter at 90 degrees.
  • the acute angle formed by the dichroic mirror and the optical axis of the first optical path is 30-60 degrees.
  • the dichroic mirror bisects the right angle formed by the excitation filter and the fluorescence filter, that is, the dichroic mirror is aligned with the optical axis of the first optical path. 45-degree angle.
  • the stable excitation light is guided to an area of at least 3 mm 2 in the field of view including the target object.
  • the fluorescence emission caused by the excitation light is guided to the image acquisition device to provide a resolution of at least 1 ⁇ m for the image of the field of view.
  • the reflector is a prism; preferably, it is a pentaprism.
  • the reflector is a plane mirror.
  • the miniature fluorescence microscope includes a light intensity detector, a feedback circuit and a drive circuit, wherein the light intensity detector is used to detect the light intensity of the excitation source and transmit it to the feedback circuit, so The feedback circuit sends a signal to the drive circuit to ensure the stability of the excitation source.
  • the excitation source, the light intensity detector, the feedback circuit and the drive circuit are arranged on a lighting circuit board.
  • the excitation light of the traditional microscope usually changes continuously with the irradiation time, which makes the imaging have unstable parameters.
  • the present invention adopts a stable excitation source to ensure that the light intensity is stable, and the excitation source is stabilized through detection-feedback-compensation, and the phenomenon of continuous signal attenuation caused by the progressive attenuation of excitation light is avoided.
  • the excitation source and the image acquisition device are arranged on the top of the housing, and the objective lens is arranged on the bottom of the housing.
  • the excitation source and the image acquisition device are arranged on the top of the shell, so that the circuit is arranged on the upper part of the shell, so that the animal is not easy to scratch and damage the key components.
  • the excitation source is a light emitting diode.
  • the light emitting diode is a monochromatic light emitting diode.
  • the excitation source is a laser diode.
  • the excitation source is flash type illumination.
  • the excitation source is a constant light intensity output.
  • the excitation filter is configured as a band pass filter.
  • the function of the dichroic mirror in the first optical path is to only allow light that meets a certain wavelength condition to pass through without changing the propagation direction of the light; the function of the dichroic mirror in the second optical path is based on the spectrum
  • the characteristic sorts out the fluorescence generated by excitation and changes its light propagation direction, while filtering out the short-wavelength excitation light directly reflected by the sample.
  • the emission filter is configured as a band pass filter.
  • the excitation filter, the dichroic mirror and the emission filter are collectively referred to as a fluorescence module.
  • the fluorescent module is replaceable.
  • the illumination mode of the excitation source is Köhler illumination.
  • the image capture device includes a photosensitive element.
  • the photosensitive element of the image acquisition device is a CMOS image sensor.
  • the image acquisition device is connected to the computer in a wired and/or wireless manner.
  • the optical path of the microscope of the present invention can be applied to the bright field optical path of a common microscope, wherein the excitation filter is replaced by a horizontal polarizer, the dichroic mirror is replaced by a vertical polarizer, and the emission filter is replaced by a horizontal polarizer.
  • the light sheet can be replaced with a half mirror.
  • the beneficial effect of the present invention is that the portable miniature fluorescence microscope of the present invention is suitable for use in scientific research and medical fields.
  • the new optical path ensures high-quality imaging effects and reduces assembly difficulty and cost.
  • the reasonable structure design ensures stable use. It is more convenient to set up on the body of experimental animals, which solves the current technical problems in the field of neuroscience in a true sense, and greatly promotes the development of the Chinese Brain Project and the world's neuroscience.
  • Fig. 1 is a schematic diagram of the structure of a portable miniature fluorescence microscope in an embodiment of the present invention.
  • Fig. 2 is a working principle diagram of the fluorescence module of the portable miniature fluorescence microscope in an embodiment of the present invention.
  • Fig. 3 is a schematic diagram of the first optical path in an embodiment of the present invention.
  • Fig. 4 is a schematic diagram of a second optical path in an embodiment of the present invention.
  • Fig. 5 is a schematic diagram of the structure of a portable miniature fluorescence microscope in another embodiment of the present invention.
  • Fig. 6 is a schematic diagram of a second optical path in which the reflector is a pentaprism in an embodiment of the present invention.
  • FIG. 7 is a schematic diagram of the second optical path of replacing the transmitting filter by coating the receiving surface of the pentaprism with a coating film in an embodiment of the present invention.
  • the inventors developed a portable miniature fluorescence microscope for the first time.
  • the miniature fluorescence microscope uses an aspheric lens with excellent optical imaging capabilities as the objective lens to expand the field of view. It also improves the resolution and greatly optimizes the imaging capabilities of the micro-microscope.
  • the micro-fluorescence microscope uses different optical elements to form a new optical path, which solves the problem of the long optical path caused by the use of an aspheric lens as the objective lens.
  • the quality distribution of the equipment is balanced to ensure the stable use of the equipment, and the present invention is completed on this basis.
  • the microscope includes a small portable housing.
  • the housing is provided with several elements constituting the first optical path and the second optical path, including an excitation source configured to provide excitation light, an objective lens, an image acquisition device, and the like.
  • the first optical path includes two end points, one end point is the excitation source and the other end point is the objective lens, and an excitation element configured and arranged to guide the excitation light to the objective lens is provided between the excitation source and the objective lens.
  • the second optical path includes two end points, one end point is the objective lens, the other end point is the image acquisition device, and a transmitting element is arranged between the objective lens and the image acquisition device, and the transmitting element is configured and arranged to be fluorescent light received by the objective lens Projected to the image capture device.
  • the excitation element includes an excitation filter, a dichroic mirror and a condensing lens
  • the emission element includes a dichroic mirror, an emission filter, a projection lens and a reflector.
  • the objective lens, converging lens, fluorescent module, projection lens, reflector, and image acquisition device are collectively called optical devices.
  • the fluorescence module is placed between the objective lens and the excitation source, and includes an excitation filter, a dichroic mirror and an emission filter.
  • the overall optical path of the present invention is different from the traditional microscope optical path.
  • the illumination light path is the top illumination test, the excitation light goes directly to the sample from top to bottom, and the imaging light path that the returned emitted light passes through does not completely overlap with the illumination light path.
  • the direction of the light path of the projection lens is changed, which greatly reduces the space occupied by the second optical path in the horizontal direction, and makes full use of the space in the vertical direction.
  • the optical path innovation of the present invention is mainly to solve the adverse consequences of using an aspheric lens as an objective lens, which leads to an increase in the optical path, a larger space occupied by the lens body, and uneven weight distribution.
  • the optical path design of the present invention is based on reality and meets the needs of use.
  • the self-focusing lens has many shortcomings, such as the problem of low imaging resolution and large aberration.
  • the cost of the self-focusing lens is high, which limits the popularization of micro-microscopes.
  • this embodiment uses an aspheric lens as The objective lens has good optical performance.
  • a reflector is used to change the linear optical path into a polyline optical path, which reduces the space of the optical path and facilitates the miniaturization of the equipment.
  • the design idea of the present invention can be understood as: changing the objective lens type to obtain a larger magnification, a wider field of view observation range and higher imaging resolution; redesigning the optical path to compensate for the optical path increase caused by changing the objective lens ,
  • the lens body occupies more space and the weight distribution is uneven, these undesirable consequences; modify the shape design according to the optical path, while ensuring the imaging quality, it also enhances the convenience of use and the stability of the entire imaging system.
