CN108387519A - Microscopic system is just set in up-conversion luminescence near-infrared and the multi-functional wide field of colour imaging - Google Patents

Abstract

The invention discloses a kind of up-conversion luminescence near-infrareds and the multi-functional wide field of colour imaging just to set microscopic system, the microscopic system integrally disposed successively from the bottom to top in fluorescence microscope mirror body has the penetrating object lens of near-infrared just setting, exciter filter turntable, dichroscope, emit filter wheel, second adjustable speculum and high sensitivity detector, it is provided with color camera just setting the side in fluorescence microscope mirror body positioned at the second adjustable speculum, the side for being located at installation dichroscope position in the outside for just setting fluorescence microscope mirror body is sequentially installed with the first adjustable speculum, collimator and extender mirror and laser, white light source is set to the side of the first adjustable speculum.This system is excited by near infrared light, emitted using the visible light or near infrared light of highly-sensitive detector detection probe, the colour imaging of sample, the final material for realizing probe, cell or real-time, non-intrusion type the highly sensitive imaging of biological tissue can be realized by switching simultaneously.

Description

Versatile widefield upright microscope system for upconversion luminescence near-infrared and color imaging

technical field

The invention belongs to the technical field of fluorescence imaging, and in particular relates to an up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system.

Background technique

Optical microscope is a kind of microscope that uses optical lens to produce image magnification effect. Upright microscope is a kind of optical microscope. Under the observation of transmitted light, the light source reaches the sample from the bottom of the fuselage through the condenser lens, and then passes through the objective lens above the sample. Then it reaches the observer's eyes or other imaging instruments through mirrors and lenses. The advantage of upright microscopes is that they are simple in structure and easy to modify. Fluorescence microscopy refers to techniques that use fluorescence to produce an image. The sample is illuminated with light of a specific wavelength, or band, which is absorbed by the fluorophores, causing them to emit light of a longer wavelength, eg, a different color than the absorbed light. By using a spectral emission filter, this illumination light is separated from the much weaker emitted fluorescence. In recent years, in biological research, fluorescent labels have been widely used to calibrate biomolecules, making fluorescence microscopy more important. Optical Instruments. Fluorescence microscopy imaging system is widely used in cell biology research and is one of the necessary tools for cell level research.

Near-infrared fluorescence imaging is a kind of optical imaging. The wavelength range of near-infrared light source is 700-900 nm, which is the first non-visible light region discovered by people. There are many problems in the imaging of 400-700 nm in the visible light region, such as being affected by biological The effects of absorption and scattering of endogenous substances in tissues, such as aerobic and anoxygenated hemoglobin, melanin, water and bilirubin, etc., on optical imaging. Then in the near-infrared region, the absorption, scattering and autofluorescence background of the tissue are very low, and the near-infrared light source can reach the maximum penetration depth in the biological tissue, and can perform deep tissue imaging, so this band range is called "near-infrared tissue". Transparent window". Up-conversion fluorescence imaging is based on up-conversion fluorescent probes, which are different from other fluorescent probes. They can emit short-wavelength fluorescence under the excitation of long-wavelength external light sources. This unique luminescent property makes up-conversion fluorescent probes perform fluorescence During imaging, the autofluorescence interference of biological tissues can be largely avoided, and the photostability of the upconversion fluorescent probe is also high, which is not prone to fluorescence quenching and other problems, and is suitable for long-term fluorescence imaging observation of organisms.

Fluorescence microscopy system has relatively low cost, fast imaging speed, and high sensitivity. Both near-infrared light and up-conversion luminescence have the advantages of good imaging depth and low noise. The micro-imaging microscope system is very necessary. At the same time, due to the need for co-localization with pathological detection such as HE staining, simple switching to realize in-situ color imaging, near-infrared luminescence and up-conversion luminescence imaging is of great significance for cell-level research.

Contents of the invention

In order to overcome the deficiencies in the prior art, the present invention provides an up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system.

