CN113391437A - Fluorescence microscope imaging system and fluorescence imaging method thereof - Google Patents
Fluorescence microscope imaging system and fluorescence imaging method thereof Download PDFInfo
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- 238000000799 fluorescence microscopy Methods 0.000 title claims abstract description 37
- 238000003384 imaging method Methods 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims description 21
- 230000005284 excitation Effects 0.000 claims abstract description 112
- 238000002073 fluorescence micrograph Methods 0.000 claims abstract description 41
- 238000012632 fluorescent imaging Methods 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims description 12
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
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- 102100039872 Inner centromere protein Human genes 0.000 description 2
- 102000029749 Microtubule Human genes 0.000 description 2
- 108091022875 Microtubule Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000004688 microtubule Anatomy 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
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- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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Abstract
The invention discloses a fluorescence microscope imaging system. The object carrying platform is used for placing a target object to be observed; the variable wavelength fluorescent light source provides an excitation light beam corresponding to different excitation wavelengths; the user input interface provides a wavelength range and a wavelength adjusting value of the user input excitation light beam; the control unit adjusts an excitation wave long of an excitation beam of the variable wavelength fluorescent light source by using the medium; and a fluorescence imaging unit for generating a plurality of fluorescence images; when the control unit adjusts the excitation wavelength long of the excitation light beam of the fluorescent light source with the variable wavelength according to the wavelength range and the wavelength adjustment value, the fluorescent imaging unit can capture a plurality of fluorescent images of the emission light beam under different wavelengths. The calculating unit calculates the definition of the plurality of fluorescence images. The determining unit determines the clearest fluorescence image. The invention has the advantages that: the clearest fluorescence images of the emitted light beams shot under the field of view of the fluorescence microscope under different wavelengths are displayed on a screen after image splicing, so that interested areas can be conveniently researched.
Description
Technical Field
The invention relates to the technical field of picture imaging, in particular to a fluorescence imaging system based on a fluorescence microscope picture and a fluorescence imaging method thereof.
Background
The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
The fluorescence microscope is used for researching images of various organisms or cells in a micro state, has wide application range, and is the most important instrument in the fields of biology and medicine. The fluorescence microscope is a tool for observing microscopic objects, the field of view of the fluorescence microscope is very limited, only a small area can be observed at the same time, and for a relatively large object or a slightly large area, the whole appearance of the fluorescence microscope cannot be directly obtained from the fluorescence microscope.
In addition, since the fluorescence microscope is a technique that requires light to see the inside of an object and is different in the excitation beam suitable for each object, it is difficult to directly obtain a clear photograph because of the structure of the conventional fluorescence microscope itself. Without a clear figure, all subsequent studies became meaningless.
It should be noted that the above background description is only for the sake of clarity and complete description of the technical solutions of the present invention and for the understanding of those skilled in the art. Such solutions are not considered to be known to the person skilled in the art merely because they have been set forth in the background section of the invention.
Disclosure of Invention
In order to overcome the drawbacks of the prior art, embodiments of the present invention provide a fluorescent microscope imaging system capable of viewing the texture and the overview of different areas of an object under study in a panoramic manner with an excitation light beam having different wavelengths and an emission light beam having different wavelengths, a method for generating video thereof, and a method for generating video thereof.
