JPH01502131A - Detection of evanescent wave background fluorescence/absorbance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Evanescent wave background fluorescence/absorbance detection Background of the invention field of invention The present invention relates to biological culture and fermentation media in bioreactors, etc. Methods and apparatus for the detection and measurement of optical properties consisting of It relates to the measurement of continuous phase fluorescence and/or absorbance in situ.

Detailed description of related technology The fluorescence of NADH or NADPH in cells is an indicator of cell concentration in the culture medium. It acts as an indicator as well as a good indicator of the metabolic status of cells in culture. It has been shown that Decatur. Some papers cover both microorganisms and yeast. This demonstrates the value of this type of in-situ measurement applying culture.

For example, "Culture" by Dublis B. Armiger (W, B, Armiger) Use fluorescent light to thicken Escherichia coli) "Analysis and control of fermentation of Fed-Batch produced" Biote Technology Bulletin 84. See Washington D.C. 1984. these The equipment for performing the measurements was from Malvern, Pennsylvania. Fluco from Biochem Technology Inc. It can be entered as the roMeasurej™ system.

From an economic point of view, alcoholized molasses or It would be beneficial to use complex nutrients such as grains at lower cost. but However, these introduce additional background fluorescence. If the medium does not change during culture If a fluorescent component is included, background (bank ground) correction will change the reading at the zero point. This is easily done by subtracting from all subsequent readings. Fluorescence from the medium hits the cells If the cells are displaced by use of It becomes more difficult to correct for changes online. This is a continuous sample for batch cultures by removing cells and measuring the fluorescence of the medium. can be done. For continuous cultivation or by adding nutrients stepwise during cultivation. Background correction is more difficult when adding.

It is possible to measure how much the fluorescence of the medium changes during fermentation. and as a result measurements can be made regarding the metabolic status and growth rate of the cells. It would be beneficial to do so. What is needed is an effective separation of cell fluorescence from soluble materials. It is a means to do so.

Optical sensors for fermentation or tissue culture provide information about intracellular substances and conditions. It is possible to give. Such information is based on existing online sensors, temperature, - more precise based on real intracellular information rather than dissolved enzyme, off-gas analysis This will allow for greater control. It was obtained from recombinant DNA and cell fusion techniques It will lead to better scaling and commercialization of products. Currently in optical background Since there is no easy way to compensate for changes in to work best.

The early research using optical sensors for intracellular metabolism described below was in 1957. This is because the fluorescence of baker's yeast is the same as that of NADH. The fluorescence of yeast with incomplete fermentation can be reduced by adding ethanol or glucose to the suspension. Duysen and Ameich et al. This is the year when Amesz observed it. Later, cultured fluorescence was developed in situ. field) and aerobic/anaerobic conversion in continuous culture. Harrison and Cha developed equipment that could be monitored. nce) was created. By using similar equipment, a firefly installed on the fermenter Optical instruments can measure changes in intracellular NADH and can be useful for process control. Bumphrey and his collaborators and others disclosed. Zabriskie and Hua+pb rey) describes the linear relationship between the logarithm of culture fluorescence and the logarithm of cell concentration. Disclosed. Ristroph 7X; f−x−−≦4’JZ (Candida utilis) was studied.

The concentration of intracellular NADH measured by culture fluorescence in fermentation depends on the number of cells, each This means that it is a function of the energy levels within the cell and the level of metabolic activity. disclosed their research. The mathematical formula obtained from these studies is as follows.

F(t) = (Y t/x (1+m(t))) ×(t) +E(t)X( t) is the cell concentration. The term in the keratin fuco is the fluorescent product, which is the property of the species. Depending on the constant component Yf7X, which is a characteristic of the aircraft, and changes in the level of metabolic action. It consists of a variable component m(t) that changes. The last term E(t) is Light is primarily concerned with environmental or background fluorescence. evident So, if E(t) varies during the fermentation, then the total fluorescence measured Obtaining information about cells from a cell is difficult or otherwise impossible. . Continuous or bunch-fed fermentation or cell culture eliminates the problem. It only makes things worse. In those techniques, additional variables include information regarding concentration. To respond to information (to be incorporated).