The present invention is achieved through the following technical solutions: an up-conversion luminescent near-infrared and color imaging multi-functional wide-field upright microscope system, the multi-functional wide-field upright microscope system is mainly composed of an upright fluorescence microscope body, a laser , collimating beam expander, white light source, first adjustable mirror, dichroic mirror, excitation filter turret, near-infrared transparent objective lens, emission filter turret, second adjustable mirror, high-sensitivity detection It is composed of a filter and a color camera; in the upright fluorescent microscope body, a near-infrared transparent objective lens, an excitation filter wheel, a dichroic mirror, an emission filter wheel, a second adjustable mirror and A high-sensitivity detector, a color camera is installed on the side of the second adjustable mirror in the body of the upright fluorescence microscope, and a first adjustable mirror is installed on the side of the body of the upright fluorescence microscope on the side where the dichroic mirror is installed. The reflector, the collimating beam expander and the laser are adjusted, and the white light source is arranged above the first adjustable reflector.

Excitation Filter Carousel contains Excitation Filters, Emission Filter Carousel contains Emission Filters, Excitation and Emission Filters are Short Pass, Band Pass or Long Pass piece.

The laser adopts a laser with a center wavelength of 980nm, 808nm or 730nm. The white light source adopts mercury lamp or LED lamp. The high-sensitivity detector adopts sCMOS detector or refrigerated EMCCD detector.

The function of each part in the upright microscope system of the present invention is:

1. The lens body of the upright fluorescence microscope is used as the connection and physical fixation of each component.

2. The laser is used as the excitation light source for near-infrared luminescence imaging and up-conversion luminescence imaging.

3. The collimating beam expander is used for the collimation and beam expansion of the laser.

4. The white light source is used as the emission light source of the excitation light.

5. The first adjustable reflector can switch the light source, such as laser light source and white light source.

6. The dichroic mirror can reflect the excitation light to the sample to excite it, and at the same time can transmit the emission light so that the emission light can be received by the detector.

7. The excitation filter turntable contains excitation filters, which can selectively transmit light through lasers, mercury lamps, and LED light sources as excitation light.

8. The near-infrared transparent objective lens is a lens group specially used for infrared light.

9. The emission filter turret contains emission filters, which can selectively transmit light through the target.

10. The second adjustable mirror can be used to freely switch between the high-sensitivity detector and the color camera.

11. A high-sensitivity detector is used to collect the fluorescence emitted by the sample after excitation.

12. The color camera is used for color imaging in the visible light band.

Upright Fluorescence Microscope: Nikon Ni-U is a scientific research grade upright microscope with strong expandability. It adopts an infinity optical system and can be easily equipped with other accessories. The system is equipped with double-layer fluorescence turntables, each layer of fluorescence turntable has six empty filter blocks, and the system is also equipped with dual camera ports and dual light source interfaces to meet the application of bioluminescent materials and up-conversion luminescent materials at the same time. For the main optical components, such as the objective lens, a flat-field fluorescent objective lens with a higher near-infrared transmittance, for example greater than 70%, is selected. In addition, under the premise of ensuring that the imaging is not affected, the optical components that block the infrared light in the optical path are removed, so as to maximize the pass rate of the entire optical system in the near-infrared light.

The laser adopts high power and large core diameter multimode fiber to achieve stronger excitation light intensity. The optical fiber adopts multimode optical fiber with a core diameter of 200um, and a collimating beam expander is added to achieve uniform field of view illumination. In addition to the precise filter combination and high-transmittance objective lens of the camera, in order to obtain a fluorescence image with a high signal-to-noise ratio, the system also needs to be equipped with a high-sensitivity detector, such as EMCCD or sCMOS.

Dichroic mirrors are different from ordinary fluorescent dichroic mirrors. The present invention uses a short-pass filter, that is, light shorter than the split wavelength passes through, while light longer than the split wavelength is reflected. After the reflected near-infrared light irradiates the sample, the fluorescence excited by the sample passes through the first blocking filter, allowing the light of 400nm to 850nm to pass through, and the light of other wavelengths is filtered out to avoid interference to the background signal. The OD value of the dichroic mirror and the first blocking filter is required to be at least greater than 6 to increase the signal-to-noise ratio. What the upright microscope system among the present invention adopts is the ZT1064rdc-sp dichroic mirror and ET850SP-2P of U.S. Chroma Company, and spectrogram is as shown in Figure 2, and wherein ZT1064rdc-sp is gray smooth line, and ET850SP-2P is Black jagged lines.