The embodiment of the application discloses: a fluorescence microscope imaging system comprises a carrying platform, a variable wavelength fluorescence light source, a user input interface, a control unit, a fluorescence imaging unit, a calculation unit and a decision unit. The object carrying platform is used for placing an object to be observed, and a fluorescent substance is added to the object; the variable wavelength fluorescent light source is positioned above the object platform and used for providing an excitation light beam corresponding to different excitation wavelengths to the object, wherein after the excitation light beam irradiates on the fluorescent substance added to the object, the fluorescent substance is excited to emit an emission light beam corresponding to different excitation wavelengths; the user input interface is used for providing a wavelength range and a wavelength adjusting value of the user input excitation light beam; the control unit is coupled to the variable wavelength fluorescent light source and the user input interface, and is used for adjusting the fire wavelength of the excitation light beam of the variable wavelength fluorescent light source according to the wavelength range and the wavelength adjustment value input by a user; the fluorescence imaging unit is used for shooting the target object so as to generate a plurality of fluorescence images in each second; when the control unit adjusts the excited Rich of the excitation light beam of the fluorescent light source with the variable wavelength according to the wavelength range and the wavelength adjustment value, the fluorescent imaging unit can capture a plurality of fluorescent images of the emission light beam under different wavelengths. The calculating unit is used for calculating the definition of the plurality of fluorescence images. The determining unit determines a clearest fluorescence image from the plurality of fluorescence images. The video generation unit is used for connecting the plurality of clearest fluorescence images determined by the calculation unit in series into a video, wherein the plurality of clearest fluorescence images respectively correspond to different wavelengths of the emitted light beams.
Further, the control unit may adjust the excitation beam of the wavelength-variable fluorescent light source first to a maximum excitation wavelength, and adjust the excitation beam of the wavelength-variable fluorescent light source according to the wavelength adjustment value after every predetermined time interval until adjusting the excitation beam of the wavelength-variable fluorescent light source to a minimum excitation wavelength.
Further, a life-pin light has an excitation wavelength of 460nm to 550nm and an emission wavelength of 590nm, light has an excitation wavelength of 420nm to 485nm, an emission wavelength of 515nm, an excitation wavelength of light of interest of probe of 395nm to 415nm, an emission wavelength of 455nm, an excitation wavelength of 330nm to 400nm, an emission wavelength of ultraviolet light of interest of 425 nm.
Further, the fluorescence imaging unit divides the target into mxn regions, and takes p fluorescence block images for each region, wherein the p fluorescence block images of each region are respectively captured at the same wavelength of the emission beam.
Further, the determining unit first defaults a first fluorescence image as a main image to be finally output, then, for each region, the calculating unit calculates the sharpness of p fluorescence block images, and if the sharpness of a specific fluorescence block image is greater than the highest sharpness of the region on the main image, the highest sharpness value is updated, and meanwhile, the determining unit replaces the default fluorescence block image of the region on the main image with the specific fluorescence block image.
The embodiment of the application discloses: a fluorescence imaging method is suitable for a fluorescence microscope imaging system which comprises a variable wavelength fluorescence light source for providing an excitation light beam to a target added with a fluorescent substance and a fluorescence imaging unit for shooting the target to generate a plurality of fluorescence images in each second. The method comprises the following steps: providing a user input interface for inputting a wavelength range and a wavelength adjustment value of the excitation light beam to a user; adjusting an excited wave length of the excitation light beam of the fluorescent light source with variable wavelength according to the wavelength range and the wavelength adjustment value input by the user; when the excited Rich wavelength of the excitation light beam of the fluorescent light source with the variable wavelength is adjusted according to the wavelength range and the wavelength adjustment value, controlling the fluorescent imaging unit to capture a plurality of fluorescent images of the emission light beam under different wavelengths; and calculating the definition of the plurality of fluorescence images; determining a clearest fluorescence image from the plurality of fluorescence images.
Further, the method further comprises: adjusting the excitation light beam of the variable wavelength fluorescent light source to correspond to a maximum excitation wavelength, and adjusting the excitation light beam of the variable wavelength fluorescent light source according to the wavelength adjustment value after every predetermined time interval until the excitation light beam of the variable wavelength fluorescent light source is adjusted to correspond to a minimum excitation wavelength.
Further, a life-pin light has an excitation wavelength of 460nm to 550nm and an emission wavelength of 590nm, light has an excitation wavelength of 420nm to 485nm, an emission wavelength of 515nm, an excitation wavelength of light of interest of probe of 395nm to 415nm, an emission wavelength of 455nm, an excitation wavelength of 330nm to 400nm, an emission wavelength of ultraviolet light of interest of 425 nm.