Corrections for or where E(t) is low must be made empirically. Most of all published studies have used synthetic media where there was. Economic factors dictate when scaling up fermentation and cell culture for commercialization. Natural nutrients such as molasses or fetal cow serum - and they are naturally fluorescent. and therefore contribute to the background value - their use will be. background When checking some of the assumptions used in the correction, e.g. the background fluorescence of the molasses and We found indications that fluorescence in yeast cells does not add linearly. rear online There is a need for a method to measure the background of media fluorescence in real time, i.e. fermentation or One sensor or This points out the need for a method using sensors.

Fluorescent media and simultaneous cell separation without physically separating cells from the media This means that a method such as fluorescing is needed. There is. According to the invention, evanescent wave phenomena are used to meet this need. It is being

Hikarijiri Absolution A non-reflective interface between two optically transparent media in which the light beam has a different index of refraction. When all the waves are reflected, an evanescent wave phenomenon as shown in FIG. 1 exists.

The light beam 11 is totally reflected from this type of surface, which is different from the mirror surface, and it is As if passing about half of the wavelength into a medium 12 with low density such as an aqueous medium. act. Reference numeral 15 is a part of the light beam 11 within the medium 15 with low density. It is an Ebane 7 cent wave. Reference numeral 16 is a measuring arrow indicating the distance and the light beam 1 1 wavelength.

If it were to be reflected off a mirror surface, it would be Figures 1 and 2 schematically show that the reflected beam is placed at It is shown in This configuration has been shown experimentally, and it shows that evanescent waves exist. This is one of the proofs of this. This part of the light beam has many characteristics of standing waves parallel to the surface. It has quality. Figure 3 shows how the intensity decreases with distance from the surface. It shows.

In Fig. 3, N is the incident wave, R is the reflected wave, and θ is the critical angle θ. at larger angles of incidence be. Z is the less dense medium measured from the interface with the more dense medium is the distance axis. Eo is the value of light at zero depth in a less dense medium. This is the initial magnitude of the electric field component. dp is the depth of penetration and its value at the surface e It is defined as the distance required for an electric field to lower −r. What is the dpO value? The density of the two media is directly related to the wavelength in the medium and inversely proportional to the angle of incidence. The percentage of refractive index has been reached. The maximum intensity of the Ebane 7 cent wave occurs at the surface; decreases exponentially with distance from

It is absorbed by a substance with a certain color and if that substance is fluorescent If so, it excites the substance so that it emits fluorescence. At the relevant 340 nm wavelength , this evanescent wave sweeps over an area of 1 square centimeter. The volume in liters would be 1.7Xl0-" liters.2.5ω length For an optical fiber with a diameter of 200 μm, the swept volume is 1.3Xl0 −” It must be Lindor.

The present invention allows light to flow from an interface between two optically transparent media with different refractive indices throughout the entire system. evanescent formed in a less dense medium when This separation can be achieved by using the properties of waves. waves in water stratification Since only about 1/3 to 1/2 of the wavelength penetrates into the optical waveguide, intact cells are It is not possible to be in the volume of a fluid swept by a springant wave. This is based on my observation that this is unlikely. Therefore, the present invention It measures the fluorescence of a medium without interference from light or the fluorescence of molecules in a solution. Ru.

The same concept is used to measure the optical absorbance of media independent of cells and special substances. It is commonly used for This means that the computer system required to analyze the data greatly reduces program complexity and therefore easier control of fermentation and tissue culture Eggplant.

At normal concentrations of cells in bioreactors, as mentioned above, the cells This means that there will be a fluid in this small volume even for a given time. Impossible.

Furthermore, for example, at an excitation wavelength of 280 nm, only evanescent waves exist in the liquid phase with approximately 1 10-170 nm penetration, even if cells are stationary just on the surface of the optical waveguide. Even when It will not interact with the Evanescent 7 cent wave. In this way, it interacts with light waves. By limiting the volume that can be created to that within the evanescent wave, the present invention It virtually proves the separation of intracellular fluorescence or absorbance from the quality fluorescence or absorbance. Ru.

The purpose of the present invention is to enable intracellular optical measurements under a wider variety of culture conditions. is to facilitate the development of fermentation and cell culture. Because it's online There are methods to continuously compensate for changes in the environment or background in real time.

Furthermore, it is an object of the present invention to reduce the optical effects of cell contents from the optical effects of the culture medium. It is to provide separation.