  • magnification focal length of the projection lens/focal length of the objective lens.
  • the focal length of the objective lens is limited by the aspheric lens and is not easy to shorten, but the focal length of the projection lens can be easily made longer.
  • the problem of the lengthening of the second optical path caused by the lengthening of the focal length of the projection lens has been solved by the optimization of the optical path design of the present invention. Therefore, in a specific embodiment, it is allowed to increase the focal length of the projection lens to obtain a greater magnification.
  • an image acquisition device with high pixel density is selected, and a larger magnification can improve the resolution.
  • emission light and the term “fluorescence” are the same concept and can be used interchangeably.
  • the microscope of this embodiment includes an excitation source 1, which generates excitation light 13, which is guided by an optical device to a target sample object for imaging the target sample object.
  • the excitation source 1, the condensing lens 2, the fluorescent module, and the objective lens 7 constitute a first optical path; the objective lens 7, the fluorescent module, the projection lens 9, the reflector 10, and the image acquisition device 12 constitute a second optical path.
  • the objective lens 7 is an intermediate medium between the first optical path and the second optical path. Among them, the objective lens 7 is arranged between the dichroic mirror 4 and the sample 6 to be observed.
  • the first optical path is the illumination light path, which is mainly used to guide the excitation light 13 to the sample 6 to be observed, so that the light intensity of the excitation light 13 is uniform.
  • the excitation source 1, the condensing lens 2, the excitation filter 3, the dichroic mirror 4 and the objective lens 7 are arranged vertically from top to bottom, which means that the excitation light 13 from the excitation source 1 passes through the excitation filter 3 and The dichroic mirror 4 is projected onto the objective lens 7 to form a first optical path.
  • the optical axis of the first optical path is a vertical straight line, and the optical axis of the first optical path is perpendicular to the sample 6 to be observed.
  • the dichroic mirror 4 in the present invention is a filter that passes short wavelengths and reflects long wavelengths.
  • the acute angle formed by the dichroic mirror 4 and the optical axis of the first optical path is 30-60 degrees.
  • the excitation filter 3 is perpendicular to the emission filter 8 at 90 degrees.
  • the dichroic mirror 4 bisects the right angle formed by the excitation filter 3 and the emission filter 8, that is, the dichroic mirror 4 forms an angle of 45 degrees with the optical axis of the first optical path.
  • the dichroic filter in the prior art reflects the excitation light generated by the excitation source, so the excitation source is placed on the side. However, after the excitation light 13 generated by the excitation source 1 of the present invention passes through the excitation filter 3, the dichroic filter refracts the excitation source 1. This design makes the excitation source 1 at the top and the first optical path as a whole In a straight line.
  • the microscope can be used to observe artificially synthesized fluorescent substances or fluorescent proteins expressed by organisms, such as green fluorescent protein (GFP).
  • the fluorescent sample 6 is irradiated with suitable excitation light 13, and the excited emission light, that is, the fluorescence 11, is captured by the image acquisition device 12, and the image acquisition is completed.
  • GFP green fluorescent protein
  • the excitation source 1 in this embodiment is coupled to the illumination circuit board on the top of the microscope.
  • the excitation source 1 can be, but is not limited to, a solid state light emitting diode (LED).
  • the excitation source 1 can be configured as a blue light-emitting diode with an emission spectrum in the range of 465nm-485nm, which can effectively excite the green fluorescent protein without photobleaching at low power.
  • the chromophore group In order to reduce the damage caused by the high-power excitation light 13 irradiating fluorescent substances or biological tissues, this embodiment uses a narrow-spectrum light-emitting diode as the excitation source 1 to improve the excitation efficiency of the fluorescent group, and the power of the excitation source 1 is set to 1. -5mW range. In this way, the photodamage and photothermal effects of sample 6 will be significantly reduced, reducing the impact of phototoxicity on the experiment.
  • the excitation source 1 can be set to flash type illumination. Because most studies have shown that for fluorescent substances, such as green fluorescent protein, irradiated with high-power excitation light 13 for a long time, photobleaching will occur, causing the fluorescent substances to lose their ability to fluoresce.
  • the flash-type illumination converts the continuous long-term exposure of sample 6 into intermittent exposure, effectively preventing the occurrence of photobleaching and reducing phototoxicity.
  • the luminous frequency and phase of the flash illumination of the excitation source 1 are coupled with the sampling rate and phase of the image acquisition to realize the acquisition of each frame of image, and the sample 6 will be effectively excited.
  • the excitation source 1 uses a constant light intensity output. For this reason, a light intensity detector is also provided on the circuit board where the excitation source 1 is located to monitor the light intensity of the LED in real time. When the luminous efficiency of the LED decreases with the increase of temperature, the light intensity detector will capture this information and transmit it to the feedback circuit, and increase the drive current to ensure that the LED light intensity output is constant. As the power continues to increase , The heat production and heat dissipation of the LED will reach a dynamic equilibrium state, at this time the power will no longer rise, and the light intensity output will always remain constant.
  • the excitation source 1 with constant light output provides the excitation light 13, which can ensure that the intensity of the excitation light 13 remains unchanged, thereby ensuring the consistency of the signal acquisition conditions during long-term imaging, and avoiding the signal caused by the progressive attenuation of the excitation light 13 The illusion of continuous attenuation. There is no need to perform light attenuation correction processing with large errors on the experimental data later.
  • a converging lens 2 is arranged between the excitation source 1 and the excitation filter 3.
  • a spherical lens can be selected as the converging lens 2, for example, a spherical lens made of K9 material with a refractive index of 1.5163 and a diameter of 2.5-4.0 mm can be selected. Reduce the size and weight of the device.
  • the fluorescence module is configured as follows: excitation filter 3470nm, dichroic mirror 4500nm, emission filter 8525nm.
  • the excitation light 13 emitted by the excitation source 1 first passes through the excitation filter 3 for the first filtering.
  • the excitation filter 3 is configured as a band pass filter to filter out stray light with a wavelength other than 470 ⁇ 10 nm.
  • the filtered excitation light 13 is directed to the front surface of the dichroic mirror 4. At this time, the dichroic lens only allows light with a wavelength in the range of 325-480 nm to pass.
  • the excitation light 13 transmitted through the dichroic mirror 4 is irradiated on the sample 6 to excite emission light with a longer wavelength, that is, fluorescence 11.
  • the emitted light is directed upward to the rear surface of the dichroic mirror 4, and at this time, the dichroic mirror 4 reflects the emitted light whose reflection wavelength is in the range of 520-610 nm.
  • the function of the dichroic mirror 4 is to sort out the excited fluorescence 11 according to the spectral characteristics and change its light propagation direction, while filtering out the short-wavelength excitation light 13 directly reflected by the sample 6.
  • the emission filter 8 is configured as a band pass filter, which only allows light with a wavelength within 525 ⁇ 15 mm to pass through, and filters the emitted light again. The filtered emission light passes through the reflector 10 and reaches the image acquisition device 12 to complete fluorescence imaging.
  • the fluorescent module in this embodiment can be replaced according to actual needs. For example, to observe red fluorescent protein (RPF), replace the fluorescent module and the supporting excitation source 1 with a component group corresponding to a longer wavelength.
