CN110161671B - Dark field, bright field, phase contrast and fluorescence multimode synchronous imaging microscopic imaging device - Google Patents
Dark field, bright field, phase contrast and fluorescence multimode synchronous imaging microscopic imaging device Download PDFInfo
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Abstract
The invention discloses a dark field, bright field, phase contrast and fluorescence multi-mode synchronous imaging microscopic imaging device which is provided with a sample table, wherein a focus of an objective lens of the sample table is used for placing a sample, one side of the sample table is provided with a light beam emitting unit formed by arranging a plurality of light sources with different wavelengths, and the other side of the sample table is sequentially provided with a light beam processing unit, a light beam amplifying unit used for amplifying light beams so as to ensure that light spots are completely irradiated on the light beam filtering unit, a light beam filtering unit and a light beam receiving unit. The invention is suitable for biological observation and detection, is convenient to use, has low cost and good imaging effect.
Description
Technical Field
The invention relates to an imaging device, in particular to a dark field, bright field, phase contrast and fluorescence multimode synchronous imaging microscopic imaging device.
Background
Optical microscopy is a ubiquitous tool in different disciplines, providing detailed visualization of materials and biological specimens. Over the past few decades, the constant progress of microscopy has introduced many new imaging modalities. However, bright field, dark field and phase contrast microscopes still represent the most common and widely used non-staining imaging methods. Bright Field (BF) microscopes provide images by mapping the intensity modulation of light passing through a sample. Although it is the simplest and most common form of microscope, it is not suitable for viewing translucent samples, such as unlabeled cells and thin tissue samples, because these samples do not exhibit strong attenuation under visible light. Dark Field (DF) microscopy can generate high contrast images of thin objects, sensitive to sample edges. The DF microscope is illuminated with oblique light beyond the maximum angle that the optical imaging system can capture, thereby minimizing the unscattered background while collecting scattered light from the sample. Phase contrast microscopes, such as Zernike and Differential Interference Contrast (DIC) microscopes, provide images by presenting the optical phase retardation of light illuminating a sample as an intensity distribution. In the paper "Quantitative phase imaging using a partial detection imaging" (OPTIC LETTERS/Vol.37, No.19/October 1,2012), a non-interferometric technique is described for Quantitative phase contrast imaging, but only a single imaging mode can be achieved.
Although bright field, dark field and phase contrast images provide complementary information of the specimen, it is not feasible to acquire these images simultaneously in a conventional microscope because each modality requires a different optical arrangement and dedicated optical elements. In addition, the switching between imaging modes is accompanied by waste of time and additional optical elements, and is not energy-efficient. In the articles "Real-time imaging in a light-emitting diode array" (Journal of biological Optics 19(10),106002(October 2014)) and microscopical refocusing and dark-field imaging using a single LED array (October 15,2011/vol.36, No.20/Optics LEDs), a method for realizing multimode illumination imaging using an LED array has been proposed, but neither of them can solve the problem of synchronous imaging, and the light source needs to be frequently switched to obtain the desired effect.
In addition, the existing fluorescence detection microscope in the market has single function, and only can realize the fluorescence detection of specific substances.
Disclosure of Invention
The invention aims to solve the problems and provides a dark field, bright field, phase contrast and fluorescence multimode synchronous imaging microimaging device, which is suitable for biological observation and detection, is convenient to use and low in cost and has a good imaging effect.
In order to achieve the purpose, the invention adopts the following technical scheme: a dark field, bright field, phase contrast and fluorescence multi-mode synchronous imaging microscopic imaging device is provided with a sample table, wherein a sample is placed at the focus of an objective lens of the sample table, a light beam emitting unit formed by arranging a plurality of light sources with different wavelengths is arranged on one side of the sample table, and a light beam processing unit, a light beam amplifying unit used for amplifying light beams so as to ensure that light spots are completely irradiated on the light beam filtering unit, a light beam filtering unit and a light beam receiving unit are sequentially arranged on the other side of the sample table.
Further: the light beam emission unit is provided with a 405nm laser, a 488nm laser, a 532nm laser and a 638nm laser; the 405nm laser is incident to the sample stage at an incident angle of less than 48.6 degrees; the 488nm laser and the 405nm laser are symmetrical about an optical axis; the 532nm laser is incident to the sample stage at an incident angle of more than 48.6 degrees; the 638nm laser and the 532nm laser are symmetrical about an optical axis.
Further: the beam processing unit consists of an objective lens with the magnification of 20 times and the numerical aperture NA of 0.75.
Further: the magnifying power of the light beam magnifying unit is 1.67, and the light beam magnifying unit is composed of a first lens with the focal length of 30mm, a second lens with the focal length of 50mm and a diaphragm, wherein the first lens is arranged on one side, the second lens is arranged on the other side, and the diaphragm is arranged on an image space focal plane of the first lens.