Furthermore, it is an object of the present invention to change the culture medium without interference from changes in cell contents. The goal is to lead to change in the world.

Furthermore, it is an object of the present invention to maintain cell contents without interference from changes in the culture medium. The goal is to lead to change in the world.

A still further object of the invention is to reduce the cost of monitoring cell culture fluorescence. That's true.

A still further object of the present invention is to isolate background or environmental fluorescence, off-line. Since no empirical measurements need to be made, the expansion of the medium in fermentation is a matter of change. The goal is to shorten the time it takes to By performing a single test, the culture conditions Preliminary optimization will be done with specific addition of media components.

It is still a further object of the invention that shear forces or other mechanical or chemical Provide information on the extent of cell rupture, regardless of the cause. That is.

According to the invention, the effect of Stirling force (stirring force) on the integrated cells is Operations when measured online in real time and data subsequently analyzed Corrections can be made during the fermentation operation rather than afterwards.

A still further object of the present invention is to provide optical fibers for directing other non-fluorescent intracellular substances. The ability to correct for background, allowing the use of absorbance methods and resulting in continuous double beam It is equivalent to the spectrophotometer needle.

A still further object of the invention is to of individual nutrients in the culture medium through improved ability to induce specific changes. The goal is to better control the concentration.

A still further object of the present invention is to greatly improve the products of fermentation and microculture. more precise control based on the metabolic state within the cell, reducing the time and expense of scale-up. new recombinant DNA and tissue culture technologies through more efficient fermentation and tissue culture techniques. The aim is to promote the commercialization of cell fusion technology.

Shaped plate ■Sara Umbrella Store FIG. 1 is a schematic diagram showing the evanescent wave phenomenon, in which a light beam 11 is connected to an optical fiber. It passes through a waveguide 13 like this and traverses the path from left to right.

FIG. 2 is an enlarged view of the circular portion 2 of FIG.

Figure 3 shows the change in evanescent wave intensity according to the distance from the interface between two media. FIG.

FIG. 4 is a partial schematic cross-sectional front view of one variation of the invention.

5 is a cross-sectional side view of the embodiment shown in FIG. 4, taken along transverse line 5--5 of FIG. The section cut along the line is viewed from the right.

FIG. 6 is a partial schematic diagram showing another embodiment of the present invention.

7 is a cross-sectional side view of the embodiment shown in FIG. 6 taken along line 7--7 of FIG. The cut section is viewed from the right.

FIG. 8 is a partially schematic cross-sectional front view showing a preferred embodiment of the present invention, in which the optical waveguide is A flat plate 1316 and a barrier 1312 confine the liquid to one end of the waveguide. .

FIG. 9 is a front view of disk 1328 taken along line 9 of FIG.

FIG. 10 is a cross-sectional side view of the embodiment shown in FIG. The section cut along line 10 is viewed from the right.

FIG. 11 is an enlarged view of region 11 in FIG.

FIG. 12 is a schematic diagram of the embodiment shown in FIG. 8, showing the path of the rays and the data processing. It shows the stages.

FIG. 13 shows an alternative implementation of the design contained within element 1331 of FIG. 1 is a schematic illustration of the contents of an example light source and detector housing.

FIG. 14 is a partial view of the embodiment shown in FIG. 13, along line 'a14' in FIG. Looking at the removed part from the right.

Details 1st mouth sucking The embodiment of the invention shown in Figure 4.5 allows an evanescent wave of light to stimulate the fluorescence of a solution. a metal housing 4 surrounding an optical fiber 44 used therein for pumping; By immersing the lightweight rod 40 consisting of 1 into a liquid, a solution containing a specific substance is created. provides a relatively simple means of measuring background fluorescence. Housing 41 defines the chamber 47 and contains the solution to be tested within the chamber 47. Liquid flows through opening 46 in housing 41.

The portion of the optical fiber 44 within the chamber 47 has an opaque sheath 42 and a transparent cloth. 43 has been removed, exposing fiber 44 directly to the solution in chamber 47. ing.

A light source of visible or invisible light, such as an incandescent lamp, for illuminating the fiber 44; A light source 1301 such as a laser, an excitation beam lens 1302, an excitation beam filter 13 03 etc. are arranged as shown in FIG.