Excitation and emission filters: In order to further distinguish the emission light of different wavelengths, a second blocking filter is added to the system, the wavelengths are ET550/50M, ET630/75M and ET811/80M, and the spectral diagrams are shown in Figure 3 and Figure 4 and Figure 5, where the black line is the first blocking filter. In addition, since the conventional fluorescent microscope light source uses a mercury lamp, the heat is relatively high, so the light path usually has a heat shield. The heat shield will block near-infrared and infrared light, so this system also removes the relevant optical components in the fluorescence light path of the microscope. The excitation filter turret and emission filter turret can replace the filters arbitrarily, and the filters can also be switched freely during the imaging process.

The beneficial effects of the present invention are: the present invention provides an up-conversion luminescence near-infrared imaging and color microscopic imaging system, which can utilize the characteristics of near-infrared light, up-conversion luminescence, high signal-to-noise ratio, and low noise, and through near-infrared Optical excitation, using a high-sensitivity detector to detect the visible light or near-infrared light emission of the probe, and at the same time, the color imaging of the sample can be realized by switching, and finally realize the real-time, non-invasive high-sensitivity imaging of the material, cells or biological tissues of the probe . The microscopic system in the present invention can emit excitation light by performing real-time fluorescence with high signal-to-noise ratio on materials, cells, tissues, and living animals labeled with normal fluorescent probes, near-infrared fluorescent probes, and up-conversion luminescent probes. Microscopic imaging, while co-localization color imaging can be performed using a color camera without moving the sample and moving the lens. In the present invention, by rationally matching the spectral range of the filter, selecting a high-transmittance microscope objective lens and a high OD value filter, and equipped with a high-sensitivity detector, the up-conversion with a high signal-to-noise ratio can also be achieved under wide-field illumination. Fluorescence image.

The present invention is based on an upright microscope, by transforming the optical components of the system, adding high-sensitivity detectors for visible light and near-infrared light and a color imaging system, adding high-power lasers, and adding a high-efficiency filter system, and constructing a set that can carry out the above Upright microscope systems for conversion imaging, near-infrared imaging, and color imaging. Compared with the previous microscopic imaging system, the present invention has high sensitivity and high signal-to-noise ratio, and is an integrated microscopic imaging device capable of imaging visible light, near-infrared light and up-conversion luminescence.

Compared with the prior art, the imaging system in the present invention also has the following advantages: 1. For the first time, it realizes the integration of ordinary fluorescence imaging, near-infrared microscopic imaging technology, up-conversion in vivo fluorescent microscopic imaging technology and color imaging technology In one microscope system; 2. It can be freely switched to ordinary fluorescence imaging mode, near-infrared imaging mode, up-conversion luminescence imaging mode and color imaging mode during the imaging process, so as to achieve co-localization of different imaging results; 3. Near-infrared imaging mode Infrared microscopy and up-conversion intravital fluorescence microscopy have higher signal-to-noise ratios.

Description of drawings

Fig. 1 is the system optical path schematic diagram of upright microscope system of the present invention;

Fig. 2 is the spectrogram of dichroic mirror and the first blocking filter among the present invention;

Fig. 3 is ET550/50m optical filter spectrogram of the present invention;

Fig. 4 is ET630/75m optical filter spectrogram of the present invention;

Fig. 5 is ET811/80m optical filter spectrogram of the present invention;

Fig. 6 is the high signal-to-noise ratio up-converted fluorescence image obtained by adopting the upright microscope system of the present invention;

In the figure: 1-upright fluorescence microscope body; 2-laser; 3-collimating beam expander; 4-white light source; 5-first adjustable mirror; 6-dichroic mirror; 7-excitation filter 8-near-infrared transparent objective lens; 9-emission filter turret; 10-second adjustable mirror; 11-high sensitivity detector; 12-color camera.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