Further: the light beam filtering unit is provided with two groups of lens arrays A, one group of filter plate arrays and one group of lens arrays B which are sequentially arranged; each group of lens array A consists of four sub-lenses A which are distributed in a shape of Chinese character 'tian' and are used for dividing light beams into 4 light beams which are correspondingly emitted to four windows of the filter array; the filter array consists of four sub-filters which are distributed in a shape of Chinese character 'tian' and used for filtering the received light corresponding to different wavelengths, so that each window only transmits the light with the corresponding wavelength and emits the light to the lens array B; the lens array B is composed of four sub-lenses B, is distributed in a shape of Chinese character 'tian', and is used for focusing four light beams on an imaging light beam receiving unit formed by an image sensor respectively.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention contains four sets of light sources with different wavelengths, and the four sets of light sources are grouped in pairs, and each group has different incident angles, so that synchronous imaging of dark field, bright field and phase contrast can be formed, and observation of different articles and synchronous observation of various modes can be realized.
(2) The invention adopts two fluorescence wave band light sources at the same time, and can realize the detection of the specific fluorescent substance.
(3) The lens array and the filter array divide light into four beams, project the four beams to four channels of the filter, and can realize subarea display through the filtering effect, and finally form images of four areas on the image sensor.
(4) The invention can solve the problems that the traditional microscope in the market has single function and cannot synchronously realize multi-mode imaging, has simple operation and easy realization, can fill the vacancy of the multifunctional microscope in the market, and has the advantages of easy operation and low cost.
Drawings
FIG. 1 is a block diagram of a dark field, bright field, phase contrast, fluorescence multi-mode simultaneous imaging micro-imaging device.
Fig. 2 is a schematic diagram of arrangement of the sub-lenses a in the lens array a.
Fig. 3 is a schematic diagram of an arrangement of four sub-filters in a filter array.
Detailed Description
Referring to fig. 1 to 3, the dark field, bright field, phase contrast, fluorescence multi-mode synchronous imaging microscopic imaging device comprises a sample stage 2, wherein a sample is placed at the focus of an objective lens of the sample stage 2, a light beam emitting unit 1 formed by arranging a plurality of light sources with different wavelengths is arranged at one side of the sample stage, and a light beam processing unit 3, a light beam amplifying unit 4 for amplifying light beams to ensure that light spots are completely irradiated on the light beam filtering unit 5, a light beam filtering unit 5 and a light beam receiving unit 6 are sequentially arranged at the other side of the sample stage 2.
The sample stage 2 is a platform capable of moving up and down to ensure that a light beam convergent point irradiates on a sample.
In this embodiment, the beam emitting unit is used for emitting light with different wavelengths at different angles, and comprises a 405nm laser 11, a 488nm laser 12, a 532nm laser 13 and a 638nm laser 14; the 405nm laser 11 is incident to the sample stage at an incident angle smaller than 48.6 degrees, and when the device is used, the device needs to be firstly adjusted to be collimated and ensure that light rays are incident to an objective lens; the 488nm laser 12 and the 405nm laser 11 are symmetrical about an optical axis, and the using method is the same as the above, and the two groups of lasers are used for realizing bright field and phase contrast imaging; the 532nm laser 13 is incident to the sample stage at an incident angle of more than 48.6 degrees, and when the 532nm laser is used, the 532nm laser needs to be firstly adjusted to be collimated, and light is ensured to pass through the sample but not to be incident to the objective lens; the 638nm laser 14 and the 532nm laser 13 are symmetrical about the optical axis, and the method of use is the same as above.
The light beam emitting unit has two modes of operation. The first mode is as follows: and (3) turning on a 405nm laser 11, a 488nm laser 12 and a 630nm laser for detecting a fluorescent substance EGFP (enhanced green fluorescent protein), wherein the excitation wavelength of the EGFP is about 480nm, and the emission wavelength is about 530 nm. And a second mode: turning on 405nm laser, 488nm laser and 530nm laser for detecting fluorescent substance PPIX (protoporphyrin), wherein the excitation wavelength of PPIX is about 405nm, and the emission wavelength is about 638 nm.