The light emitted from the light source 1301 passes through the lens 1302, where it is made parallel, and then the light emitted from the lens 1302 is parallelized. filter 1303, where the desired wavelength (schematically depicted by the dashed arrow) ) is filtered into a monochromatic excitation beam 1326 and passes through an aperture in plate 1340. .

Excitation beam 1326 converts the wavelength light represented by excitation beam 1326 into an excitation emission beam. A micro ink mirror 1349 provided to reflect the image to the camera lens 1358 reach.

Here, beam 1326 is focused and enters directly from the proximal end of optical fiber 44; The proximal end of the optical fiber 44 may extend from the metal housing 41 if necessary. . As mentioned above, excitation beam 1326 propagates within optical fiber 44 to the end, e.g. For example, it reaches an optical trap 45 made of black silicon rubber.

The evanescent wave portion of the pump beam 1326 propagates down the optical fiber 44 and exits the optical fiber 44. very close to fiber 44 (i.e. within 172 to 1/3 wavelength as described above) ) meet the solution molecules in the chamber 47 and encourage the solution to fluoresce as much as possible. wake up

Such fluorescent light enters the optical fiber 44 and propagates to the proximal end as a luminescent beam 1327. , where it exits the optical fiber 44 and is delivered to the excitation emission beam lens 1358 and focused. Ru. Beam 1327 then passes through a guikro ink mirror 1349 and It is formed to pass the wavelength of light emitted by the fluorescence of the solution being measured. ing.

The emitted beam is then emitted by an electron beam, e.g. from a multiplier phototube in counting mode. Detection system 1323 is hit.

The detection system 1323 is equipped with a filter or a monochromator. Therefore, it responds selectively to the wavelength of the emitted light. Electronic detection system 1323 Generates a signal to be sent to the quinine ratio amplifier 1344. Reference detector 13502, excitation Detects the intensity of beam 1326 and sends it to lock-in ratio amplifier 1344 signal, where the two signals compensate for the excitation beam 13260 intensity changes. The same processing as before is performed.

The signal from the lock-in ratio amplifier 1344 is converted into an analog-to-digital converter. 1345, where the digital display panel or signal processor 13 46. Preferably (the solution and certain substances in it) an additional digital signal 135 from a known device for reading bulk fluorescence (containing 2 is generated and similarly sent to the display panel and processor. Signal 1352 is one example For example, trademark (Fluoromeasure) manufactured by Biochem Technology Co., Ltd. (Marube) The light is emitted from a fluorometer at the University of Pennsylvania, Pennsylvania. Data of signal 1345 is subtracted from the data in signal 1352, the resulting data is sent to member 1351. fluctuations in the solute fluorescence are subtracted from the bulk fluorescence, so that the It represents fluorescence.

Figure 6.7 shows another embodiment, a chamber with a pair of open lowers 2.73. one that utilizes the optical fiber 62 provided within the housing 61 that forms the optical fiber 67; It is.

The slurry (muddy fluid) that is the object of the optical measurement of the present invention is introduced from the input lower 2. and is discharged from the output lower 3. Preferably, chamber 67 has laminar flow. It is recommended that the design be made as follows.

As in the embodiment shown in FIGS. 4 and 5, the light from the light a 1301 is transmitted through the lens 130. 2. It passes through the filter 1303 and is guided to the end of the optical fiber 62. however , which strengthens the evanescent waves associated with light propagating straight through the optical fiber 62. In order to Use rather than have. This is incident on the optical fiber 62 along the axis Block out the light.

In this configuration, reference detector 1350 is carried by excitation beam 1326. The light source 1301 is arranged along a different optical path than the light source 1301. This is the light source It is possible to detect changes in the intensity of the signal and supply it to the lock-in ratio amplifier 1344 as before This is to ensure that

At the end of the optical fiber 62, an electronic detection system 1323 is located and internally receive the light that propagates. Detection system 1323 detects the emitted beam 1326. The device is configured to detect the intensity of light at a wavelength. In that case, the cellular or other A useful indicator for measuring the absorbance of solutes in solution that are free from particulate matter. issue a number. The absorbance information will be related to the concentration of the solute being monitored and , a background number that may be appropriately subtracted from other absorbance readings that provide useful data. It might be.

Alternatively, the detection system 1323 detects the fluorescence emitted by the solute. assembled with a suitable filter or monochromator to detect the wavelength of light. The resulting data may be used to implement the invention shown in Figures 4 and 5. Similar to that generated by the example.

