CN101071106A - On-line cell micro observation instrument for biochemical reactor - Google Patents
On-line cell micro observation instrument for biochemical reactor Download PDFInfo
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Abstract
This invention involves an online cell microscopic observation instrument for biochemical reactor, including the main instrument of the observation, the objective lens, the observation window incident, sampling devices, external light source system. Its characteristics are: reflectance prism is installed between external light source and lens system. There is a reflecting mirror behind the objective lens and in the front of entrance window. Reflecting prism is placed in the front of annular aperture films. the upper side of the reflecting prism has a home or area array CCD image sensor. Sampling device includes sampling block, elastic components, a mobile device. The elastic element is installed between sampling block and the drive shaft of mobile devices. the gab between the front face of sampling block and observation entrance window develops the sampling pool. The external light source system of this invention can easily replace wavelength through different filter replacement; dark field of view at the same time lighting can improve resolution of observation instrument for the cells into the contrast of the image and resolution; and it is simple in structure, easy-to-use, manufacturing costs low.
Description
Technical field
The present invention relates to a kind of online cell micro observation instrument, particularly a kind of online cell microscopic morphology scope that is applied to the biochemical reaction jar.
Background technology
Cultivate when carrying out production with living cells in the bioprocesses, the cell growth is epochmaking monitoring parameter with metamorphosis, and long-term method of coming to take a sample from jar obtains by the off-line microscopic examination.Along with the progress of electronic technology, can be converted into digital signal to image in recent years, carry out Computer Processing, obtain the growth and the metamorphosis information that quantize thus.But above method is the off-line interval sampling, and shortage dynamic observes, special anti-living contaminants is the matter of utmost importance of process operation as the animal cell large-scale incubation, therefore can not frequently take a sample, bring the observation dynamic poor thus, the problem of process analysis procedure analysis difficulty.
According to above operating position, the online cell micro observation instrument that is inserted into fermentation tank must meet following basic demand: 1) the clear differentiation bacterium of the resolution of micro observation instrument and visual field energy, fungi and zooblast, dead cell and living cells, and do corresponding morphological analysis; 2) because the incubation cell concentration changes, micro observation instrument must have corresponding Cmax boundary; 3) must overcome micro-probe and insert camera lens surface thalli growth or the absorption that forms behind the fermentation tank for a long time; 4) the micro observation instrument structure helps the sterilization and the sterile working of bio-reactor; 5) micro observation instrument must have conversion of real-time digital picture intelligence and the processing power that meets the reactor requirement.
Germany Dr Christoph Bittner has described a kind of online microscope equipment (original position microscope: the new process of biomass on-line determination in the incubation of fluorescence excitation method in the doctorate paper in 1994, Dr Christoph Bittner, Hannover, 1994).Its groundwork principle as shown in Figure 1, the monochromatic ultraviolet light that laser sends reflexes to object lens through lens combination L2, L3 by double-colored eyeglass again behind lens L1, aperture spatial filtering, be projected to cell surface in the retort chamber by object lens then, because fluorescent effect, the fluorescence that cell produces converges through object lens, double-colored eyeglass and catoptron are again through imaging in behind the filter plate on the synchronous video camera.Because therefore the cell high speed rotating in the retort requires the time shutter of synchronous video camera very short, generally be no more than 2ms (may the needs adjustment) according to the difference of enlargement factor.The advantage of the method is not for needing the complicated mechanical sampler.But this scheme adopts the bright field lighting system, and is bad for the contrast and the resolution of image that hyaline cell becomes; Again owing to adopt high-speed photography, the mode that single wavelength fluorescent excites, it is higher in use to change the excitation wavelength cost.