  • RPF red fluorescent protein
  • the function of the reflector 10 is to change the light receiving position of the image acquisition device 12.
  • the main advantage is that when the optical path is long, the space occupation in the horizontal direction can be reduced, and the mass distribution of the entire device of the present invention can be balanced. According to actual needs, the orientation of the reflector 10 can be changed to change the position of the image acquisition device 12.
  • the reflector 10 is a plane mirror.
  • the illumination method of the present invention adopts Köhler illumination, which aims to ensure the uniformity of the excitation light 13 irradiated on the sample 6 to be observed.
  • the mutual distance positions are set to complete the first optical path.
  • the light emitted by the excitation source 1 passes through the condensing lens 2, it converges and forms an image under the condensing lens 2, and the position of the image just falls on the focal point of the upper surface of the objective lens 7.
  • the convergent imaging light continues to propagate downwards, passes through the objective lens 7, and forms parallel light in multiple directions, uniformly irradiating the observed sample 6 to achieve Kohler illumination.
  • the aperture parameters such as the field of view 5 diaphragm and the aperture diaphragm in the Köhler illumination are limited by the size of the inner wall of the device. Unless special needs are required, the maximum clear aperture is generally used.
  • the excitation light 13 emitted by the excitation source 1 can irradiate the sample 6 with parallel beams through the Koehler illumination.
  • This effect is realized by the LED excitation source 1, the condensing lens 2 and the objective lens 7.
  • the key point to realize the Köhler illumination is that the real image of the excitation source 1 just falls on the focal point of the upper surface of the objective lens 7.
  • the field of view 5 diaphragm, the aperture diaphragm is limited by the inner wall of the microscope and cannot be adjusted at will.
  • the second optical path is the imaging optical path, which is used to guide the emitted light to the image acquisition device 12, and at the same time enlarge the image of the observed sample to achieve the expected resolution, and the detailed information of the observed sample 6 can be clearly identified.
  • a projection lens and a reflector 10 are arranged between the emission filter 8 and the image acquisition device 12.
  • an aspheric lens is selected as the objective lens 7.
  • the luminous point located on the focal plane of the lower surface of the objective lens 7 emits emitted light, that is, the fluorescence 11, enters the objective lens 7, passes through the rear hole of the objective lens 7, and is emitted as parallel light.
  • the emitted parallel light is sorted by the dichroic mirror 4 and reflected to change the light path direction, and then filtered by the emission filter 8 to reach the projection lens 9.
  • the emitted light converges at one point on the image acquisition device 12 (here configured as a photosensitive element) to form a clear image.
  • the function of the reflector 10 is to change the direction of the light emitted from the projection lens 9 and to make rational use of the space structure.
  • the objective lens 7 of the present invention uses an aspheric lens instead of a self-focusing lens.
  • a self-focusing lens is used as the objective lens 7, but the self-focusing lens has the disadvantages of serious aberrations and a narrow field of view 5, resulting in distortion of the image and a small field of view.
  • the diameter of the self-focusing lens cannot be increased arbitrarily, and the self-focusing lens with a diameter greater than 3 mm cannot be produced basically.
  • the density gradient of the self-focusing lens is difficult to control.
  • the finished lens only has the central part of the lens, and the image can be imaged within about half of the diameter. This limits the field of view of the miniature fluorescence microscope.
  • the imaging has aberrations, and the farther to the edge of the lens, the aberrations become more serious, which further restricts the size of the field of view 5 and also causes poor resolution.
  • Some devices use a self-focusing lens as the objective lens 7, mainly because of its short focal length (less than 2 mm), which facilitates shortening the optical path length.
  • shortcomings such as small field of view 5 and serious aberration are inevitable.
  • the aspheric mirror used in the present invention has a mature manufacturing process, and aspheric mirrors of different specifications can be designed and produced according to requirements to cope with different actual imaging conditions. Therefore, an aspheric lens with a larger diameter can be selected as the objective lens 7 to expand the range of the field of view 5.
  • an aspheric lens with a diameter of 3 mm or 5 mm can be selected, and the range of the field of view 5 is more than 10 times larger than that of a self-focusing lens.
  • the aberration of an aspheric lens is better than that of an ordinary spherical lens and far better than a self-focusing lens.
  • a 0.5NA aspheric lens can provide an imaging resolution of 1 ⁇ m, which is 2.5 times higher than a self-focusing lens. Therefore, regardless of the range of the field of view 5 or the imaging resolution, the imaging effect of using an aspheric lens as the objective lens 7 is far better than that of a self-focusing lens.
  • the invention mainly uses a newly designed optical path to solve it. Since the aspheric lens uses the refractive index and surface curvature of the lens to achieve the refractive effect, the existence of surface curvature does not allow lenses with larger diameters (greater than 3mm) to have shorter focal lengths (less than 1.5mm).
  • the aspheric lens has a relatively longer focal length when used as the objective lens 7.
  • the reason is that, in order to make full use of the numerical aperture of the objective lens 7 and the pixel density of the photosensitive element, it is necessary to ensure that the imaging has a sufficient magnification to achieve the expected imaging resolution.
  • the magnification in the embodiment can be simply calculated as the ratio of the focal length of the projection lens 9 to the objective lens 7. When an aspheric lens is used as the objective lens 7, the focal length becomes longer without reducing the magnification.
  • the focal length of the projection lens 9 must be increased, that is, the distance from the projection lens 9 to the image capture device 12 is increased.
  • the focal length of the projection lens 9 will increase exponentially as the focal length of the objective lens 7 becomes longer, so the length of the second optical path increases significantly.
  • the increase of the optical path will increase the occupied space of the entire device, which is not convenient for miniaturization.
  • the newly designed optical path greatly reduces the space occupied by the second optical path in the horizontal direction, makes full use of the space in the vertical direction, and solves the aforementioned problems.
  • the photosensitive element of the image acquisition device 12 in this embodiment may be, but is not limited to, a CMOS image sensor.
  • the emitted light is reflected by the reflector 10 and focused on the CMOS sensor, and the image is collected by the CMOS sensor and transmitted to the computer.
  • a variety of methods are used to transmit data, including the use of reliable data transmission links to transmit data. It also includes the use of TV signals, FM signals, amplitude modulation signals, and phase modulation signals to transmit images wirelessly.
  • the image transmitted from the CMOS sensor to the computer can include two versions, wired and wireless, to meet different experimental requirements.
  • the behavior of the observation object is on the two-dimensional ground, or when it is relatively simple (such as rats, rabbits, birds), use the wired version.
  • the data transmission speed is fast, its data pass rate is at least 400Mb/s, can collect data continuously for a long time, realize the sampling rate of VGA frame at least 60 frames per second.
  • the wireless version can be used.
  • the advantage is that the observation object can move freely in a large space.
  • the observation object carries a small battery to power the instrument, and the continuous data collection time depends on the size of the battery carried by the observation object, which can basically meet the needs of use.
  • the wireless data transmission rate is at least 300Mb/s, and the sampling rate of at least VGA frame is 30 frames/sec.
  • the excitation source 1 of the first optical path and the image acquisition device 12 of the second optical path are both located at the top of the housing, and the objective lens 7 is located at the bottom of the housing. Therefore, most of the circuits, especially the circuit components of the excitation source 1, are arranged on the top which cannot be touched by animals.