Further, the method further comprises: the target is divided into mxn regions, and for each region, p fluorescence block images are captured, wherein the p fluorescence block images of each region are captured at the same wavelength of the emission beam.
Further, the method further comprises: firstly, defaulting a first fluorescence image as a final output main image, and then calculating the definition of p fluorescence block images aiming at each region; and if the definition of a specific fluorescence block image is larger than the highest definition of the area on the main image, updating the highest definition value, and replacing the default fluorescence block image of the area on the main image by the specific fluorescence block image.
By means of the technical scheme, the invention has the following beneficial effects: the clearest fluorescence image obtained by shooting different excitation beams and different Rich photophracters under the field of view of the fluorescence microscope is displayed on a screen after image splicing, so that the whole view of a researched object under a microscopic field of view can be conveniently checked, and an interested area can be conveniently researched.
In order to make the aforementioned and other objects, features and advantages of the invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic view of a fluorescence microscopy imaging system according to a first embodiment of the invention.
FIG. 2 is a block diagram of a fluorescence microscope imaging system in a first embodiment of the invention.
Fig. 3 is a schematic diagram of an image taken by a fluorescence imaging unit.
FIG. 4 is a flow chart of a method of controlling a fluorescence microscope imaging system in an embodiment of the invention.
Reference numerals of the above figures: 10. a fluorescence microscope imaging system; 110. a carrier platform; 120. a variable wavelength fluorescent light source; 130. a user input interface; 140. a control unit; 150. a fluorescence imaging unit; 160. a calculation unit; 170. a determination unit; OB1, target; EX, excitation beam; EM, emitting a light beam; RANGE, wavelength RANGE; Δ λ, wavelength adjustment value; λ _ MAX, maximum wavelength; λ _ MIN, minimum wavelength; AREA 11-AREA 35, AREA; s410, S420, S430, S440, S450 and steps.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that, in the description of the present invention, the terms "first", "second", and the like are used for descriptive purposes only and for distinguishing similar objects, and no precedence between the two is considered as indicating or implying relative importance. In addition, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
Please refer to fig. 1 and fig. 2. Fig. 1 is a schematic diagram of a fluorescence microscope imaging system 10 in a first embodiment of the invention, and fig. 2 is a block diagram of a fluorescence microscope imaging system 10 in a first embodiment of the invention. As shown in fig. 1 and 2, a fluorescence microscope imaging system 10 includes a stage 110, a variable wavelength fluorescence light source 120, a user input interface 130, a control unit 140, a fluorescence imaging unit 150, a calculation unit 160, and a determination unit 170. The object stage 110 is used for placing an object OB1 to be observed, and a fluorescent substance is added to the object OB 1; the variable wavelength fluorescent light source 120 is located above the stage 110 and is configured to provide an excitation light beam EX corresponding to different excitation wavelengths to the object OB1, wherein when the excitation light beam EX impinges on the fluorescent material added to the object OB1, the fluorescent material is excited to emit an emission light beam EM corresponding to the different excitation wavelengths. The user input interface 130 is used for providing a wavelength RANGE of the user input excitation beam EX and a wavelength adjustment value Δ λ, wherein the wavelength RANGE includes a maximum wavelength λ _ MAX and a minimum wavelength λ _ MIN. The control unit 140 is coupled to the variable wavelength fluorescent light source 120 and the user input interface 130, and configured to adjust the excitation wavelength of the excitation beam EX of the variable wavelength fluorescent light source 120 according to the wavelength RANGE and the wavelength adjustment value Δ λ input by the user. The fluorescence imaging unit 150 is used to capture the object OB1 to generate a plurality of fluorescence images per second. When the control unit 140 adjusts the excited Rich wavelength of the excitation beam EX of the fluorescent light source 120 with variable wavelength according to the wavelength RANGE RANGE and the wavelength adjustment value Δ λ, the fluorescent imaging unit 150 can capture a plurality of fluorescent images of the emission beam EM under different emission wavelengths. The calculating unit 160 is coupled to the fluorescence imaging unit 150 for calculating the sharpness of the plurality of fluorescence images. The determining unit 170 is coupled to the calculating unit 160 for determining a clearest fluorescence image from the plurality of fluorescence images. However, this is only an example and not a limitation of the present invention.