It should be noted that the light beam emitting unit 1 includes four sets of light sources with different wavelengths, and the emergent light of each set of light source is collimated light with a wavelength λ1、λ2、λ3、λ4. (corresponding to 630nm laser 13, 488nm laser 12, 405nm laser 11 and 530nm laser 14, respectively) wherein1、λ2、λ3Light sources of wavelength, λ2、λ3、λ4The light sources with one group of wavelengths are turned on only one group of light sources, i.e. lambda1Or λ4The light source of the wavelength is turned off. When starting lambda1、λ2、λ3Light source of wavelength, λ1Light source of wavelength for dark field imaging, lambda2、λ3Light sources of wavelength for bright field, phase contrast, fluorescence imaging, where λ2The light source with wavelength is used as fluorescence excitation light source, and the wavelength of fluorescence emission light is lambda4(ii) a When starting lambda2、λ3、λ4Light source of wavelength, λ4Light source of wavelength for dark field imaging, lambda2、λ3Light sources of wavelength for bright field, phase contrast, fluorescence imaging, where λ3The light source with wavelength is used as fluorescence excitation light source, and the wavelength of fluorescence emission light is lambda1. When the light source is arranged, lambda is ensured2、λ3When the light of the wavelength light source irradiates the sample, the angle formed by the light of the wavelength light source and the optical axis of the objective lens is +/-alpha, 0<α<θNA,θNAThe numerical aperture of the objective lens is in accordance with θNA＝sin-1NA, where θ NA is the numerical aperture angle and NA is the numerical aperture of the objective lens; and, λ is required to be ensured1、λ4When the light of the wavelength light source irradiates the sample, the angle beta, beta is formed with the optical axis of the objective lens>θNA。
In this embodiment, the beam processing unit 3 is composed of an objective lens with a magnification of 20 times and a numerical aperture NA of 0.75.
In this embodiment, the magnification of the light beam magnifying unit 4 is 1.67, and the light beam magnifying unit is composed of a first lens 41 with a focal length of 30mm, a second lens 42 with a focal length of 50mm, and a diaphragm, wherein the first lens 41 is disposed on one side, the second lens 42 is disposed on the other side, and the diaphragm 43 is disposed on the image focal plane of the first lens, that is, the object focal plane of the second lens. The beam amplifying unit 4 receives the emitted light (in the figure, the light (i) is a bright field or phase contrast light path, and the light (ii) is a dark field light path), and focuses the light at one point thereafter.
In this embodiment, the light beam filtering unit 5 has two sets of lens arrays a51, one set of filter arrays 52 and one set of lens arrays B53 arranged in sequence; each group of lens array A51 consists of four sub-lenses A which are distributed in a shape of Chinese character tian for dividing the light beam into 4 light beams which are correspondingly emitted to four windows of the filter array 52; the filter array 52 is composed of four sub-filters, distributed in a shape of a Chinese character tian, and used for filtering the received light corresponding to different wavelengths, so that each window only transmits the light of the corresponding wavelength and emits the light to the lens array B53; the lens array B53 is composed of four sub-lenses B distributed in a shape of a Chinese character 'tian' for focusing four light beams on the imaging beam receiving unit 6 formed by the image sensor, respectively.
In the filter plates distributed in a shape of Chinese character 'tian', each filter plate and the sub-lens units in the lens array form four windows in a one-to-one correspondence mode, and the wavelength acted by the four windows is lambda1、λ2、λ3、λ4Let the images finally focused on the four areas of the image sensor (light beam receiving unit 6) be λ respectively1、λ2、λ3、λ4The image of (2).
The light beam receiving unit 6 is used for receiving the finally formed image, and the four areas respectively correspond to images with the wavelengths of 532nm, 408nm, 488nm and 630 nm. In the first working mode, a first region is EGFP fluorescence imaging, a second region and a third region are bright field and phase contrast imaging, and a fourth region is dark field imaging; and in the second working mode, the first region is dark field imaging, the second and third regions are bright field and phase contrast imaging, and the fourth region is PPIX fluorescence imaging.
It is emphasized that the length or width of the image sensor is larger than the dimension of each side of the filter array, the target surface of the image sensor is divided into four regions, which are distributed in a shape of Chinese character 'tian', and one region is used for receiving lambda1Optical signal of wavelength, region two for receiving lambda2Optical signal of wavelength, region three-way for receiving lambda3Optical signal of wavelength, region four for receiving lambda4An optical signal of a wavelength. When lambda is1、λ2、λ3When the light source of the wavelength is turned on, the first region is used for dark field imaging, the second region and the third region are used for bright field and phase contrast imaging, and the fourth region is used for lambda2Lambda excited by fluorescent sample4Corresponding fluorescence imaging; when lambda is2、λ3、λ4When the light source of wavelength is turned on, the first region is used for lambda3Lambda excited by fluorescent sample1Corresponding fluorescence imaging, area two and area three for bright field and phaseAnd the lining imaging and the area four are used for dark field imaging. Establishing X-Y coordinates for the receiving surface of the image sensor, wherein X represents a horizontal axis coordinate, Y represents a vertical axis coordinate, and the light intensity distribution of the second area and the third area is Iλ2(x, y) and Iλ3(x, y) a light intensity distribution of bright field imaging of IBF(x, y) the intensity distribution of the phase contrast imaging is IDPC(x, y) wherein IBF(x, y) and IDPC(x, y) are respectively represented by formula IBF(x，y)＝Iλ2(x，y)+Iλ3(x，y)，
Thus obtaining the product. When lambda is1When the light source with the wavelength is used for dark field imaging, the light intensity distribution of the dark field imaging is IDF(x,y)＝Iλ1(x, y) a fluorescence imaging light intensity distribution of IFL＝Iλ4(x, y); when lambda is4When the light source with the wavelength is used for dark field imaging, the light intensity distribution of the dark field imaging is IDF(x,y)＝Iλ4(x, y) a fluorescence imaging light intensity distribution of IFL＝Iλ1(x,y)。
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment, and all technical solutions belonging to the principle of the present invention belong to the protection scope of the present invention. Modifications to the disclosed 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.