  • the lens focusing method is no longer a rotating device housing part, but directly adjusts the projection from the outside.
  • the horizontal position of the lens 9 changes the distance between the projection lens 9 and the image acquisition device 12 to complete focusing, which is more convenient and does not involve external circuits.
  • the portable mini-fluorescence microscope of this embodiment is small in size and light in weight.
  • the size can even be as small as 8cm 3 , and the weight can be as light as about 3.5g. It has good imaging effects and high resolution.
  • This instrument can be used in brain sciences. The research can be set up on the heads of experimental animals. When the awake animals are free to move, the activity of the marked nerve tissue or other types of cells in the animal’s brain can be observed through fluorescence imaging; the blood flow changes in the blood vessels can be directly observed, so It can be directly used to detect the dynamic changes of superficial blood vessel blood flow in medical clinical patients.
  • the microscope in this embodiment is roughly the same as the microscope in embodiment 1.
  • the excitation filter 3 in embodiment 1 is replaced with a horizontal polarizer
  • the dichroic mirror 4 is replaced with For the vertical polarizer, replace the emission filter 8 with a half mirror.
  • the microscope in this embodiment is roughly the same as the microscope in embodiment 1. The difference is that in this embodiment, the position of the emission filter 8 is moved before the image acquisition device 12, after the reflector 10, and placed horizontally. As shown in FIG. 5, such an arrangement can make the horizontal movement space of the projection lens 9 larger, which is more convenient for focusing.
  • the position of the emission filter 8 of the present invention is not particularly limited.
  • the function of the emission filter 8 is only filtering, and it can be placed before the image acquisition device 12 and the second light after the dichroic mirror 4. Anywhere on the road.
  • the microscope in this embodiment is roughly the same as that in embodiment 1.
  • the difference is that the inner surface of the reflector 10 in this embodiment is coated with a layer of coating, which can filter fluorescence, and its effect It is equivalent to the emission filter 8. Therefore, in this embodiment, the emission filter 8 can be removed to simplify the components.
  • the number of coating layers there is no particular limitation on the number of coating layers, and it can be one, two or more layers.
  • the flat mirror plated with the coating in this embodiment can function as a reflector and an emission filter at the same time, and a separately added emission filter can be omitted, which saves space and reduces weight.
  • the microscope in this embodiment is roughly the same as the microscope in embodiment 1.
  • the reflector 10 in this embodiment is a prism, such as a pentaprism (as shown in FIG. 6).
  • the prism can be used as a reflector. It has a longer optical path and higher fluorescence reflection efficiency.
  • the shape of the pentaprism can be changed according to the angle of the dichroic mirror.
  • the microscope in this embodiment is roughly the same as that in embodiment 5.
