CN108982433B - Optical layer cutting device adopting advanced optical interference microscopy - Google Patents
Optical layer cutting device adopting advanced optical interference microscopy Download PDFInfo
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- CN108982433B CN108982433B CN201710416404.2A CN201710416404A CN108982433B CN 108982433 B CN108982433 B CN 108982433B CN 201710416404 A CN201710416404 A CN 201710416404A CN 108982433 B CN108982433 B CN 108982433B
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
- G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
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Abstract
An optical sectioning device using advanced optical interference microscopy, comprising: a beam splitter capable of splitting an incident beam into a reflected beam and a transmitted beam; a broadband light source device for providing the incident light beam; the reference end unit is used for enabling the transmission light beam to return to the optical splitter through an adjustable optical path; a short wave light source device; a first dichroic beam splitter having a first side facing the short-wave light source device and a third side facing the beam splitter, and being capable of being opaque to light beams having a wavelength shorter than a predetermined wavelength; an objective lens having a parallel light side to face the second side of the first dichroic splitter; a sample carrying unit facing a condensing side of the objective lens; and a projection lens and a sensing unit for receiving an output beam of the beam splitter.
Description
Technical Field
The present invention relates to an optical layer cutting device, and more particularly, to an optical layer cutting device using advanced optical interference microscopy.
Background
When performing tumor resection, it is often necessary to wait for the physician to check with cryosection (cryosection) to determine whether the tumor has been completely removed, which is time consuming and may not be able to completely confirm that all the tumor tissue has been removed due to the time constraint.
During the process of freezing and slicing, for the sample with much moisture, the ice crystal (crystal ice) generated after freezing can destroy the tissue structure; in the fat-rich (fat) sample, at the freezing and solidifying temperature of general tissues (about-20 ℃), the fat tissues are easy to fall off from the section because the fat tissues are not frozen and solidified, and the section tissues are incomplete.
Optical Coherence Tomography (OCT) is an emerging optical imaging technology in recent years, which works on the principle similar to that of ultrasound but has a higher resolution than ultrasound, and mainly uses the difference of light reflection, absorption and scattering abilities of tissues and the principle of optical interference to image and resolve a sample. Because the tissue can be directly scanned at normal temperature without freezing and slicing procedures, the structural distortion (morphological artifacts) generated when the multi-moisture or multi-fat tissue is frozen and sliced can be avoided to maintain the integrity of the tissue sample, thereby improving the accuracy of pathological examination and contributing to shortening the operation time and improving the operation effect. However, since the depth of field (depth of focus) of the optical imaging technique is too large, the tissue must be physically sliced (thickness is usually 4-5 μm) during the examination to avoid overlapping the tissue images at different depths, so as to obtain a clear image.
Known techniques, such as U.S. Pat. No. 4, 9185357, 2 entitled "Optical tissue section Using full field Optical coherence tomography", disclose a full field OCT apparatus for viewing tissue slices, characterized by: includes a full-field imaging interferometer and an optical dividing imaging system. The depth of field is improved by an optical interferometer, and a structural image in the tissue can be directly obtained, so that the transverse resolution and the longitudinal resolution of the image can reach within 1 mu m, and a tissue fixation (fixation) procedure is omitted. Such as: freezing or paraffin (paraffin) coating, and solid section (sectioning) procedures.
In addition, the conventional H & E stained section (H & E section) uses two dyes, hematoxylin (hematoxylin) and eosin (eosin), to respectively stain the nucleus (nucleus) and cytoplasm (cytoplasm) with bluish purple and pink colors, however, the obtained image shows bluish purple signal of the nucleus, and details in the nucleus are not easily revealed. Such as: nucleolus (nucleolus) or heterochromatin (heterochromotin), which is a patent that complements detailed images of the nucleus by fluorescent staining to meet the needs of pathological examinations.