As with the previous embodiment, the signal from the detection system 1323 is 1344 and then output directly to a display panel or processor 1346. The output is output to, for example, a chart recorder 1353 as shown in the figure. Ru.

By using evanescent wave development, FIGS. As shown in Figure 11, the sequential mode quickly alternates for virtually simultaneous reading. It is possible to measure both absorbance and fluorescence. Regarding the plane plate 1316 as a waveguide However, the device housed within the detector sheath 1332 is Real-time measurement of several variables that helps measure the instantaneous concentrations of various components of a product. 1312 or for use in a reactor fermentation vessel 1312. It is configured.

The detector cladding 1332 is a conventional pipe extending from the wall of the reactor vessel 1312. It is disposed within the loop-shaped mounting hole 1362. The end of the mounting port 1362 is the grommet 1 333, and the grommet is threaded to engage with cover 1332. It is attached to the opening 1362 and thereby secured to the reactor wall 1312.

The length of the mounting port 1362 and the length of the detector housing 1332 into the reaction mixer 1314 Due to the desired depth of penetration beyond the wall 1312, the rubbery O-ring 1311 is inserted into one of the three O-ring grooves 1361, and the housing 1 332 retaining sleeve 1354 is watertightly sealed within mounting bay 1362.

A threaded waveguide plate mounting sleeve 1336 engages a retaining sleeve 1354. to firmly hold waveguide plate 1316 in place. Choose from several lengths Insert 1335 having one length with a waveguide plate 1316 12 into the reaction mixer 1314 a predetermined distance into the reaction mixer 1314. Extend the leaves 1336.

A fluorescent enclosure 1308 extends outward from the vessel wall 1312 and is attached to the insert 13. It is screwed together with 35. Among the aforementioned elements are optical fiber elements 1305, 13 17. 1319 and 1320 are included, and the element is directed toward the waveguide plate 1316. or transmit light from there. Optical fiber element 1305.1317. 1319.1320 is defined by the fluorescent enclosure cover retainer bezel 1309. It passes through the fluorescent enclosure cover 1307 which is held in place.

The light source 1301, lens 1302, filter 1303 and aperture 1340 are - As mentioned above in i. The excitation beam optical chopper 1325 is schematically shown in FIG. The optical path through the focused excitation beam 1326 is shown schematically in FIG. and evanescent wave pumping fiber optic 1305 and fiber optic 1320. They are interfering with each other in succession. As shown in more detail in Figure 9, the excitation beam light beam The chopper 1325 includes a disk 1328 on which a semicircular mirror 1329 is placed. The pump beam 1326 is connected to the other half of the disk 1328 through an optical fiber 1. It has an opening 1342 wide enough to allow passage in 320 directions.

Chopper disk 1328 is rotated by shaft 133o. disk 1328 Upon rotation, excitation beam 1326 is directed into one of two optical paths. excitation beam 1326 is projected onto mirror 1329, the beam follows optical path 1326A. , toward the optical fiber 13o5. Alternately, the disks 1328 are connected to the excitation beam 13 26 is rotated to a position where it is projected into aperture 1342, it connects optical fiber 132o Pass in the direction of.

The optical fiber 1305 receives the pump beam from a sealed, light-tight housing 1331. 1326A to the fluorescent detector enclosure 1332. Optical fiber 1305 Complete with flexible transparent craft 1306 and flexible opaque outer device 304 It typically consists of several individual fibers coated with a fiber. transparent clarinet The refractive index of the fiber 1306 is slightly less than that of the optical fiber 1305.

After passing through grommet 1333, the individual optical fibers of optical fiber 13o5 are It passes through an expanded mounting block 1355, as shown in FIG. light Fiber mounting block 1355 withstands sterilization temperatures of approximately 140”C. Injection molding of plastic, such as polynurphon, is preferred.

of optical fiber 1305 connected to prism 131o along an opaque surface. The individual fibers are cut perpendicular to the longitudinal axis of the fiber. mounty The blocking block 1355 allows light to be incident at right angles to the opaque surface of the prism 1310. The fiber bundle 1305 is held in this way. The plate in contact with the plane plate waveguide 1316 The angle of the opaque surface of 1310 with respect to the side of the rhythm aligns the light beam 1326A with the plane This is the angle that leads to the plate waveguide 1316 at an angle greater than the critical angle.