  • this embodiment is coated with a coating on the surface of the prism, specifically, a coating 14 (such as As shown in FIG. 7), the coating film 14 can filter fluorescence, and its effect is equivalent to that of the emission filter 8. Therefore, in this embodiment, the emission filter 8 can be removed to simplify the components.
  • the number of layers of the coating film 14 is also not particularly limited, and may be one layer, two layers or multiple layers.
  • the coated prism in this embodiment can function as a reflector and an emission filter at the same time, and a separately added emission filter can be omitted, saving space and reducing weight.

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Abstract

本发明涉及一种便携式微型荧光显微镜，其包括第一光学路径和第二光学路径，第一光学路径一端为激励源，另一端为物镜，激励源和物镜之间设置有被配置和布置为将激发光引导至物镜的激发元件；第二光学路径一端为物镜，另一端为图像采集装置，物镜和图像采集装置之间设置有被配置和布置为由将由物镜接收到的荧光投射到图像采集装置的发射元件；其中，物镜为非球面透镜；发射元件包括反射器，该反射器设置在图像采集装置前，用于改变图像采集装置的光接收位置。本发明在保证高质量的成像效果的同时，维持微小体积，简化组装并降低成本，合理的结构设计保证了使用的稳定性，更方便于架设在实验动物身体上。

Description

便携式微型荧光显微镜 技术领域
本发明涉及一种显微镜成像设备，具体涉及一种便携式微型荧光显微镜。
背景技术
在神经科学领域，荧光显微镜为探索神经环路结构和功能提供了极大的帮助。但传统的荧光显微镜的总重量大，占空间多，造价高，这些特性在诸多方面也限制了科学探索的前进。例如在观察记录实验动物脑部细胞活动时，由于传统荧光显微镜的体型较大，所以不得不将实验动物麻醉或者束缚起来，以保证成像的稳定性。但对清醒的，自由活动的实验动物脑部细胞活动观察实验就很难完成。所以适合架设在自由活动的实验动物头部的便携式微型荧光显微镜成为了目前的迫切需要。虽然有些微型荧光显微镜已经研发出来，填补了这部分实验技术的空缺，但是因为其照搬传统大型荧光显微镜光路，而带来成像质量差，观察范围小，材料依赖性大等多种缺陷。
传统的荧光显微镜，光路被业界广泛使用于大型荧光显微镜的设计制造。工作原理是由激励源发出的光经过激发滤光片允许特定波长的激发光通过，再经过二向色镜反射，将激发光照射在样品上，受激发光照射的样品发出发射光，即荧光。波长较长的荧光可透过二向色镜，再经过荧光滤光片滤除杂光，允许特定波长的荧光照射在检测器上。同时，发射光经过物镜和投射透镜后，在检测器上会聚成放大的像。这种光路设计虽然简单易行，但是光学路径长，占用空间多，显然只适合制造大型仪器，并不适合微型便携式设备。现有的微型设备通常延用传统大型荧光显微镜的光路设计，仅在尺寸上等比降低。然而光路的设计决定了外形结构，同时也限制了该仪器的性能。首先，在采用这种光路的情况下，为了尽量减少体积，就必须采用焦距非常短的透镜作为物镜，才能在保证足够放大倍数的情况下缩短光学路径的长度。所以，在这种光路中，必须依赖自聚焦透镜作为物镜。然而自聚焦透镜的制作工艺复杂，密度梯度的均一性难以控制，这导致了该设备的观察范围小，成像分辨率低以及图像扭曲变形等缺陷。其次，虽然该设备的外形小巧，但是投射透镜后的成像调焦结构十分不便，如果要调焦，就必须松开螺丝，旋转CMOS传感器电路板底座。CMOS电路板上有线束从设备的顶部连接到设备的侧面，给LED电路板供 电。这样的设计导致在调焦时会牵扯供电线路，并且实验动物会抓挠侧面暴露的供电线路，可能会导致电路短路或断路而损坏设备。这不仅大大降低了设备的易用性，而且增加了使用的危险性。最后，该设备使用简单的恒流LED电路，为成像提供激发光。但是LED的发光效率会随着发热导致的温度升高而降低。激发光强度的改变直接影响最终成像亮度，导致成像条件不可控，每次实验条件不一致。
上述的主要缺陷严重影响了设备的使用，并降低了设备采集的实验数据的有效性与准确性，使其不具有说服力度。因此，本领域尚缺乏一种从实际出发，根据实验需要设计光路，研发制作的新设备，使其采集的数据准确有效。
发明内容
本发明的目的在于提供一种便携式微型荧光显微镜。该便携式微型荧光显微镜可以有效克服现有技术的不足，且根据目前科学发展趋势的需要，重新设计光路，使得该光路具有适应性，该微型荧光显微镜还具有体积小、重量轻；观察视野大；成像分辨率高，像差小的特点。
为达到上述目的，本发明提供一种便携式微型荧光显微镜，具体地，所述显微镜包括第一光学路径和第二光学路径；所述第一光学路径为从激励源至物镜，且所述激励源和所述物镜之间设置有激发元件，所述激发元件被配置和布置为将从所述激励源发出的激发光引导至所述物镜；所述第二光学路径为从所述物镜至图像采集装置，且所述物镜和所述图像采集装置之间设置有发射元件，所述发射元件被配置和布置为将由所述物镜接收到的荧光投射到所述图像采集装置；其中，所述物镜为非球面透镜；所述发射元件包括反射器，所述反射器设置在所述图像采集装置前，用于改变所述图像采集装置的光接收位置。
在另一优选例中，所述显微镜包括壳体，所述第一光学路径和所述第二光学路径内置于所述壳体中。
在另一优选例中，所述物镜设置在所述二向色镜与所述被观察样品之间。
在另一优选例中，所述激励源、任选的会聚透镜、激发滤光片、二向色镜和所述物镜竖向排列。
在另一优选例中，所述第一光学路径的光轴垂直于被观察的样品。
在另一优选例中，所述激发元件包括激发滤光片和二向色镜。
在另一优选例中，所述激发元件还包括会聚透镜。
在另一优选例中，所述会聚透镜为球透镜。
在另一优选例中，所述会聚透镜设置在所述激励源和所述激发滤光片之间。
在另一优选例中，所述激发元件的数量为多个。
在另一优选例中，所述二向色镜为通短波反射长波的滤光片。
在另一优选例中，所述发射元件包括所述二向色镜，投射透镜和所述反射器。
在另一优选例中，所述发射元件的数量为多个。
在另一优选例中，所述激励源发出的激发光首先通过激发滤光片，进行第一次滤波，再通过二向色镜，进行第二次滤波，透过所述物镜，然后照射在被观察的样品上。
在另一优选例中，从样品激发出波长较长的荧光，所述荧光通过二向色镜进行第三次过滤并反射所述荧光通过所述投射透镜、所述反射器最后到达所述图像采集装置。
在另一优选例中，经所述发射滤光片过滤后的所述荧光，通过所述投射透镜后会聚。
在另一优选例中，所述发射元件还包括发射滤光片，所述荧光通过所述发射滤光片进行第四次过滤。在另一优选例中，所述发射滤光片位于所述图像采集装置之前、所述二向色镜之后的所述第二光路上的任意位置。
在另一优选例中，所述反射器表面涂有镀膜，所述镀膜用于过滤所述荧光。
在另一优选例中，所述镀膜覆盖所述反射器的表面。
在另一优选例中，所述激发滤光片与所述发射滤光片成90度垂直。
在另一优选例中，所述二向色镜与所述第一光学路径的光轴所成锐角为30-60度。