However, in this patent architecture: (1) after the short-wave light beam (such as ultraviolet light) passes through the light splitting film of the light splitter, 10-40% of the original light intensity is remained; (2) and the penetration rate of the anti-reflection coating of the optical splitter in an ultraviolet light wave band with the wavelength less than 400nm is low. Under the influence of these two factors, the fluorescence signal of the sample becomes very weak, and in order to enhance the fluorescence signal intensity, shorten the exposure time, and further increase the image capturing speed, a novel optical layer cutting device is needed in the art.
Disclosure of Invention
An objective of the present invention is to disclose an optical layer cutting device, wherein a first dichroic beam splitter is disposed between the beam splitter and a first objective lens, so as to prevent short-wave light beams irradiated onto a sample from being split by the beam splitter, thereby enhancing fluorescence signal intensity, shortening exposure time, and further increasing image capturing speed.
Another objective of the present invention is to disclose an optical layer cutting device, wherein when a fluorescent light beam emitted from a sample passes through a first dichroic beam splitter, the first dichroic beam splitter filters a short-wavelength light beam to obtain a fluorescent signal with a better contrast, so as to shorten an exposure time and further increase an image capturing speed.
Another objective of the present invention is to disclose an optical layer cutting device, wherein the sensing unit has a long-wave pass filter for further filtering the short-wave light beam to increase the intensity of the fluorescence signal, shorten the exposure time, and further increase the image capturing speed.
To achieve the above object, an optical sectioning device using advanced optical interference microscopy is provided, which has:
the beam splitter is provided with a first side edge, a second side edge, a third side edge and a fourth side edge and can enable an incident beam incident from the first side edge to be split into a reflected beam passing through the second side edge and a transmitted beam passing through the third side edge;
a broadband light source device for generating a broadband light beam to illuminate the first side of the optical splitter;
a reference end unit for returning the transmitted beam to the optical splitter via an adjustable optical path;
the short-wave light source device is used for generating a short-wave light beam;
a first dichroic beam splitter having a first side, a second side and a third side, wherein the first side faces the short wave light source device, the third side faces the second side of the beam splitter, and the first dichroic beam splitter is configured to make a light beam having a wavelength shorter than a predetermined wavelength unable to penetrate therethrough, and the wavelength of the short wave light beam is smaller than the predetermined wavelength;
a first objective lens having a parallel light side and a light-gathering side, the parallel light side facing the second side of the first dichroic beam splitter;
a sample carrying unit facing the light-gathering side of the first objective lens and used for carrying a sample dyed with a fluorescent agent;
a projection lens having an incident side and an emergent side, the incident side facing the fourth side of the beam splitter; and a sensing unit facing the light-emitting side of the projection lens.
In one embodiment, the reference terminal unit includes: an optical path retarder having a first side and a second side, the first side facing the third side of the beam splitter; a second objective lens having a parallel light side and a light-gathering side, the parallel light side facing the second side of the optical path retarder; and a reflector facing the light-gathering side of the second objective lens for reflecting the transmitted light beam, wherein the optical path retarder is used for adjusting the adjustable optical path to make the adjustable optical path symmetrical to a sample optical path formed by the sample bearing unit, the first objective lens and the first dichroic beam splitter.
In one embodiment, the broadband light source device and the short-wave light source device both comprise a light source; or a light source and a grating; or a light source, a grating and a turning mirror with adjustable inclination angle; or an LED strip-shaped distributed light source.
In one embodiment, the sample-holding unit further has a white light source to provide a suitable transmission brightness for the first objective lens, the white light source includes a white LED, a white halogen lamp, or a tungsten lamp.