As a result, the light beam 1326A is confined in the plane plate waveguide and connected to the waveguide 1316. Evanescent waves can be generated at the interface between the waveguide and the aliction medium in contact with it. Probably.

Prism 1310 is rectangular and has the same refractive index as plane plate waveguide 1316.

of the excitation beam 1326 during its transmission from the optical fiber 1305 to the prism 1310. Intensity loss occurs when the sides of prism 1310 are larger than the diameter of optical fiber 1305. By having also the optical fiber 1305 and the prism 1310 equal to The surface of the inclined prism 1310 covered with the liquid 1324 having a minimized by having a transient interface.

The flat transparent side of the prism 1310 connects the prism to the end face of the plane plate waveguide 1316. Use a transparent adhesive having the same refractive index as 1310 and the flat plate waveguide 1316. It is joined by The actual angle of the inclined surface of prism 1310 is 1310 and the refractive index of the materials used in the planar plate waveguide 1316. Plane The plate waveguide 1316 has a circular cross section and is made of a bubble-free and undistorted material, such as quartz. It is made. The two parallel surfaces 1337 and 1338 are highly optically polished. true parallel within normal manufacturing tolerances. Cylindrical shape of plane plate waveguide 1316 The side wall is significantly taller than its diameter. The plane plate waveguide 1316 extends along the side wall. The sound is prevented by an opaque seal 1356 of PTFE polymer (e.g. Teflon, etc.). It is sealed to prevent light loss and act as an encapsulant. Plane plate waveguide The front surface 1338 of the tube 1316 is coated with a very thin, hard transparent layer, such as a welded diamond. , for example, Surlyn (manufactured by DuPont). waveguide The thickness of the tube coating 1315 is such that the evanescent waves 1339 are within the medium 1314. has no effect on penetration. Waveguide coating 1315 coats medium 131 Preventing the adhesion of products on cells created by impurities and particulate matter 1313 in 4. is desirable. The waveguide coating 1315 also coats the planar plate waveguide 1316. Prevents damage such as scratches to the front surface 1338.

The evanescent wave 1339 is generated by the excitation beam 1326 inside the plane plate waveguide 1316. This occurs when the light is repeatedly reflected between the two non-specular surfaces 1337 and 1338. Ru. The evanescent wave 1339 generated in this way is transmitted through the plane plate waveguide 1316. It penetrates through the front surface 1338 and into the media 1314 under supervision. As mentioned above, The evanescent wave is approximately half the wavelength of the excitation beam 1326 into the medium 1314 under monitoring. It can only penetrate up to a distance. Discrete particulate material such as cells appearing in the medium 1314 1313 does not substantially affect the evanescent wave 1339.

As shown in FIG. 8.12, the opposite end of the planar plate waveguide 1316 has its distal end 13 A prism arrangement 21341 similar to prism 1310 is provided along 37. Ru. The thickness of the plane plate waveguide and the distance between the prisms 1310 and 13411 are determined by the projected light beam. beam 1326A is refracted by an integral number of times and exits from prism 1341. is being done. The ends of the individual fibers of optical fiber 1317 are Cut at right angles to the axis. The mounting block 1355 is The fiber bundle 13 is arranged such that the longitudinal axis of the fiber bundle 13 is perpendicular to the opaque surface of the prism 1341. Hold 17.

An alternative structure is to use a planar waveguide 1316 and a prism 1310.134. 1 may be assembled as a single unit. Furthermore, the relationships illustrated here Instead, the surfaces of prisms 1310 and 1341 are drawn onto the surface of waveguide 13160. The slopes of the prisms may be recessed into the surface of the waveguide. Furthermore, other As an example, the two diametrically opposing ends of the waveguide plate may be beveled and the prism 131 It may serve to perform the same function as a 0°1340 bevel.