在另一优选例中，所述二向色镜平分所述激发滤光片与所述荧光滤光片所成的直角，即所述二向色镜与所述第一光学路径的光轴成45度角。
在另一优选例中，将稳定的所述激发光引导到包括目标对象的视场中的至少3mm 2的区域。
在另一优选例中，由所述激发光引起的荧光发射引导向所述图像采集装置，为视场的图像提供至少1μm的分辨率。
在另一优选例中，所述反射器为棱镜；优选地，为五棱镜。
在另一优选例中，所述反射器为平面镜。
在另一优选例中，所述微型荧光显微镜包括光强检测器、反馈电路和驱动电路，其中，所述光强检测器用于检测所述激励源的光强并传递至所述反馈电路，所述反馈电路发送信号至所述驱动电路保证所述激励源的稳定。
在另一优选例中，所述激励源、所述光强检测器、所述反馈电路和所述驱动电路设置在照明电路板上。
传统显微镜的激发光通常随照射时间而不断变化，使得成像时具有不稳定的参数。与之相比，本发明采用稳定激励源，保证光强稳定不变，通过检测-反馈-补偿使得激励源稳定，避免了激发光进行性衰减造成的信号持续衰减现象。
在另一优选例中，所述激励源和所述图像采集装置设置在外壳顶部，所述物镜设置在壳体底端。将激励源和图像采集装置设置在外壳顶部，使得电路集中在外壳上部布置，从而使动物不易抓挠和破坏关键部件。
在另一优选例中，所述激励源为发光二极管。
在另一优选例中，所述发光二极管为单色发光二极管。
在另一优选例中，所述激励源为激光二极管。
在另一优选例中，所述激励源为闪光式照明。
在另一优选例中，所述激励源为恒定光强输出。
在另一优选例中，所述激发滤光片配置为带通滤光片。
所述二向色镜在第一光学路径中的作用是仅允许满足一定波长条件的光通过，并不改变光的传播方向；所述二向色镜在第二光学路径中的作用是按光谱特性分拣出激发产生的荧光并改变它的光传播方向，同时滤除样品直接反射出的短波长激发光。
在另一优选例中，所述发射滤光片配置为带通滤波器。
在另一优选例中，所述激发滤光片，所述二向色镜和所述发射滤光片统称为荧光模块。
在另一优选例中，所述荧光模块是可更换的。
在另一优选例中，所述激励源的照明方式为柯勒照明。
在另一优选例中，所述图像采集装置包括感光元件。
在另一优选例中，所述图像采集装置的感光元件为CMOS图像传感器。
在另一优选例中，所述图像采集装置通过有线和/或无线的方式与电脑通信连 接。
在另一优选例中，本发明的显微镜的光路可应用于普通显微镜的明场光路，其中，将激发滤光片替换为水平偏振片，将二向色镜替换为垂直偏振片，将发射滤光片替换为半透半反镜即可。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本发明的有益效果是本发明的便携式微型荧光显微镜适合使用在科学研究和医疗领域，新的光路保证了高质量的成像效果同时也降低了组装难度和成本，合理的结构设计保证了使用的稳定性，更方便于架设在实验动物身体上，真正意义上解决了目前神经科学领域的技术难题，大力推动了中国脑计划和世界神经科学的发展。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图做简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明的一个实施例中的便携式微型荧光显微镜的结构示意图。
图2为本发明的一个实施例中的便携式微型荧光显微镜的荧光模块的工作原理图。
图3为本发明的一个实施例中的第一光学路径的示意图。
图4为本发明的一个实施例中的第二光学路径的示意图。
图5为本发明的另一个实施例中的便携式微型荧光显微镜的结构示意图。
图6为本发明的一个实施例中的反射器为五棱镜的第二光学路径的示意图。
图7为本发明的一个实施例中的通过在五棱镜的接收面涂覆镀膜来代替发射滤光片的第二光学路径的示意图。
各附图中。各标示如下：
1-激励源；
2-会聚透镜；
3-激发滤光片；
4-二向色镜；
5-视场；
6-样品；
7-物镜；
8-发射滤光片；
9-投射透镜；
10-反射器；
11-荧光；
12-图像采集装置；
13-激发光；
14-镀膜。
具体实施方式
本发明人经过广泛而深入的研究，通过大量研究，首次开发了一种便携式微型荧光显微镜，与现有技术相比，该微型荧光显微镜使用光学成像能力出色的非球面镜作为物镜，扩大了视场范围，同时也提升了分辨率，极大的优化了微型显微镜的成像能力；此外，该微型荧光显微镜使用不同的光学元件形成新的光路，解决了用非球面镜作为物镜而导致的光学路径变长的问题，同时又平衡了设备的质量分布，保证设备的稳定使用，在此基础上完成了本发明。
该显微镜包括尺寸较小的便携式外壳。该外壳内设置有构成第一光学路径和第二光学路径的数个元件，包括配置为提供激发光的激励源、物镜和图像采集装置等。第一光学路径包括两个端点，一个端点为激励源，另一端点为物镜，并且在激励源和物镜之间设置有被配置和布置为将激发光引导至物镜的激发元件。第二光学路径包括两个端点，一个端点为物镜，另一端点为图像采集装置，并且在物镜和图像采集装置之间设置有发射元件，该发射元件被配置和布置为由物镜接收到的荧光投射 到图像采集装置。其中，激发元件包括激发滤光片、二向色镜和会聚透镜；发射元件包括二向色镜、发射滤光片、投射透镜和反射器。
物镜、会聚透镜、荧光模块、投射透镜、反射器和图像采集装置整体称为光学装置。荧光模块置于物镜和激励源之间，包括激发滤光片、二向色镜和发射滤光片。
本发明的总体光路与传统的显微镜光路不同。首先，照明光路为顶照试，激发光从上往下直达样品，返回的发射光经过的成像光路不与照明光路完全重合，这一点需要“通短波反射长波”的二向色镜的功能实现。此外，在成像光路中，由于反射器的加入，改变了射出投射透镜的光路走向，极大地缩减了第二光学路径在水平方向上的空间占用体积，充分利用了竖直方向上的空间。同时也将本身质量较大的图像采集装置模块，从整个装置的侧面移到装置的顶部，平衡了整个装置的质量分布，提升了设备的稳定性，便于实际使用。本发明的光路创新，主要是为了解决使用非球面镜作为物镜，而导致光学路径增长，镜体占用空间变大，重量分布不均匀的不良后果。本发明的光路设计是从实际出发，满足了使用需要。
此外，传统的荧光显微镜在小型化时通常采用自聚焦透镜，其主要原因是用来解决小型化之后光学路径长度不足的困难。但是自聚焦透镜具有较多缺点，例如成像分辨率小、像差较大的问题，同时自聚焦透镜成本较高，限制了微型显微镜的普及，与之相比，本实施例采用非球面透镜作为物镜，具有较好的光学性能，同时为了保证足够的光学路径长度，采用反射器将直线光学路径变为折线光学路径，减少了光路的空间，利于设备的微型化。
本发明的设计思路可以被理解为：更换物镜类型，从而获得更大放大倍数，更广的视野观察范围和更高的成像分辨率；重新设计光路，为了弥补因更换物镜而导致的光学路径增长，镜体占用空间变大，重量分布不均匀，这些不良后果；根据光路修改外形设计，在保证成像质量的同时也增强使用的便利性和整个成像系统的稳定性。
放大倍数可以表述为：放大倍数＝投射透镜的焦距/物镜的焦距。物镜的焦距由非球面镜限制，不易缩短，但投射透镜的焦距很容易做长。投射透镜的焦距变长导致的第二光学路径变长的问题已经由本发明的光路设计优化解决，所以，在特定的实施例中，允许增大投射透镜的焦距来获得更大的放大倍数。另外，在某些实施例中，选用高像素密集度的图像采集装置，更大的放大倍数可以提升分辨率。
术语
在本申请中，术语“发射光”和术语“荧光”为同一概念，可互换使用。
以下将结合附图对本发明的较佳实施例进行详细说明，以便更清楚理解本发明的目的、特点和优点。应理解的是，附图所示的实施例并不是对本发明范围的限制，而只是为了说明本发明技术方案的实质精神。此外，附图为示意图，因此本发明装置和设备的并不受所述示意图的尺寸或比例限制。
需要说明的是，在本专利的权利要求和说明书中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
实施例1