Note that fluorescence is a light-cooling luminescence phenomenon. When a fluorescent substance is irradiated with incident light (typically, ultraviolet light or X-ray) of a certain wavelength, the fluorescent substance absorbs light energy and enters an excited state, and emits outgoing light.
In a possible embodiment, control unit 140 first adjusts excitation beam EX of variable wavelength fluorescent light source 120 with respect to a maximum excitation wavelength λ _ MAX as desired, and adjusts excitation beam EX of variable wavelength fluorescent light source 120 with respect to wavelength adjustment value Δ λ after every predetermined time interval until excitation beam EX of variable wavelength fluorescent light source 120 is adjusted to a minimum excitation wavelength λ _ MIN.
For example, the user can set the maximum wavelength λ _ MAX of the wavelength RANGE of the excitation light beam to 500nm, set the minimum wavelength λ _ MIN to 350nm, and set the wavelength adjustment value Δ λ to 50nm, so that the control unit 140 first adjusts the excitation light beam EX of the wavelength-variable fluorescent light source 120 such that the maximum wavelength λ _ MAX is 500nm, and then the fluorescence imaging unit 150 captures a plurality of fluorescence images of the target object OB1 with the emission wavelength of the emission light beam EM of 590 nm; then, after every predetermined time interval (for example, every 30 seconds or after the fluorescence imaging unit 150 has captured all images of the object OB 1), the control unit 140 adjusts the excitation beam EX of the wavelength-variable fluorescence light source 120 according to the wavelength adjustment value Δ λ, and sequentially decrements the excitation beam EX to 450nm and 400nm … until the minimum excitation beam EX _ MIN of the excitation beam EX of the wavelength-variable fluorescence light source 120 is adjusted. The fluorescence imaging unit 150 then likewise acquires a plurality of fluorescence images of the object OB1 corresponding to the emission beam EM having wavelengths of 590nm, 515nm, 455nm, 425 nm. The maximum wavelength, the minimum wavelength, the wavelength adjustment value and the predetermined time interval are only exemplary illustrations and are not limitations of the present invention. The setting values can be designed into different values according to actual requirements, and the scope of the invention is also covered by the invention.
In a possible embodiment, an excitation wavelength of 460nm to 550nm and an emission wavelength of 590nm, light with respect to an excitation wavelength of 420nm to 485nm and an emission wavelength of 515nm, of light with respect to an object of interest, an excitation wavelength of 395nm to 415nm, an emission wavelength of 455nm, an ultraviolet light with respect to an excitation wavelength of 330nm to 400nm and an emission wavelength of 425 nm. However, this is only an example and not a limitation of the present invention.
For example, three components of a human tumor cell can be stained with different fluorescent substances, DNA is stained blue, INCENP protein is stained green, microtubules are stained purple, each fluorescence image is photographed with the fluorescence imaging unit 150 by using the corresponding excitation filter and emission filter separately, and finally a complete fluorescence image is superimposed, which can clearly distinguish the DNA stained blue, the INCENP protein stained green and the microtubules stained purple.
Referring to fig. 3, fig. 3 is a schematic diagram of an image captured by the fluorescence imaging unit 150. As shown in fig. 3, the fluorescence imaging unit 150 divides the object OB1 into mxn regions (e.g., 3x5 regions AREA 11-AREA 35, each region having a size of 16 × 16), and takes p fluorescence block images for each region (e.g., the first region AREA11), wherein the p fluorescence block images of each region are respectively captured at the same wavelength of the excitation beam. In the above example, when the maximum wavelength λ _ MAX of the excitation light beam EX is 500nm, the minimum wavelength λ _ MIN is 350nm, and the wavelength adjustment value Δ λ is 50nm (4 different excitation wavelengths 500nm, 450nm, 400nm, and 350nm, corresponding to different emission wavelengths 590nm, 515nm, 455nm, and 425nm, respectively), a fluorescence block image of 800(4 × 200) first regions AREA11 is obtained in a single session. By analogy, 800 images of the fluorescence block of the second AREA12 are captured for the second AREA12 until all the AREAs (including 3x5 AREAs AREA 11-AREA 35) are completely captured for 200 images of the fluorescence block under different emission wavelengths. That is, mxnxp phosphor images are obtained in total for each of the excitation beam EX and the emission beam EM having different wavelengths, and in the above example, 5 × 3 × 200 phosphor images are obtained in total for each of the excitation beam EX and the emission beam EM having different wavelengths.