There is patent to introduce a kind of micro observation instrument (US6,809,862) in the U.S. in 2004 based on on-line sampling.As shown in Figure 2, sample window 4 moves around, and when sample window 4 moved to microscope 16 and position, illuminating lamp source, the cell solution in the retort 10 just flowed into observation chamber 12; When sample window 4 moves to other positions, the cell in the observation chamber 12 is molten write relatively stable so that microexamination.Convenient in order to change microlens, as shown in Figure 3 specialized designs a cleaning chambers.Pulling pull bar 2 makes microlens and sample window 4 arrive the cleaning chambers positions, and cleaning chambers chamber wall is porose 8, can be used for introducing steam or thimerosal and carries out disinfection or clean.In this position, can change the microscope camera lens easily simultaneously.But this scheme adopts the bright field lighting system, and is bad for the contrast and the resolution of image that hyaline cell becomes; Because illuminator is worked, be not easy to change light source, toggle lights excitation wavelength again in retort, can't distinguish different types of cell, as dead cell and living cells; And frequent sampling and sterilization cause the hidden danger of microbiological contamination can for online biological culture.
German patent DE 10350243 had been introduced a kind of SLD of utilization (Super LightDiode) illumination in 2004, the online cell micro observation instrument of Multimode Fiber (multimode optical fiber) transmission, synchronous high-speed shooting.The light that laser or light emitting diode send shines the cell in the retort (reactor) in multimode optical fiber is transferred to retort, the light of cell reflection is imaged on the ccd video camera after object lens converge.Because cell is motion constantly in retort, therefore requires the time shutter of video camera smaller, could catch the cell image in moment.The video camera picture shot is sent into Computer Processing.This scheme adopts the bright field lighting system, and is bad for the contrast and the resolution of image that hyaline cell becomes; Again owing to must adopt high-speed camera under the situation faster in the retort stirring rate owing to there is not the sampler restriction depth of field, therefore the same with scheme shown in Figure 1 to the depth of field influence can't eliminate; Adopt the SLD (Super Light Diode superbright light emitting diode) of complexity simultaneously owing to its light source, therefore the light source driving controller more complicated is changed light source, is switched excitation wavelength cost height and relatively more difficult.
Summary of the invention
Technical matters to be solved by this invention is to adopt dark field illumination to improve contrast and the resolution of online cell micro observation instrument for image that cell is become; A kind of external light source system of replaceable wavelength is provided; Mechanical sampling device simple in structure, the online cell micro observation instrument that is used for biochemical reactor of easy and simple to handle and low cost of manufacture.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of online cell micro observation instrument that is used for biochemical reactor comprises the scope main body, and object lens are observed entrance window, sampler, external light source system; The scope body front end is provided with the observation entrance window, observe the entrance window front portion sampler is set, in the scope main body, observe the entrance window back and place object lens and external light source system, be characterized in: between external light source system and the object lens reflecting prism is set, the object lens back is provided with catoptron before the observation entrance window, the annular diaphragm sheet is placed in the reflecting prism front, and top, reflecting prism side is equipped with CCD or array image sensor; Sampler is by the sampling piece, and flexible member, mobile device constitute, and wherein, is connected flexible member between the driving shaft of sampling piece and mobile device, sampling piece front end face with observe gap formation between the entrance window pond of sampling.
The external light source system is by light source, condenser, and interchangeable color filter 7 is formed; Light source is Halogen lamp LED or LED lamp, and the light of its emission focuses on through condenser and produces required wave band light through interchangeable color filter after the back forms parallel beam.
The scope main body is connected with retort by set nut; Adopt the O-ring seal sealing between scope main body and the retort.
One of mobile device is by driving shaft, connecting link, the sampling pull bar, flexible member is formed, the connecting link that driving shaft wherein is housed is connected with the sampling pull bar by screw, the sampling pull bar seals by welding in the junction with flexible member and is connected, and an other end face of flexible member also is connected with the scope main body by welding in the junction simultaneously.
Mobile device two by driving shaft, motor, sampler barrel is formed, wherein sampler barrel is connected the front end of scope main body, the sampler barrel rear end is equipped with motor, motor output shaft connects driving shaft.
The sampling piece is made of three different-diameter right cylinders, and is made by stainless steel or sapphire material.
CCD or array image sensor are given the image acquisition and processing unit through opto-electronic conversion with digital data transmission, and the collection processing unit is sent result into Computer Analysis, show or storage.