如图1和2所示，本实施例的显微镜包括激励源1，激励源1产生激发光13，激发光13由光学装置引导至目标样本对象，用于对目标样本对象成像。激励源1、会聚透镜2、荧光模块和物镜7构成第一光学路径；物镜7、荧光模块、投射透镜9和反射器10、图像采集装置12构成第二光学路径。物镜7为第一光学路径和第二光学路径的中间媒介。其中，物镜7配置在二向色镜4与被观察样品6之间。
如图3所示，第一光学路径为照明光路，主要是为了将激发光13引导至被观察样品6上，使激发光13的光强均匀一致。
激励源1、会聚透镜2、激发滤光片3、二向色镜4和物镜7从上到下依次竖向排列，也就是说来自激励源1的激发光13依次通过激发滤光片3和二向色镜4后投射到物镜7上形成第一光学路径。第一光学路径的光轴为一竖向直线，且第一光 学路径的光轴垂直于被观察的样品6。
本发明中的二向色镜4为通短波反射长波的滤光片。二向色镜4与第一光学路径的光轴所成锐角为30-60度。优选地，激发滤光片3与发射滤光片8成90度垂直。二向色镜4平分激发滤光片3与发射滤光片8所成的直角，即二向色镜4与第一光学路径的光轴成45度角。
现有技术中的二向色滤光镜对由激励源产生的激发光是反射，因此其激励源是侧置的。但是本发明的由激励源1产生的激发光13经过激发滤光片3后，二向色滤光镜对激励源1是折射，这样的设计，使得激励源1位于顶部，第一光学路径整体成一条直线。
在一个实施例中，该显微镜可用于观察人工合成的荧光物质或生物体表达的荧光蛋白，例如绿色荧光蛋白(GFP)。用合适的激发光13照射荧光样品6，激发出的发射光，即荧光11，被图像采集装置12捕捉到，完成图像采集。
在该实施例中的激励源1耦合至显微镜顶部的照明电路板。激励源1可为，但不限于固态发光二极管(LED)。
对于绿色荧光蛋白的观察，在一个实施例中，可将激励源1配置为发射光谱为465nm-485nm范围的蓝色发光二极管，保证低功率不发生光漂白的情况下，有效的激发绿色荧光蛋白上的发色基团。为了减少大功率激发光13照射荧光物质或生物组织而产生的损伤，本实施例采用窄光谱发光二极管作为激励源1，提高对荧光基团的激发效率，将激励源1的功率设定在1-5mW范围内。如此，样品6的光损伤和光热效应都会明显降低，减少光毒性对实验的影响。
激励源1可被设置为闪光式照明。因为多数研究表明，对荧光物质，如绿色荧光蛋白，用大功率激发光13长时间照射，会发生光漂白现象，导致荧光物质失去发荧光能力。闪光式照明，将对样品6的连续长时间曝光转变为间断式曝光，有效防止光漂白的发生，并且降低光毒性。激励源1闪光式照明的发光频率和相位与图像采集的采样率和相位相互耦合，实现每一帧图像的采集，样品6都会被有效的激发。
激励源1采用恒定光强输出。为此，激励源1所在电路板上还设置有光强探测器，实时监测LED的光强。当LED随着温度的升高发光效率降低时，光强探测器会捕捉到这一信息并传递给反馈电路，加大驱动电流从而保证LED光强输出恒定不变，随着功率的不断变大，LED的产热和散热会达到一个动态平衡的状态，此时功率不 会再上升，光强输出始终保持恒定。采用恒定光强输出的激励源1提供激发光13，可以保证激发光13的强度不变，从而确保在长时间成像过程中信号采集条件的一致性，避免了激发光13进行性衰减造成的信号持续衰减的假象。不需要后期对实验数据做误差较大的光衰校正处理。
在激励源1和激发滤光片3之间配置会聚透镜2，可以选取球透镜作为会聚透镜2，例如可选取K9材料制成的，具有1.5163折射率，直径2.5-4.0mm的球透镜，以减小装置的体积和重量。
为了对绿色荧光蛋白进行成像观察，荧光模块做如下配置：激发滤光片3470nm，二向色镜4500nm，发射滤光片8525nm。激励源1发出的激发光13首先通过激发滤光片3，进行第一次滤波。激发滤光片3配置为带通滤光片，将波长在470±10nm以外的杂光滤除。过滤后的激发光13射向二向色镜4的前表面，此时二向色透镜仅允许波长在325-480nm范围内的光通过。透过二向色镜4的激发光13照射在样品6上，激发出波长较长的发射光，即荧光11。发射光向上射向二向色镜4的后表面，此时二向色镜4将反射波长在520-610nm范围内的发射光反射。二向色镜4的作用是按光谱特性分拣出激发产生的荧光11并改变它的光传播方向，同时滤除样品6直接反射出的短波长激发光13。发射滤光片8配置为带通滤波器，仅允许波长在525±15mm以内的光通过，对发射光再次过滤。过滤后的发射光经过反射器10抵达图像采集装置12，完成荧光成像。
本实施例中的荧光模块可根据实际的需要进行更换，如观察红色荧光蛋白(RPF)，将荧光模块和配套激励源1换成对应于更长波长性质的元件组即可。
其中，反射器10的作用是改变图像采集装置12的光接收位置。主要优点是可以在在光学路径较长时，减少水平方向上的空间占用，平衡本发明整个设备的质量分布。根据实际的需要，可选择改变反射器10的朝向来改变图像采集装置12的位置。在本实施例中，反射器10为平面镜。
本发明的照明方式采用柯勒照明，目的是保证激发光13照射在被观察样品6上的均一性。
根据会聚透镜2和物镜7的大小和焦距，设置好相互间的距离位置，完成第一光学路径。激励源1发出的光经过会聚透镜2后，在会聚透镜2下方会聚成像，该像的位置刚好落在物镜7的上表面的焦点上。在满足上述位置条件的情况下，会聚成像的光继续向下传播，穿过物镜7，将形成多个方向的平行光，均匀的照射在被 观察的样品6上，实现柯勒照明。柯勒照明中的视场5光阑，孔径光阑等孔径参数由设备的内壁大小限定，除非特殊需要，一般采用最大通光孔径。
本发明通过柯勒照明实现激励源1发出的激发光13对样品6产生平行光束的照射。该效果由LED激励源1，会聚透镜2和物镜7三者共同实现。实现柯勒照明的关键点在于：激励源1的实像刚好落在物镜7的上表面的焦点上。视场5光阑，孔径光阑由显微镜的内壁限定，不可随意调整。
第二光学路径为成像光路，用于将发射光引导至图像采集装置12，同时放大被观察样本的像，达到预计的分辨率，能够清晰辨识被观察样品6的细节信息。
如图4所示，在发射滤光片8和图像采集装置12之间配置投射透镜和反射器10。
在本发明的实施例中，选用非球面镜作为物镜7。在被观察样品6上，位于物镜7下表面焦平面的发光点发出发射光，即荧光11，进入物镜7，穿出物镜7后孔，以平行光射出。射出的平行光经过二向色镜4的分拣且反射改变光路走向，再经过发射滤光片8的过滤后，到达投射透镜9。平行光穿过投射透镜9后，射出的光线在图像采集装置12(此处配置为感光元件)上会聚于一点，形成清晰的像。反射器10的作用是改变从投射透镜9射出的光线走向，合理利用空间结构。
本发明的物镜7采用非球面透镜而不是自聚焦透镜。在现有技术中，自聚焦透镜被用来作为物镜7，但是自聚焦透镜具有像差严重、视场5狭小的缺点，导致成像扭曲变形，且视野范围较小。由于制造工艺的限制，自聚焦透镜的直径不可任意增大，直径大于3mm的自聚焦透镜，基本不可生产。并且，自聚焦透镜的密度梯度，难以控制，成品透镜仅透镜中央部分，约直径一半的范围内可以成像。这限制了微型荧光显微镜的视场范围。同时，由于密度梯度延径向的非线性变化，导致成像有像差，且越往透镜边缘，像差越严重，又进一步限制了视场5大小，也导致分辨率差。有些设备选用自聚焦透镜作为物镜7，主要是因为其焦距较短(小于2mm)，便于缩短光学路径长度。但是视场5范围小，像差严重等缺点是不可避免的。