It should be noted that m, n, and p are only exemplary and not limiting. The setting values can be designed into different values according to actual requirements, and the scope of the invention is also covered by the invention.
It should be noted that, in the above embodiment, each of the mxn regions is captured by using the same wavelength RANGE and the same wavelength adjustment value Δ λ to capture the fluorescence block image, which is only an example and is not a limitation of the present invention. In other embodiments, different wavelength RANGEs RANGE (including the maximum wavelength λ _ MAX and the minimum wavelength λ _ MIN) or different wavelength adjustment values Δ λ may be used for capturing the fluorescence block image in different regions. In this case, different wavelength RANGEs RANGE (including the maximum wavelength λ _ MAX and the minimum wavelength λ _ MIN) or different wavelength adjustment values Δ λ must be input for the respective regions. For example, the maximum wavelength λ _ MAX of the AREA11 may be set to 500nm, the minimum wavelength λ _ MIN may be set to 350nm, the wavelength adjustment value Δ λ may be set to 50nm, the maximum wavelength λ _ MAX of the AREA12 may be set to 500nm, the minimum wavelength λ _ MIN may be set to 400nm, the wavelength adjustment value Δ λ may be set to 100nm, the maximum wavelength λ _ MAX of the AREA13 may be set to 450nm, the minimum wavelength λ _ MIN may be set to 350nm, the wavelength adjustment value Δ λ may be set to 50nm, and so on, so that different AREAs may correspond to different wavelength RANGEs RANGE rage (different maximum wavelength λ _ MAX and minimum wavelength λ _ MIN) and/or different wavelength adjustment values Δ λ.
In one possible embodiment, the wavelength RANGE of the excitation beam EX may include a maximum wavelength λ _ MAX and a decreasing number N. For example, the user may set the maximum wavelength of the wavelength RANGE of the excitation light beam EX to 500nm, set the number of times N to 3, and set the wavelength adjustment value Δ λ to 50nm, so the control unit 140 may first condition the excitation wavelength of the excitation light beam EX of the variable-wavelength fluorescent light source 120, and at this time, the fluorescence imaging unit 150 may capture a plurality of fluorescence images of the object OB1 corresponding to the emission light beam with the wavelength of 590 nm; then, after every predetermined time interval (e.g., every 30 seconds or after all images of the object OB1 are captured by the fluorescence imaging unit 150), the control unit 140 adjusts the excitation beam EX of the wavelength-variable fluorescence light source 120 according to the wavelength adjustment value Δ λ, and adjusts the excitation beam EX sequentially to 450nm and 400nm … until the corresponding wavelength is adjusted to 350nm (the total number of times of decrease is 3).
In another possible embodiment, the wavelength RANGE of the excitation beam EX may include a minimum wavelength λ _ MIN and an incremental number, and is also within the scope of the present invention.