In one embodiment, the sensing unit includes a second dichroic beam splitter, a color two-dimensional photosensitive device, a long-pass filter, and a monochrome two-dimensional photosensitive device, the second dichroic beam splitter having a first side, a second side, and a third side, the first side facing the projection lens to reflect the fluorescent light beam and the white light beam through the third side and image them on the color two-dimensional photosensitive device, and to transmit the broadband light beam through the second side and image them on the monochrome two-dimensional photosensitive device; or the sensing unit comprises a turnable turning mirror, a color two-dimensional photosensitive assembly, a long-wave pass filter and a monochrome two-dimensional photosensitive assembly, when the turnable turning mirror is turned over, the white light beam is imaged on the color two-dimensional photosensitive assembly, when the turnable turning mirror is turned over, the broadband light beam and the fluorescent light beam are imaged on the monochrome two-dimensional photosensitive assembly sequentially, and the long-wave pass filter is arranged between the projection lens and the turnable turning mirror, between the projection lens and the second dichroic beam splitter, between the turnable turning mirror and the monochrome two-dimensional photosensitive assembly or between the second dichroic beam splitter and the monochrome two-dimensional photosensitive assembly.
In one embodiment, when the wavelength range of the broadband light beam is 470nm to 800nm, the wavelength range of the short-wave light beam is 365nm to 460nm, the operating wavelength range of the optical splitter is 400nm to 800nm, and the cut-off wavelength ranges of the first dichroic beam splitter and the long-wave pass filter are 400nm to 470 nm.
In one embodiment, when the wavelength range of the broadband light beam is 650nm to 1000nm, the wavelength range of the short-wave light beam is 365nm to 630nm, and the operating wavelength range of the optical splitter is 400nm to 1000nm, the cut-off wavelength ranges of the first dichroic beam splitter, the second dichroic beam splitter and the long-wave pass filter are 400nm to 650 nm.
In one embodiment, a first polarizer is further disposed between the broadband light source device and the optical splitter, the monochromatic two-dimensional photosensitive component further has a second polarizer in front of it, a first quarter-wave plate is further provided between the first objective lens and the first dichroic beam splitter, a second quarter-wave plate is further disposed between the optical path retarder and the second objective lens, the first polarizer has a first polarization direction, the second polarizer has a second polarization direction, the first polarization direction and the second polarization direction are perpendicular to each other, the first quarter-wave plate has a first optic axis direction, the second quarter-wave plate has a second optic axis direction, the first optical axis direction and the second optical axis direction are both between the first polarization direction and the second polarization direction, and are used for providing an effect of enhancing interference efficiency and image quality.
In an embodiment, the apparatus further comprises an information processing device for executing an image processing program.
In one embodiment, the reference end unit further has an axial stage, and the sample bearing unit further has a three-dimensional moving stage, so that the information processing apparatus can calculate a three-dimensional image of the sample by moving the second objective lens and the reflecting mirror of the reference end unit through the axial stage, moving the sample dyed with the fluorescent agent through the three-dimensional moving stage, and modulating the optical path retarder.
Drawings
FIG. 1 is a schematic structural diagram of an optical sectioning device using advanced optical interference microscopy according to an embodiment of the present invention;
FIG. 2 is a schematic illustration of the splitting and focusing process of FIG. 1;
FIG. 3 is a schematic diagram of an embodiment of a reference terminal unit of FIG. 1;
FIG. 4 is a schematic diagram of the light collection and post-merger projection process of FIG. 1;
FIG. 5a is a schematic structural diagram of an embodiment of the sensing unit of FIG. 1;
FIG. 5b is a schematic structural diagram of another embodiment of the sensing unit of FIG. 1;
FIG. 5c is a schematic structural diagram of another embodiment of the sensing unit of FIG. 1;
FIG. 5d is a schematic structural diagram of a further embodiment of the sensing unit of FIG. 1;
FIG. 6 is a schematic structural diagram of an optical sectioning device using advanced optical interference microscopy according to another embodiment of the present invention;
FIG. 7 is a schematic structural diagram of an optical sectioning device using advanced optical interference microscopy according to yet another embodiment of the present invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
Referring to fig. 1 to fig. 2, fig. 1 is a schematic structural diagram of an optical layer cutting device using advanced optical interference microscopy according to an embodiment of the present invention, and fig. 2 is a schematic diagram of a splitting and focusing process of fig. 1.