The invention has the beneficial effects as follows:
(1) after light source sends the light of parallel beam through interchangeable different color filters generation specific bands, handle the parallel beam that the light of institute's incident is converted to annular by the annular diaphragm sheet, and form the cell solution of inside, formed sampling pond between an illuminating ray irradiation sampling piece with big annular aperture angle and the scope view window through catoptron.Because illuminating ray is that encircling cell solution carries out incident illumination, its light does not return and enters object lens.Therefore the visual field is a dark field.The reflection ray that has only cell in the irradiated solution or particle enters object lens after project on CCD or the array image sensor after being coated with 45 degree reflecting prisms reflections of reflectance coating.Therefore, external light source of the present invention system is by replacing different color filters, can changing wavelength easily; Dark field illumination can improve contrast and the resolution of scope for image that hyaline cell becomes simultaneously;
(2) sampler is by the sampling piece, flexible member, mobile device constitutes, the end face of sampling piece and the clearance space between the scope view window have formed a sampling pond that isolates with retort, take a sample this moment solution cell and interior relative isolation of solution cell of retort in the pond,, be not convenient to observe with the stirrer motion of retort.And mobile device is simple in structure, and is easy to use, low cost of manufacture.
Description of drawings
Fig. 1 is the structure principle chart of the online microscope equipment of fluorescence excitation method;
Fig. 2 is a U.S. Pat 6,809, the optical principle synoptic diagram of 862 micro observation instrument;
Fig. 3 is a U.S. Pat 6,809, the sampling cavity of 862 micro observation instrument, cleaning chambers structural representation;
Fig. 4 is illumination optical of the present invention and imaging optical path synoptic diagram;
Fig. 5 is one of a sampler embodiment of the present invention structural representation;
Fig. 6 is two structural representations of sampler embodiment of the present invention;
Fig. 7, the 8th, the work synoptic diagram in sampling pond.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done into one explanation.
As shown in Figure 4, the online cell micro observation instrument that is used for biochemical reactor of the present invention comprises scope main body 43, catoptron 23, object lens 24 are observed entrance window 22, sampler 40, external light source system 60, reflecting prism 25, annular diaphragm sheet 26, CCD or array image sensor 30.
The scope body front end is provided with observes entrance window 22, observes entrance window 22 front portions sampler 40 is set, and in the scope main body 43, observes entrance window 22 back and places object lens 24 and external light source system 60.
Between external light source system 60 and the object lens 24 reflecting prism 25 is set, object lens 24 back are provided with catoptron 23 before the observation entrance window 22, and annular diaphragm sheet 26 is placed in reflecting prism 25 fronts, and top, reflecting prism 25 side is equipped with CCD or array image sensor 30.
External light source system 60 is by light source 28, condenser 29, and interchangeable color filter 27 is formed; Halogen lamp LED, LED lamp can be adopted in lamp source 28, and the light of its emission focuses on the back through condenser 29 and forms the parallel beam outgoing.Parallel beam is handled the parallel beam that the light of institute's incident is converted to annular by annular diaphragm sheet 26 after seeing through the light of replaceable color filter 27 generation specific bands.The annular parallel beam forms the cell solution of inside, formed sampling pond between an illuminating ray irradiation sampling piece 21 with big annular aperture angle and the scope view window 22 through catoptron 23.Because illuminating ray is that encircling cell solution carries out incident illumination, its light does not return and enters object lens 24.Therefore the visual field is a dark field.The reflection ray that has only cell in the irradiated solution or particle enters object lens 24 after project on CCD or the array image sensor 30 after being coated with 45 degree reflecting prisms, 25 reflections of reflectance coating.After 30 opto-electronic conversion, become digital data transmission and give image acquisition and processing unit 31.Image acquisition and processing unit 31 send computing machine 32 to analyze result, shows or storage.
As Fig. 5, shown in 6, sampler 40 is by sampling piece 21, flexible member 38, mobile device constitutes, wherein, is connected flexible member 38 between the driving shaft 51 of sampling piece 21 and mobile device, sampling piece 21 front end faces and the gap formation between the observation entrance window 22 pond 50 of sampling.