In the determination process, firstly, the determining unit 370 firstly defaults a first image as a main image to be finally output, then calculates the sharpness of p fluorescence block images for each region calculating unit 360, and if the sharpness of a specific fluorescence block image is greater than the highest sharpness of the region on the main image, updates the highest sharpness value, and simultaneously replaces the default fluorescence block image of the region on the main image with the specific fluorescence block image. For example, in the case of an excitation wavelength of 500nm and an emission wavelength of 590nm, the determining unit 370 first defaults the first fluorescence block images of all the AREAs AREA 11-AREA 35 to be the final output main image, where the first fluorescence block images of all the AREAs correspond to the emission wavelength of 590 nm. Then, for the first AREA11, the calculating unit 360 calculates the sharpness of 200 fluorescence block images, and if the sharpness of a specific fluorescence block image is greater than the highest sharpness of the first AREA11 on the main image, the highest sharpness value is updated, and the determining unit 370 replaces the default fluorescence block image (i.e., the first fluorescence block image) of the first AREA11 on the main image with the specific fluorescence block image. When the sharpness of all of the 200 phosphor images of the first AREA11 is compared, the determining unit 370 determines a sharpest phosphor image. By analogy, for all the AREAs AREA11 to AREA35, the calculating unit 360 calculates the sharpness of 200 fluorescence block images, and the final determining unit 370 determines the clearest fluorescence block image of each of the AREAs AREA11 to AREA35, and combines the clearest fluorescence block images into the final output main image. Thus, there is one clearest main image at each wavelength, and in the above example, the present invention will generate 4 clearest main images (4 wavelengths). Each main image is spliced by the clearest AREAs AREA 11-AREA 35.
Referring to fig. 4, fig. 4 is a flowchart of an light imaging method suitable for a fluorescence microscope imaging system according to an embodiment of the invention. The fluorescence microscope imaging system comprises a variable wavelength fluorescence light source for providing an excitation light beam to a target object and a fluorescence imaging unit for shooting the target object to generate a plurality of fluorescence images in each second.
The method comprises the following steps:
s410: providing a user input interface for inputting a wavelength range and a wavelength adjustment value of the excitation light beam to a user;
s420: adjusting an excited wave length of the excitation light beam of the fluorescent light source with variable wavelength according to the wavelength range and the wavelength adjustment value input by the user;
s430: when the excited Rich wavelength of the excitation light beam of the fluorescent light source with the variable wavelength is adjusted according to the wavelength range and the wavelength adjustment value, controlling the fluorescent imaging unit to capture a plurality of fluorescent images of the emission light beam under different wavelengths;
s440: calculating the definition of the plurality of fluorescence images;
s450: determining a clearest fluorescence image from the plurality of fluorescence images.
By means of the technical scheme, the invention has the following beneficial effects: the clearest images of the emitted light beams with different wavelengths shot under the microscope field of view are displayed on a screen after being spliced, and a video is generated, so that the overall view of a researched object under the microscopic field of view can be conveniently checked, and an interested area can be conveniently researched.
The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (10)
1. A fluorescence microscopy imaging system, comprising:
the object carrying platform is used for placing an object to be observed, and a fluorescent substance is added to the object;
a variable wavelength fluorescent light source located above the object stage for providing an excitation beam corresponding to different excitation wavelengths to the object, wherein when the excitation beam is irradiated on the fluorescent substance added to the object, the fluorescent substance is excited to emit an emission beam corresponding to different emission wavelengths;
a user input interface for providing a user with a wavelength range and a wavelength adjustment value for inputting the excitation beam, wherein the wavelength range includes a maximum wavelength and a minimum wavelength;
a control unit, coupled to the plurality of fluorescence filter modules and the user input interface, for adjusting an excited wavelength of the excitation light beam of the variable wavelength fluorescence light source according to the wavelength range and the wavelength adjustment value input by the user; and
a fluorescence imaging unit for photographing the object to generate a plurality of fluorescence images per second; when the control unit conditioners the excitation wavelength for the excitation light beam of the variable wavelength fluorescent light source according to the wavelength range and the wavelength adjustment value, the fluorescence imaging unit can capture a plurality of fluorescence images of the emission light beam under different wavelengths;
a calculating unit, coupled to the fluorescence imaging unit, for calculating the sharpness of the plurality of fluorescence images; and
a determining unit, coupled to the calculating unit, for determining a clearest fluorescence image from the plurality of fluorescence images.