Claims (4)
1. A dark field, bright field, phase contrast and fluorescence multimode synchronous imaging microscopic imaging device is characterized by comprising a sample table (2), wherein a focus of an objective lens of the sample table (2) is used for placing a sample, one side of the sample table is provided with a light beam emitting unit (1) formed by arranging a plurality of light sources with different wavelengths, and the other side of the sample table (2) is sequentially provided with a light beam processing unit (3), a light beam amplifying unit (4) used for amplifying light beams so as to ensure that light spots are completely irradiated on the light beam filtering unit (5), a light beam filtering unit (5) and a light beam receiving unit (6);
the light beam emission unit (1) comprises four sets of light sources with different wavelengths, the wavelengths of the light sources in each set are lambda 1, lambda 2, lambda 3 and lambda 4 respectively, when the light of the light sources with the wavelengths of lambda 2 and lambda 3 irradiates a sample, the light source forms a symmetrical angle +/-alpha with the optical axis of the objective lens, 0< alpha < theta NA, the corresponding relation between the theta NA and the numerical aperture of the objective lens is theta NA = sin-1NA, wherein the theta NA is a numerical aperture angle, and the NA is the numerical aperture of the objective lens; when the light of the light source with the wavelength of lambda 1 and lambda 4 irradiates the sample, a certain angle beta is formed between the light source and the optical axis of the objective lens, wherein the angle beta is larger than theta NA;
the light beam filtering unit (5) is provided with two groups of lens arrays A (51), one group of filter plate arrays (52) and one group of lens arrays B (53) which are sequentially arranged; each group of lens array A (51) consists of four sub-lenses A which are distributed in a shape of Chinese character 'tian' and are used for dividing light beams into 4 light beams which are correspondingly emitted to four windows of the filter array (52); the filter array (52) consists of four sub-filters which are distributed in a shape of Chinese character 'tian', the wavelengths acted by the four sub-filters are lambda 1, lambda 2, lambda 3 and lambda 4, so that images finally focused on four areas of the image sensor are lambda 1, lambda 2, lambda 3 and lambda 4 respectively, the filter array is used for filtering received light corresponding to different wavelengths, and each window only transmits the light with the corresponding wavelength and emits the light to the lens array B (53); the lens array B (53) is composed of four sub-lenses B, is distributed in a shape of Chinese character 'tian', and is used for focusing four light beams on an imaging light beam receiving unit (6) formed by an image sensor;
wherein the light source with the wavelength of lambda 3 is used as a fluorescence excitation light source, the light source with the wavelength of lambda 1 or lambda 2 is used as a fluorescence excitation light source, and the wavelength of the fluorescence emission light is lambda 4.
2. The dark field, bright field, phase contrast, fluorescence multi-mode simultaneous imaging microscopy imaging device of claim 1, wherein: the beam emitting unit has a 405nm laser (11), a 488nm laser (12), a 532nm laser (13), a 638nm laser (14); the 405nm laser (11) is incident to the sample stage at an incidence angle of less than 48.6 degrees; the 488nm laser (12) and the 405nm laser (11) are symmetrical about an optical axis; the 532nm laser (13) is incident to the sample stage at an incident angle of more than 48.6 degrees; the 638nm laser (14) and the 532nm laser (13) are symmetrical about an optical axis.
3. The dark field, bright field, phase contrast, fluorescence multi-mode simultaneous imaging microscopy imaging device of claim 1, wherein: the light beam processing unit (3) is composed of an objective lens with the magnification of 20 times and the numerical aperture NA of 0.75.
4. The dark field, bright field, phase contrast, fluorescence multi-mode simultaneous imaging microscopy imaging device of claim 1, wherein: the magnifying power of the light beam magnifying unit (4) is 1.67, and the light beam magnifying unit is composed of a first lens (41) with the focal length of 30mm, a second lens (42) with the focal length of 50mm and a diaphragm, wherein the first lens (41) is arranged on one side, the second lens (42) is arranged on the other side, and the diaphragm (43) is arranged on the image space focal plane of the first lens.
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