As shown in the figure, the optical layer cutting device using advanced optical interference microscopy of the present embodiment includes: a broadband light source apparatus 100; a short wave light source device 200; a beam splitter 300; a first dichroic beamsplitter 400; a first objective lens 500; a sample carrying unit 600; a projection lens 700; a reference side unit 800; and a sensing unit 900.
The broadband light source device 100 is used for generating a broadband light beam 10, and the traveling direction of the broadband light beam is indicated by a hollow head arrow; the short-wave light source device 200 is used for generating a short-wave light beam 20, and the traveling direction of the short-wave light beam is indicated by a solid arrow; the sample carrying unit 600 is configured to carry a sample 610 dyed with a fluorescent agent, the fluorescent agent in the sample 610 dyed with the fluorescent agent is irradiated by the short-wave light beam 20 to release a fluorescent light beam 30, and a traveling direction of the fluorescent light beam 30 is indicated by a dashed arrow; the sample-holding unit 600 further has a white light source 620 to provide a proper transmission brightness for the first objective lens 500, and the traveling direction of a white light beam 40 emitted from the white light source 620 is indicated by a blank arrow.
The optical splitter 300 has a first side S301, a second side S302, a third side S303 and a fourth side S304; the broadband light source device 100 is configured to generate a broadband light beam 10 to illuminate the first side S301 of the optical splitter 300, and the optical splitter 300 is capable of splitting the broadband light beam 10 incident from the first side S301 to be reflected by the second side S302 and transmitted by the third side S303.
The short-wave light source device 200 is configured to generate a short-wave light beam 20, the first dichroic splitter 400 has a first side S401, a second side S402, and a third side S403, the first side S401 faces the short-wave light source device 200 and reflects the short-wave light beam 20, and the third side S403 faces the second side S302 of the splitter 300 and is configured to transmit the broadband light beam 10.
The first objective lens 500 has a parallel light side S501 and a light-gathering side S502, wherein the parallel light side S501 faces the second side S402 of the first dichroic splitter 400; the sample carrying unit 600 faces the light-gathering side S502 of the first objective 500 and is used for carrying a sample 610 dyed with a fluorescent agent, and the sample carrying unit 600, the first objective 500 and the first dichroic splitter 400 form a sample optical path.
The projection lens 700 has an incident side S701 and an emergent side S702, the incident side S701 faces the fourth side S304 of the beam splitter 300; the sensing unit 900 faces the light exit side S702 of the projection lens 700.
The white light source 620 includes, for example but not limited to, a white LED, a white halogen lamp, or a tungsten lamp, the first objective 500 has a working wavelength range of, for example but not limited to, 350-1000 nm, and the broadband light source apparatus 100 and the short-wave light source apparatus 200 both include a light source; or a light source and a grating; or a light source, a grating and a turning mirror with adjustable inclination angle; or an LED strip-shaped distributed light source (not shown), which is well known in the art, and therefore will not be described again.
Please refer to fig. 3, which is a schematic structural diagram of an embodiment of the reference end unit of fig. 1.
As shown, the reference terminal unit 800 includes: an optical path retarder 810; a second objective lens 820 and a reflector 830.
The optical path retarder 810, the second objective lens 820 and the reflector 830 form an adjustable optical path, and the reference end unit 800 is used for returning the broadband light beam 10 to the optical splitter 300 (not shown) through the adjustable optical path.
The optical path retarder 810 has a first side S811 and a second side S812, wherein the first side S811 faces the third side S303 of the optical splitter 300 (not shown); the second objective lens 820 having a parallel light side S821 and a light-gathering side S822, the parallel light side S821 facing the second side S812 of the optical path retarder 810; the reflector 830 faces the condensing side S822 of the second objective lens 820 for reflecting the broadband light beam 10. The second objective lens 820 has an operating wavelength range of, for example, but not limited to, 350-1000 nm.