2. The fluorescence microscope imaging system of claim 1, wherein the control unit includes a previous conditioning of the excitation beam of the variable wavelength fluorescent light source with respect to a maximum excitation wavelength, and conditioning the excitation beam of the variable wavelength fluorescent light source according to the wavelength adjustment value after every predetermined time interval until conditioning of the excitation beam of the variable wavelength fluorescent light source with respect to a minimum excitation wavelength is performed.
3. A fluorescence microscope imaging system according to claim 1, wherein light is of an excitation wavelength of 460nm to 550nm and an emission wavelength of 590nm, light is of an excitation wavelength of 420nm to 485nm and an emission wavelength of 515nm, as appropriate, with respect to light of an excitation wavelength of 395nm to 415nm and an emission wavelength of 455nm, ultraviolet light is of an excitation wavelength of 330nm to 400nm and an emission wavelength of 425 nm.
4. The fluorescence microscopy imaging system of claim 1, wherein the fluorescence imaging unit divides the object into mxn regions, and p fluorescence block images are taken for each region, wherein the p fluorescence block images of each region are respectively captured at the same wavelength of the emission beam.
5. The fluorescence microscope imaging system according to claim 4, wherein the determining unit defaults a first fluorescence image as a main image of a final output, and then for each region, the calculating unit calculates the sharpness of p fluorescence block images, and updates the highest sharpness value if the sharpness of a specific fluorescence block image is higher than the highest sharpness of the region on the main image, while the determining unit replaces the default fluorescence block image of the region on the main image with the specific fluorescence block image.
6. A fluorescence imaging method for a fluorescence microscope imaging system including a variable wavelength fluorescence light source for providing an excitation light beam to a target to which a fluorescent substance is added and a fluorescence imaging unit for photographing the target to generate a plurality of fluorescence images per second, the method comprising the steps of:
providing a user input interface for inputting a wavelength range and a wavelength adjustment value of the excitation light beam to a user;
adjusting an excited wave length of the excitation light beam of the fluorescent light source with variable wavelength according to the wavelength range and the wavelength adjustment value input by the user;
when the excited Rich wavelength of the excitation light beam of the fluorescent light source with the variable wavelength is adjusted according to the wavelength range and the wavelength adjustment value, controlling the fluorescent imaging unit to capture a plurality of fluorescent images of the emission light beam under different wavelengths;
calculating the definition of the plurality of fluorescence images; and
determining a clearest fluorescence image from the plurality of fluorescence images.
7. The fluorescence imaging method of claim 6, wherein adjusting the excitation wavelength of the excitation beam of the variable wavelength fluorescence light source according to the wavelength range and the wavelength adjustment value input by the user further comprises:
adjusting the excitation light beam of the variable wavelength fluorescent light source to correspond to a maximum excitation wavelength, and adjusting the excitation light beam of the variable wavelength fluorescent light source according to the wavelength adjustment value after every predetermined time interval until the excitation light beam of the variable wavelength fluorescent light source is adjusted to correspond to a minimum excitation wavelength.
8. A fluorescence imaging method according to claim 6, characterized in that light of the excitation wavelength of 460nm to 550nm and an emission wavelength of 590nm, light is applied with respect to an excitation wavelength of 420nm to 485nm and an emission wavelength of 515nm, while violet light has an excitation wavelength of 395nm to 415nm and an emission wavelength of 455nm, as should be mentioned, ultraviolet light has an excitation wavelength of 330nm to 400nm and an emission wavelength of 425 nm.
9. The fluorescence imaging method of claim 6, further comprising the steps of:
the target is divided into mxn regions, and for each region, p fluorescence block images are captured, wherein the p fluorescence block images of each region are captured at the same wavelength of the emission beam.
10. The fluorescence imaging method of claim 9, further comprising the steps of:
firstly, defaulting a first fluorescence image as a final output main image, and then calculating the definition of p fluorescence block images aiming at each region;
if the definition of a specific fluorescence block image is larger than the highest definition of the area on the main image, updating the highest definition value, and replacing the default fluorescence block image of the area on the main image by the specific fluorescence block image.
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