本发明采用的非球面镜，制造工艺成熟，可根据需要，设计并生产出不同规格的非球面镜，以应对不同的实际成像情况。因此可选用直径较大的非球面镜作为物镜7，来扩大视场5范围，在一些实施例中，可选用直径3mm或5mm的非球面镜， 视场5范围比自聚焦透镜大10倍以上。非球面镜的像差优于普通球面镜，更远胜于自聚焦透镜。在一个实施例中，0.5NA的非球面镜可提供1μm的成像分辨率，比自聚焦透镜提升2.5倍。因此，不管从视场5范围还是成像分辨率的角度考量，使用非球面镜作为物镜7的成像效果远优于自聚焦透镜。对于非球面镜的焦距比自聚焦透镜长的问题，本发明中主要用新设计的光路予以解决。由于非球面镜是利用透镜的折射率和表面弧度来达到屈光效果的，所以表面弧度的存在，不允许较大直径(大于3mm)的透镜拥有较短的焦距(小于1.5mm)。因此，非球面镜与自聚焦透镜相比，作为物镜7时，焦距会相对比较长。物镜7的焦距变长，带来的影响是，光学路径随之变长，尤其是第二光学路径的长度会明显增加。其原因是，为了充分利用物镜7的数值孔径和感光元件的像素密集度，必须保证成像的有足够的放大倍数，才能达到预期的成像分辨率。实施例中的放大倍数可简单计算为投射透镜9与物镜7的焦距之比。当使用非球面镜做作为物镜7时，焦距变长，而放大倍数不降低，必须增加投射透镜9的焦距，即从投射透镜9射出的光汇聚到图像采集装置12的距离增加。并且投射透镜9的焦距会随着物镜7的焦距变长而成倍的增加，所以第二光学路径的长度明显增加。传统设计中，光学路径的增加会增大整个设备的占用空间，不便于小型化。然而，新设计的光路极大的缩减了第二光学路径在水平方向上的空间占用体积，充分利用了竖直方向上的空间，解决了上述问题。
本实施例中的图像采集装置12的感光元件可为，但不限于CMOS图像传感器，发射光经反射器10反射后聚焦在CMOS传感器上，其图像由CMOS传感器采集后传输至电脑。根据需要，适用多种方式传输数据，包括使用可靠数据传输链路传送数据。还包括用电视信号，调频信号，调幅信号，调相信号的方式无线传输图像。
本实施例中图像由CMOS传感器传输至电脑可包括有线和无线两种版本，以满足不同的实验需求。当观察对象的行为活动是在二维地面，或比较简单时(如鼠，兔子，鸟)，使用有线版本即可。其优点是数据传输速度快，其数据通过率至少为400Mb/s，可连续长时间采集数据，实现VGA画幅至少60帧每秒的采样率。当观察对象的行为活动是在三维空间，或比较复杂时(如猴，猫，狗等)，则可使用无线版本，优点是观察对象可在大空间内自由活动。观察对象携带小型电池给仪器供电，可连续采集数据的时间根据观察对象背负电池的大小而定，基本可满足使用需求。无线数据传输率至少300Mb/s，至少VGA画幅30帧/秒的采样率。使用可靠传输的数据链路层，确保数据传输过程中的可靠性，避免因电磁干扰导致图像受损。
此外，在本实施例中第一光学路径的激励源1，以及第二光学路径的图像采集装置12均位于外壳的顶部，而物镜7则位于外壳的底部。因此对于大部分线路，特别是激励源1的线路部件均设置在动物无法接触的顶部。
由于本发明中第二光学路径设计在侧面，而且在第二光学路径的投射透镜9后设置有反射器10，因此，透镜调焦方式不再是旋转设备外壳零件，而是直接从外部调整投射透镜9的水平位置，改变投射透镜9到图像采集装置12之间的间距，完成调焦，更方便，不会牵扯外部线路。
本实施例的便携式微型荧光显微镜的体积很小，重量很轻，大小甚至可以小到8cm 3，重量可以轻到3.5g左右，且成像效果好，分辨率高，这种仪器可以用于脑科学的研究，可以架设在实验动物的头上，在清醒动物自由活动时，通过荧光成像观察动物脑中被标记的神经组织或其它类型细胞的活动；又可以直接观察血管血流的变化情况，所以可以直接用于检测医学临床患者的浅表层血管血流动态变化。
实施例2
本实施例中的显微镜与实施例1中的显微镜大致相同，与之不同的是，本实施例将实施例1中的激发滤光片3替换为水平偏振片，将二向色镜4替换为垂直偏振片，将发射滤光片8替换为半透半反镜。通过上述元件的替换，使得本实施例的显微镜可实现清晰的明场成像。依此原理，可以改装普通显微镜的明场光路。
实施例3
本实施例中的显微镜与实施例1中的显微镜大致相同，与之不同的是，本实施例将发射滤光片8的位置移至图像采集装置12之前、反射器10之后，且水平放置，如图5所示，这样的设置可以让投射透镜9的水平移动空间变大，更便于调焦。
需要说明的是，对本发明的发射滤光片8的位置没有特别的限制，发射滤光片8的作用只是滤波，其可放在图像采集装置12之前、二向色镜4之后的第二光路上的任意位置。
实施例4
本实施例中的显微镜与实施例1中的显微镜大致相同，与之不同的是，本实施例中的反射器10的内表面涂有一层镀膜，该镀膜可以起到过滤荧光的作用，其效果等同于发射滤光片8，因此，在本实施例中可以移除发射滤光8，以精简元件。对镀膜的层数没有特别的限制，可以是一层、两层或者多层。
本实施例中的镀有镀膜的平面镜可以同时起到反射器和发射滤光片的作用，可以省去单独添加的发射滤光片，节省空间和减轻重量。
实施例5
本实施例中的显微镜与实施例1中的显微镜大致相同，与之不同的是，本实施例中的反射器10为棱镜，例如五棱镜(如图6所示)，使用棱镜作为反射器可以拥有更长的光学路径，以及更高的荧光反射效率。五棱镜的形状可以根据二向色镜的角度而改变。
实施例6
本实施例中的显微镜与实施例5中的显微镜大致相同，与之不同的是，本实施例在棱镜表面镀有镀膜，具体地，在五棱镜的光接收面上镀有一层镀膜14(如图7所示)，该镀膜14可以起到过滤荧光的作用，其效果等同于发射滤光片8，因此，在本实施例中可以移除发射滤光8，以精简元件。镀膜14的层数也没有特别的限制，可以是一层、两层或者多层。
本实施例中的镀有镀膜的棱镜可以同时起到反射器和发射滤光片的作用，可以省去单独添加的发射滤光片，节省空间和减轻重量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种便携式微型荧光显微镜，其特征在于，所述显微镜包括第一光学路径和第二光学路径；
    所述第一光学路径为从激励源至物镜，且所述激励源和所述物镜之间设置有激发元件，所述激发元件被配置和布置为将从所述激励源发出的激发光引导至所述物镜；
    所述第二光学路径为从所述物镜至图像采集装置，且所述物镜和所述图像采集装置之间设置有发射元件，所述发射元件被配置和布置为将由所述物镜接收到的荧光投射到所述图像采集装置；
    其中，所述物镜为非球面透镜；
    所述发射元件包括反射器，所述反射器设置在所述图像采集装置前，用于改变所述图像采集装置的光接收位置。
  2. 根据权利要求1所述的便携式微型荧光显微镜，其特征在于，所述激发元件包括激发滤光片和二向色镜；所述发射元件包括所述二向色镜和投射透镜。
  3. 根据权利要求2所述的便携式微型荧光显微镜，其特征在于，所述激励源发出的激发光首先通过所述激发滤光片，进行第一次滤波，再通过所述二向色镜，进行第二次滤波，透过所述物镜，然后照射在被观察的样品上。
  4. 根据权利要求2所述的便携式微型荧光显微镜，其特征在于，从样品激发出荧光，所述荧光通过所述二向色镜进行第三次过滤并反射所述荧光通过所述投射透镜、所述反射器最后到达所述图像采集装置。
  5. 根据权利要求4所述的便携式微型荧光显微镜，其特征在于，所述发射元件还包括发射滤光片，所述荧光通过所述发射滤光片进行第四次过滤。
  6. 根据权利要求1所述的便携式微型荧光显微镜，其特征在于，所述反射器表面涂有镀膜，所述镀膜用于过滤所述荧光。
  7. 根据权利要求1所述的便携式微型荧光显微镜，其特征在于，将稳定的所述激发光引导到包括目标对象的视场中的至少3mm 2的区域。
  8. 根据权利要求1所述的便携式微型荧光显微镜，其特征在于，由所述激发光引起的荧光发射引导向所述图像采集装置，为视场的图像提供至少1μm的分辨率。
  9. 根据权利要求1所述的便携式微型荧光显微镜，其特征在于，所述反射器为平面镜或五棱镜。
  10. 根据权利要求1所述的便携式微型荧光显微镜，其特征在于，所述微型荧光显微镜包括光强检测器、反馈电路和驱动电路，其中，所述光强检测器用于检测所述激励源的光强并传递至所述反馈电路，所述反馈电路发送信号至所述驱动电路保证所述激励源的稳定。
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