As shown in Figure 5, one of mobile device is by driving shaft 51, connecting link 46, sampling pull bar 44, flexible member 39 is formed, the connecting link 46 that driving shaft 51 wherein is housed is connected with sampling pull bar 44 by screw 47, and sampling pull bar 44 and flexible member 39 are by sealing in the welding of 9a place and being connected, and an other end face of while flexible member 39 also is connected with scope main body 43 by welding at the 9b place.
Scope main body 43 is connected with retort 41 by set nut 42.Adopt O-ring seal 45 sealings between scope main body 43 and the retort 41.Scope is observed entrance window 22 and is immersed retort 41 inside, and its material is quartz glass or sapphire.Sampling piece 21 is three different-diameter cylindrical shapes, adopts stainless steel or sapphire material to make.One end and flexible member 38 welding, an other end face can have been formed a sampling pond 50 with scope view window 22 along scope view window central axial direction move left and right.
Outwards spur sampling pull bar 44, sampling pull bar 44 promotes sampling pieces 21 by connecting link 46, flexible member 38 and moves to close view window 22 directions, and flexible member 39 is in compressive state simultaneously; Inwardly promote sampling pull bar 44, sampling pull bar 44 promotes sampling piece 21 by connecting link 46, flexible member 38 and moves to outlying observation window 22 directions, and flexible member 39 is in relaxed state simultaneously. Flexible member 38,39 all adopts stainless steel material to make, and connecting link 46, pull bar 44 also are to be made by stainless steel material simultaneously.
As Fig. 7, shown in 8, when connecting link 46 when the direction of arrow moves, connecting link 46 moves by the direction of flexible member 38 drive sampling pieces 21 towards outlying observation instrument view window 22, distance between sampling piece 21 and the scope view window 22 becomes big, at this moment the take a sample sampling pond 50 of the composition between piece 21 and the scope view window 22 is communicated with retort 41, and the interior cell solution of sampling pond cell solution and retort is communicated with.When connecting link 46 drives sampling pieces 21 when the direction near scope view window 22 moves by flexible member 38, distance between sampling piece 21 and the scope view window 22 diminishes, when reaching the limit of the position, the end face of sampling piece 21 and the clearance space between the scope view window 22 have formed a sampling pond 50 that isolates with retort 41, take a sample this moment solution cell and interior relative isolation of solution cell of retort in the pond 50,, be not convenient to observe with the stirrer motion of retort.The depth size in the sampling pond after the shaping can obtain by the distance between control sampling piece 21 end faces and the scope view window 22.
As shown in Figure 6, mobile device two by driving shaft 51, motor 48, sampler barrel 49 is formed, wherein sampler barrel 49 is connected the front end of scope main body 43, sampler barrel 49 rear ends are equipped with motor 48, motor 48 output shafts connect driving shaft 51.
Scope main body 43 is connected by set nut 42 with retort 41.Sampling pond 50 is made up of end face of sampling piece 21 and the gap between the scope view window 22.Motor 48 is a heat resisting motor, is sealed in the immersion retort solution part in the scope main body 43.When driving sampling piece 21 by flexible member 38, motor 48 moves to an extreme position along the direction that makes sampling pond 50 smaller volume, cell solution in the sampling pond 50 and the solution in the retort 41 are isolated, so the cell solution in the pond of sampling is static relatively, is convenient to observe.On the contrary, when motor 48 drives sampling pieces 21 when other direction moves, the solution in the sampling pond 50 and retort 41 interior cell solutions mix.
Embodiments of the invention one:
Be checking the present invention, use a kind of online cell micro observation instrument that is used for biochemical reactor of the present invention reorganization culture propagation process to be observed and use at microorganism reactor
Every 2h sampling once carrying out cell optical density (OD600) mensuration, dry cell weight, off-line microscope blood count in incubation, online cell micro observation instrument is observed automatically and is counted, result such as table 1 simultaneously.