As shown in Figures 1 to 6, an up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system is mainly composed of an upright fluorescence microscope body 1, a laser 2. Collimating beam expander 3, white light source 4, first adjustable reflector 5, dichroic mirror 6, excitation filter turntable 7, near-infrared transparent objective lens 8, emission filter turntable 9, second An adjustable reflector 10, a high-sensitivity detector 11, and a color camera 12 are composed; a near-infrared transparent objective lens 8, an excitation filter turntable 7, a dichroic Mirror 6, emission filter turntable 9, second adjustable reflector 10 and high-sensitivity detector 11, in the side of the second adjustable reflector 10 in the upright fluorescent microscope mirror body 1, a color camera 12 is arranged, and The outside of the mirror body 1 of the upright fluorescence microscope is located on the side where the dichroic mirror 6 is installed, and the first adjustable reflector, the collimating beam expander 3 and the laser 2 are installed in sequence, and the white light source 4 is arranged on the first adjustable reflector 5 side ends.

The excitation filter turntable 7 contains an excitation filter, and the emission filter turntable 9 contains an emission filter, and the excitation filter and the emission filter adopt a short-pass filter, a band-pass filter or a long-pass filter. filter. The laser 2 adopts a laser 2 with a center wavelength of 980nm, 808nm or 730nm. The white light source 4 adopts a mercury lamp or an LED lamp. The high-sensitivity detector 11 adopts sCMOS detector or refrigerated EMCCD detector.

The system composition of the present invention includes upright fluorescent microscope body 1, color + monochrome imaging dual ports, white LED + laser 2 dual light source interfaces, double-layer fluorescent filter block turntable and a series of excitation and emission filters for realizing functions. . This system is based on an upright fluorescent microscope Nikon Ni-U. The light source is introduced by a 980nm continuous laser fiber. After being collimated and expanded, it is uniformly injected into the microscope optical path. After being split by 6 dichroic mirrors, it is reflected through the objective lens and irradiated. After reaching the sample, the fluorescence generated by the sample and the unabsorbed excitation light pass through the objective lens and return to the dichroic mirror for 6 beam splitting, and then use a high-sensitivity camera to receive the optical signal, which is digitized and processed by computer software. At the same time, a color camera 12 is installed on the second interface of the camera, which can realize high-sensitivity imaging and acquire color signals at the same time, as shown in FIG. 1 .

Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent replacements to the technical solution of the present invention by those skilled in the art will not depart from the present invention. The essence and scope of the technical solution of the invention.

Claims (5)

1. An up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system is characterized in that: the multifunctional wide-field upright microscope system is mainly composed of upright fluorescence microscope mirror body, laser, collimator Beam expander, white light source, first tunable mirror, dichroic mirror, excitation filter turret, NIR transparent objective, emission filter turret, second tunable mirror, high sensitivity detector and color The camera is composed of: the near-infrared transparent objective lens, the excitation filter turntable, the dichroic mirror, the emission filter turntable, and the second adjustable reflector are integrated in the lens body of the upright fluorescence microscope from bottom to top. and a high-sensitivity detector, the color camera is arranged on the side of the second adjustable mirror in the upright fluorescence microscope body, and the two-way camera is installed outside the upright fluorescence microscope body The side of the position of the color mirror is sequentially installed with the first adjustable reflector, collimator beam expander and laser, and the white light source is arranged above the first adjustable reflector. 2. The up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system according to claim 1, characterized in that: the excitation filter turntable contains an excitation filter, and the emission filter The turntable contains an emission filter, and the excitation filter and the emission filter are short-pass filters, band-pass filters or long-pass filters. 3. The up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system according to claim 1 or 2, characterized in that: the laser is a laser with a central wavelength of 980nm, 808nm or 730nm. 4. The up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system according to claim 1 or 2, characterized in that: the white light source is a mercury lamp or an LED lamp. 5. The up-conversion luminescent near-infrared and color imaging multifunctional wide-field upright microscope system according to claim 1 or 2, characterized in that: the high-sensitivity detector adopts sCMOS detector or cooling EMCCD detector.