The optical path retarder 810 is configured to generate a continuous interference carrier (continuous interference carrier wave) with one or more carrier waves by, for example but not limited to, changing a displacement (displacement) of a turning mirror in a state that the adjustable optical path is symmetrical to the sample optical path, and record each pixel by a monochromatic two-dimensional photosensitive assembly 940, and extract an interference intensity image of a cross section (en-face) after calculating an intensity change generated by interference by root mean square and intensity average, wherein the interference carrier can also be generated by the three-dimensional moving platform 630. Which is a known technique and will not be described in detail herein.
Please refer to fig. 4, which is a schematic diagram illustrating a projection process after light collection and convergence in fig. 1.
As shown, the sample-carrying unit 600 can reflect the short-wave light beam 20 and the broadband light beam 10, and the fluorescence agent in the fluorescence agent-dyed sample 610 (not shown) emits a fluorescence light beam 30 after being irradiated by the short-wave light beam 20, and the white light source 620 (not shown) of the sample-carrying unit 600 also emits a white light beam 40.
The first dichroic beam splitter 400 is configured to make the light beam with a wavelength shorter than a predetermined wavelength not penetrate therethrough, and the short-wave light beam 20 has a wavelength shorter than the predetermined wavelength, that is, the short-wave light beam 20 cannot penetrate therethrough, only the broadband light beam 10, the fluorescent light beam 30 and the white light beam 40 can respectively penetrate through the first dichroic beam splitter 400.
The third side S303 of the optical splitter 300 faces the reference unit 800 for reflecting the broadband light beam 10; the second side S302 faces the first dichroic splitter 400 for respectively transmitting the broadband light beam 10, the fluorescent light beam 30 and the white light beam 40.
Since the adjustable optical path and the sample optical path are symmetrical (in between the coherent length), the broadband light beam reflected by the adjustable optical path and the broadband light beam reflected by the sample optical path are combined to generate an optical interference phenomenon, which is a known technology and is not described herein again.
The projection lens 700 has an incident side S701 and an emergent side S702, the incident side S701 faces the fourth side S304 of the optical splitter 300, and the emergent side S702 is used for projecting the broadband light beam 10, the fluorescent light beam 30 and the white light beam 40 to the sensing unit 900.
Please refer to fig. 5 a-5 b, wherein fig. 5a is a schematic structural diagram of an embodiment of the sensing unit of fig. 1; FIG. 5b is a schematic structural diagram of another embodiment of the sensing unit of FIG. 1.
The sensing unit 900 includes: a flip-flop turning mirror 915; a color two-dimensional photosensitive element 920; a long-wavelength pass filter 930 and a monochromatic two-dimensional photosensitive element 940.
The long-pass filter 930 is used for further filtering the short-wave light beam 20 (not shown), and the turning mirror 915 has a turning support (not shown) for adjusting to a turning-on state or a turning-off state, which is well known in the art and will not be described herein again.
As shown in fig. 5a, the long-wave pass filter 930 is disposed between the projection lens 700 and the reversible turning mirror 915, when the reversible turning mirror 915 is turned over (flip on), the white light beam 40 is imaged on the color two-dimensional photosensitive assembly 920, and when the reversible turning mirror 915 is turned over (flip off), the broadband light beam 10 and the fluorescent light beam 30 are imaged on the monochrome two-dimensional photosensitive assembly 940 in sequence.
As shown in fig. 5b, the long-wave pass filter 930 is disposed between the turning mirror 915 and the monochromatic two-dimensional photosensitive assembly 940, when the turning mirror 915 is turned over (flip on), the white light beam 40 is imaged on the color two-dimensional photosensitive assembly 920, and when the turning mirror 915 is turned over (flip off), the broadband light beam 10 and the fluorescent light beam 30 are imaged on the monochromatic two-dimensional photosensitive assembly 940 sequentially.