As shown in Table 1: online count results of the cell of online cell micro observation instrument and cell optical density (OD600), cell blood counting chamber count results basically identical, and bigger than normal a little when late stage of culture.Each cell concentration and fermentation time carry out the indicial equation match, linearly dependent coefficient is the highest with online cell micro observation instrument, with blood counting chamber off-line counting basically identical, and apparently higher than the result of cell optical density and dry cell weight counting method, this is because optical densitometric method and the dry weight method all can't be distinguished extremely, the solid particle in living cells and the fermentation liquor, thereby error is bigger.
Table 1 off-line, online cell count and cell optical density, dry cell weight result are relatively
| Incubation time (h) | Off-line blood counting chamber bacterium number (10 7Individual/mL) | Online cell micro observation instrument counting (107/mL) | Cell optical density OD600 | Dry cell weight DCW (g/L) |
| 0 4 6 8 10 | 0.99±0.12 3.10±0.11 6.29±0.10 12.11±0.11 16.46±0.09 | 1.2±0.08 3.12±0.09 6.31±0.10 12.27±0.10 16.50±0.11 | 8.2 8.9 10.1 12.1 13.8 | 6.1 6.6 7.2 8.3 9.2 |
| R2 (linear dependence) | 0.9908 | 0.9934 | 0.9237 | 0.9353 |
Example two of the present invention:
Be checking the present invention, use the online cell micro observation instrument that is used for biochemical reactor of the present invention recombinant animal cell (HEK293) incubation to be observed and use at the mammalian cell reactor
The every 24h sampling once carrying out of sweat off-line cell blood count.The two cell count result such as table 2.As shown in Table 2, the cell quantity count results basically identical of the blood count of off-line and online cell micro observation instrument.
Table 5L mechanical stir-reactor is cultivated the HEK293 cell density relatively
| Time | Day0 | Day1 | Day2 | Day3 | Day4 | Day5 | Day6 |
| Blood count * online the counting of 105 cellsml-1 * 105 cellsml-1 | 1.50± 0.2 1.48± 0.2 | 2.5±0.2 2.3±0.2 | 3.6±0.2 3.5±0.2 | 5.0±0.2 4.9±0.2 | 6.8±0.2 6.9±0.2 | 9.0± 0.2 8.8± 0.2 | 7.2± 0.2 7.1± 0.2 |
Claims (7)
1. online cell micro observation instrument that is used for biochemical reactor, comprise scope main body (43), object lens (24), observe entrance window (22), sampler (40), external light source system (60), scope main body (43) front end is provided with observes entrance window (22), observe entrance window (22) front portion sampler (40) is set, in the scope main body (41), observe entrance window (22) back and place object lens (24) and external light source system (60), it is characterized in that, between described external light source system (60) and the object lens (24) reflecting prism (25) is set, object lens (24) back, observe the preceding catoptron (23) that is provided with of entrance window (22), annular diaphragm sheet (26) is placed in reflecting prism (25) front, and reflecting prism (25) top, side is equipped with CCD or array image sensor (30); Described sampler (40) is by sampling piece (21), flexible member (38), mobile device constitutes, wherein, be connected flexible member (38) between sampling piece (21) and the driving shaft (51) of mobile device, sampling piece (21) front end face with observe gap formation between the entrance window (22) pond (50) of sampling.
2. the online cell micro observation instrument that is used for biochemical reactor according to claim 1 is characterized in that, described external light source system (60) is by light source (28), condenser (29), and interchangeable color filter (27) is formed; Described light source (28) is Halogen lamp LED or LED lamp, and the light of its emission focuses on through condenser (29) and produces required wave band light through interchangeable color filter (27) after the back forms parallel beam.
3. the online cell micro observation instrument that is used for biochemical reactor according to claim 1 is characterized in that, described scope main body (43) is connected with retort (41) by set nut (42); Adopt O-ring seal (45) sealing between scope main body (43) and the retort (41).