Please refer to fig. 5 c-5 d, wherein fig. 5c is a schematic structural diagram of another embodiment of the sensing unit of fig. 1; FIG. 5d is a schematic structural diagram of a further embodiment of the sensing unit of FIG. 1.
The sensing unit 900 includes: a second dichroic beamsplitter 910; a color two-dimensional photosensitive element 920; a long-wavelength pass filter 930 and a monochromatic two-dimensional photosensitive element 940, wherein the long-wavelength pass filter 930 is used to further filter the short-wavelength light beam 20 (not shown).
As shown in fig. 5c, the long-wavelength pass filter 930 is disposed between the projection lens 700 and the second dichroic beam splitter 910, and the fluorescent light beam 30 and the white light beam 40 are reflected by the second dichroic beam splitter 910 through the third side S903 and imaged on the color two-dimensional photosensitive assembly 920; and transmitting and imaging the broadband light beam 10 to the monochrome two-dimensional photosensitive element 940 via the second side S902.
As shown in fig. 5d, the long pass filter 930 is disposed between the second dichroic beam splitter 910 and the monochromatic two-dimensional photosensitive element 940, and the fluorescent light beam 30 and the white light beam 40 are reflected by the second dichroic beam splitter 910 through the third side S903 and imaged on the color two-dimensional photosensitive element 920; and transmitting and imaging the broadband light beam 10 to the monochrome two-dimensional photosensitive element 940 via the second side S902.
When the wavelength range of the broadband light beam 10 is between 470nm and 800nm, the turning mirror 915 is needed, the wavelength range of the short-wave light beam 20 (not shown) is between 365nm and 460nm, the operating wavelength range of the optical splitter 300 (not shown) is between 400nm and 800nm, and the cut-off wavelength range of the first dichroic beam splitter 400 is between 400nm and 470 nm.
When the wavelength range of the broadband light beam 10 is 650nm to 1000nm, the wavelength range of the short-wavelength light beam 20 is 365nm to 630nm, the operating wavelength range of the optical splitter 300 is 400nm to 1000nm, the turning mirror 915 or the second dichroic beam splitter 910 can be used, and the cut-off wavelength ranges of the first dichroic beam splitter 400, the second dichroic beam splitter 910 and the long-wavelength pass filter 930 are 400nm to 650 nm.
Fig. 6 is a schematic structural diagram of an optical layer cutting device using advanced optical interference microscopy according to another embodiment of the present invention.
As shown, a first polarizer 950 is further disposed between the broadband light source apparatus 100 and the optical splitter 300; the monochrome two-dimensional photosensitive element 940 further has a second polarizer 960 in front; a first quarter-wave plate 970 is further disposed between the first dichroic beam splitter 400 and the first objective lens 500; a second quarter-wave plate 980 is further disposed between the retarder 810 and the second objective lens 820.
The first polarizer 950 has a first polarization direction, the second polarizer 960 has a second polarization direction, the first quarter-wave plate 970 has a first optic axis direction, the second quarter-wave plate 980 has a second optic axis direction, the first polarizer direction and the second polarization direction are perpendicular to each other, and both the first optic axis direction and the second optic axis direction are between the first polarization direction and the second polarization direction.
The first polarizer is oriented vertically in the figure; the second polarization direction is horizontal; the first optical axis direction and the second optical axis direction are both 45 degrees, but not limited thereto. When the broadband light beam 10 generated by the broadband light source device 100 passes through the first polarizer 950 to become a vertical polarization direction, after being split by the beam splitter 300, the broadband light beam respectively passes through the second quarter-wave plate 980 of the reference end unit 800 and the first quarter-wave plate 970 between the first objective 500 and the first dichroic beam splitter 400 to become a forward circular polarization direction, after being reflected, the broadband light beam becomes a reverse circular polarization direction, after passing through the second quarter-wave plate 980 and the first quarter-wave plate 970 again, the broadband light beam becomes a horizontal polarization direction, and the second polarizer 960 in front of the monochromatic two-dimensional photosensitive assembly 940 only allows the broadband light beam 10 in the horizontal polarization direction to pass through, so as to further isolate the reflection light of the antireflection film of the beam splitter 300, thereby providing an effect of enhancing interference efficiency and image quality.