4. the online cell micro observation instrument that is used for biochemical reactor according to claim 1, it is characterized in that, described mobile device is by driving shaft (51), connecting link (46), sampling pull bar (44), flexible member (39) is formed, the connecting link (46) that driving shaft (51) wherein are housed is connected with sampling pull bar (44) by screw (47), (9a) seals by welding and is connected sampling pull bar (44) in the junction with flexible member (39), and also (9b's other end face of flexible member (39)) is connected with scope main body (43) by welding in the junction simultaneously.
5. the online cell micro observation instrument that is used for biochemical reactor according to claim 1, it is characterized in that, described mobile device is by driving shaft (51), motor (48), sampler barrel (49) is formed, wherein sampler barrel (49) is connected the front end of scope main body (43), and sampler barrel (49) rear end is equipped with motor (48), and motor (48) output shaft connects driving shaft (51).
6.. the online cell micro observation instrument that is used for biochemical reactor according to claim 1 is characterized in that described sampling piece (21) is made of three different-diameter right cylinders, and is made by stainless steel or sapphire material.
7.. the online cell micro observation instrument that is used for biochemical reactor according to claim 1, it is characterized in that, described CCD or array image sensor (30) are given image acquisition and processing unit (31) through opto-electronic conversion with digital data transmission, collection processing unit (31) is sent result into computing machine (32) analysis, shows or storage.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008154869A1 (en) * | 2007-06-18 | 2008-12-24 | Shanghai Guoqiang Bioengineering Equipment Co., Ltd | Examination microscope for biochemical reactor |
| CN103207151A (en) * | 2013-04-18 | 2013-07-17 | 上海科源电子科技有限公司 | On-line cell microscopic observer for biological reactor |
| US8785180B2 (en) | 2007-06-18 | 2014-07-22 | Shanghai Guoqiang Bioengineering Equipment Co., Ltd. | Biochemical reactor |
| CN104204771A (en) * | 2012-02-23 | 2014-12-10 | 赫斯托因德私人有限公司 | A digital imaging system for biopsy inspection |
| CN104677299A (en) * | 2013-11-29 | 2015-06-03 | 上海微电子装备有限公司 | Film detection device and method |
| CN105699379A (en) * | 2016-03-14 | 2016-06-22 | 南京市疾病预防控制中心 | Schistosoma myracidium detection device |
| CN106594553A (en) * | 2017-01-11 | 2017-04-26 | 哈尔滨理工大学 | Newborn nostril illumination device |
| CN113092346A (en) * | 2021-04-06 | 2021-07-09 | 中国水利水电科学研究院 | Algae cell counting detection system and detection method thereof |
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- 2007-06-18 CN CNB2007100421890A patent/CN100561203C/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008154869A1 (en) * | 2007-06-18 | 2008-12-24 | Shanghai Guoqiang Bioengineering Equipment Co., Ltd | Examination microscope for biochemical reactor |
| US8785180B2 (en) | 2007-06-18 | 2014-07-22 | Shanghai Guoqiang Bioengineering Equipment Co., Ltd. | Biochemical reactor |
| CN104204771A (en) * | 2012-02-23 | 2014-12-10 | 赫斯托因德私人有限公司 | A digital imaging system for biopsy inspection |
| CN103207151A (en) * | 2013-04-18 | 2013-07-17 | 上海科源电子科技有限公司 | On-line cell microscopic observer for biological reactor |
| CN104677299A (en) * | 2013-11-29 | 2015-06-03 | 上海微电子装备有限公司 | Film detection device and method |
| CN105699379A (en) * | 2016-03-14 | 2016-06-22 | 南京市疾病预防控制中心 | Schistosoma myracidium detection device |
| CN105699379B (en) * | 2016-03-14 | 2018-07-31 | 南京市疾病预防控制中心 | Schistosoma miracidium detection device |
| CN106594553A (en) * | 2017-01-11 | 2017-04-26 | 哈尔滨理工大学 | Newborn nostril illumination device |
| CN106594553B (en) * | 2017-01-11 | 2019-03-29 | 哈尔滨理工大学 | A kind of newborn nostril lighting device |
| CN113092346A (en) * | 2021-04-06 | 2021-07-09 | 中国水利水电科学研究院 | Algae cell counting detection system and detection method thereof |
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