Fig. 7 is a schematic structural diagram of an optical layer cutting device using advanced optical interference microscopy according to still another embodiment of the present invention.
As shown, the reference end unit 800 further has an axial platform 840; the sample-carrying unit 600 further has a three-dimensional moving platform 630; the optical layer cutting device using advanced optical interference microscopy further comprises an information processing device (not shown) for executing an image processing procedure.
The second objective lens 820 and the reflecting mirror 830 are moved by the axial stage 840, the optical path length modulation retarder 500 and the sample 610 dyed with the fluorescent agent is moved by the three-dimensional moving stage 630, so that the information processing device (not shown in the figure) can calculate a three-dimensional image (not shown in the figure) of the sample, which is a known technology and will not be described herein again.
Through the design disclosed above, the present invention has the following advantages:
1. according to the optical layer cutting device, the first dichroic beam splitter is arranged between the beam splitter and the first objective lens and is used for enabling short-wave light beams irradiated on a sample not to be split by the beam splitter, so that the intensity of a fluorescence signal is enhanced, the exposure time is shortened, and the image taking speed is increased.
2. According to the optical layer cutting device, when a fluorescent light beam emitted by a sample passes through the first dichroic beam splitter, the first dichroic beam splitter firstly filters a short-wave light beam to obtain a fluorescent signal with good contrast, the exposure time is shortened, and the image taking speed is increased.
3. In the optical layer cutting device, the sensing unit is provided with the long-wave pass filter, and the short-wave light beam is further filtered to enhance the intensity of a fluorescence signal, shorten the exposure time and accelerate the image taking speed.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. An optical sectioning device using advanced optical interference microscopy, comprising:
the beam splitter is provided with a first side edge, a second side edge, a third side edge and a fourth side edge and can enable an incident beam incident from the first side edge to be split into a reflected beam passing through the second side edge and a transmitted beam passing through the third side edge;
a broadband light source device for generating a broadband light beam to illuminate the first side of the optical splitter;
the reference end unit is used for enabling the transmission light beam to return to the optical splitter through an adjustable optical path;
the short-wave light source device is used for generating a short-wave light beam;
a first dichroic beam splitter having a first side, a second side, and a third side, the first side facing the short wave light source device, the third side facing the second side of the beam splitter, the first dichroic beam splitter being configured to be impenetrable by a light beam having a wavelength shorter than a predetermined wavelength, and the short wave light beam having a wavelength shorter than the predetermined wavelength;
a first objective having a parallel light side and a condensing side, the parallel light side facing the second side of the first dichroic splitter;
the sample bearing unit faces to the light condensation side of the first objective lens and is used for bearing a sample dyed with a fluorescent agent;
the projection lens is provided with an incident light side and an emergent light side, and the incident light side faces the fourth side of the light splitter; and
a sensing unit facing the light exit side of the projection lens,
the sensing unit comprises a second dichroic beam splitter, a color two-dimensional photosensitive assembly, a long-wave pass filter and a monochrome two-dimensional photosensitive assembly, wherein the second dichroic beam splitter is provided with a first side edge, a second side edge and a third side edge, the first side edge faces the projection lens so as to reflect a fluorescent light beam and a white light beam through the third side edge and image the fluorescent light beam and the white light beam on the color two-dimensional photosensitive assembly, and transmit the broadband light beam through the second side edge and image the broadband light beam on the monochrome two-dimensional photosensitive assembly; or the sensing unit includes a formula that can turn over mirror, a colour two dimension photosensitive assembly, a long wave passes through optical filter and a monochromatic two dimension photosensitive assembly, when can turn over the formula turn over mirror and turn over, white light beam image on colour two dimension photosensitive assembly, when turning over then broadband light beam with fluorescence light beam successively images on monochromatic two dimension photosensitive assembly, long wave passes through optical filter set up in projection lens with can turn over between the formula turn over mirror, between projection lens and the second dichroic beam splitter, can turn over the formula monochromatic mirror with between the two dimension photosensitive assembly or the second dichroic beam splitter with between the monochromatic two dimension photosensitive assembly.
2. The optical sectioning apparatus employing advanced optical interference microscopy according to claim 1, wherein the reference end unit comprises:
an optical path retarder having a first side and a second side, the first side facing the third side of the optical splitter;
the second objective lens is provided with a parallel light side and a light condensation side, and the parallel light side faces the second side edge of the optical path retarder; and
and the reflecting mirror faces the light-gathering side of the second objective lens and is used for reflecting the transmitted light beam, wherein the optical path retarder is used for adjusting the adjustable optical path so that the adjustable optical path is symmetrical to a sample optical path formed by the sample bearing unit, the first objective lens and the first dichroic beam splitter.
3. The optical sectioning apparatus employing advanced optical interference microscopy according to claim 1, wherein the broadband light source apparatus, the short wavelength light source apparatus, each include a light source; or a light source and a grating; or a light source, a grating and a turning mirror with adjustable inclination angle; or an LED strip-shaped distributed light source.
4. The optical sectioning apparatus according to claim 2, wherein the sample-holding unit further has a white light source including a white LED, a white halogen lamp, or a tungsten lamp to provide a proper transmittance of the first objective lens.
5. The optical sectioning device using advanced optical interference microscopy according to claim 1, wherein the wavelength range of the broadband light beam is 470nm to 800nm, the wavelength range of the short wave light beam is 365nm to 460nm, and the cut-off wavelength ranges of the first dichroic beam splitter and the long pass filter are 400nm to 470 nm.
6. The optical sectioning device using advanced optical interference microscopy according to claim 1, wherein when the wavelength of the broadband light beam is in a range of 650nm to 1000nm, the wavelength of the short-wavelength light beam is in a range of 365nm to 630nm, the operating wavelength of the beam splitter is in a range of 400nm to 1000nm, and the cut-off wavelength ranges of the first dichroic beam splitter, the second dichroic beam splitter and the long pass filter are in a range of 400nm to 650 nm.
7. The optical sectioning apparatus using advanced optical interference microscopy according to claim 2, wherein a first polarizer is further provided between the broadband light source apparatus and the beam splitter, a second polarizer is further provided in front of the monochromatic two-dimensional photosensitive assembly, a first quarter-wave plate is further provided between the first objective lens and the first dichroic beam splitter, a second quarter-wave plate is further provided between the optical retarder and the second objective lens, the first polarizer has a first polarization direction, the second polarizer has a second polarization direction, the first polarizer direction and the second polarization direction are perpendicular to each other, the first quarter-wave plate has a first optic axis direction, the second quarter-wave plate has a second optic axis direction, and the first optic axis direction and the second optic axis direction are both between the first polarization direction and the second polarization direction, for providing an effect of enhancing interference efficiency and image quality.
8. The optical sectioning apparatus using advanced optical interference microscopy according to claim 2, further comprising an information processing device for performing an image processing procedure.
9. The optical sectioning device using advanced optical interference microscopy according to claim 8, wherein the reference end unit further has an axial stage, and the sample-carrying unit further has a three-dimensional moving stage, by which the second objective lens and the reflecting mirror are moved, the optical path retarder is modulated, and the sample dyed with the fluorescent agent is moved by the three-dimensional moving stage, so that the information processing device can calculate a three-dimensional image of the sample.
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