Optical fiber 13170 Individual fibers are spliced to the slope of prism 1341 Transmission from the liquid film 1324 with a refractive index close to that of the plane plate waveguide 1316 The national interface is established. The optical fiber 1317 is an optical fiber. It passes through the fiber mounting block 1355 and has a circular cross section. optical fiber The fiber 1317 then passes through the grommet 1333 and the light source and detector housing. Enter 1331. As shown in FIG. 8, there is light in the direct optical path of the optical fiber 1317. There is a chopper assembly 1318, which is similar to the excitation light chopper 1325. The structure is as follows. Next to the optical chopper assembly 1318 in the same direction , a fermentation beam filter 1321 that removes all non-fluorescent light, and an electronic detection system. A light emitting beam lens 1322 that converges a light emitting beam 1327 on a system 1323. more guided.

FIG. 12 schematically shows the optical path of the embodiment of the invention shown in FIG. light A light beam 1326 from a source 1301 is applied to a fiber optic bundle 1305, 1320. The light is focused by lens 1302 so that the light is properly connected. Light into optical fibers and waveguides When connecting, it is necessary to pay attention to the numerical aperture (NA) of the optical fiber or waveguide. . Numerical aperture (NA) and fiber diameter or fiber bundle or crystal waveguide Matching the lens 1302 is a problem.

Fiber cladding must be carefully prevented from acting as a waveguide. No. This is achieved by covering the cladding with an opaque sheath. Uniform To obtain a uniform distribution of light, the lens is used to uniformly distribute the light at the entrance surface of the fiber or waveguide. must be restricted so that The light then passes through the slit or diaphragm. It passes through and from there leads to chopper 1325.

The chopper 1325 alternately cuts the excitation light beam 1326 to the mirror 1. By removing 329, light beam 1326 is divided into two optical paths 1326A and 13 26B.

Beam 1326A is directed through fiber bundle 1305 to prism 1310 and its It then properly connects the optical beam to the plane plate waveguide 1316, so that the waveguide The light beam is guided through multiple internal reflections. Evanescent waves are selected It is absorbed by solution components that can interact with light at the given wavelength. Ebane The incident wave optically interacts with the particles 1313 suspended within the reaction medium 1314. Does not interact or excite fluorescence. Solvents that can emit these fluorescent lights The liquid components emit their fluorescence at or near the surface of the flat plate waveguide 1316. Ru.

A portion of the emitted light is coupled to a waveguide and passes through optical path 1327A to chopper 1318. transmitted to. Light beam 1326B passes through the window through fiber bundle 132o. is transmitted to the inner surface of the flat plate waveguide 1337. This means that the light hits the surface at right angles. The light travels straight and illuminates the bulk suspension near the plane plate 1316, causing the calyx to solution 1314 and particles or cells suspended therein capable of emitting fluorescence 1 313 are excited to emit fluorescence.

A portion of the emitted fluorescent beam 1327B returns through the flat plate 1316 and enters the fiber. It enters bundle 1319 and is guided to chopper 1318. Chopper 1318 is Shin It is synchronized with the chopper 1325 through the clocker 1348, and as a result, the chopper 13 25 diverts the light beam onto path 1326A, chopper 1318 diverts the light beam onto path 13 The light passing through 27A passes through a filter 1321 and a lens 1322 to a detector 1323. Ras. When chopper 1325 diverts the light beam onto path 1326B, the chopper 1318 is the diameter! The light passing through 1327B passes through filter 1321 and lens 1322. It leads to the detector 1323.

Synchronizer 1348 also agrees the chopper position and signal processing steps. As a result, the signal processor can e.g. As in the measurement of Nescent wave fluorescence, the signal generation mode can be monopolized. process the files.

The signals from the detector 1323 and the synchronizer 1348 are, for example, AC-DC. input to the converter and linearizer 1343 and then the lock-in ratio amplifier. 1344, where the amplitude of the signal is input to a reference detector 1350. corrected for changes in excitation beam amplitude detected by the analog to a digital converter 1345, and then a digital display or signal amplifier. Output to processor 1346 and then output to digital memory or storage device 1347 It will be done.

If filter 1321 is configured to pass the emitted fluorescent wavelengths, If so, the device of the invention measures fluorescence. If the filter 1321 If the device is configured to pass the same wavelength as 26, the device will pass the light of solution 1314. Measure the optical absorbance.

If it is desired that only fluorescence and not optical absorbance be measured, use the Alternative embodiments may omit the chopper 1318 of FIG. 13.16. The result As a result, the light beam 1327A is directed to the filter 1321 and the detector 1323. Rather, it is simply captured. The fiber optic 1317 is omitted in such embodiments; Alternatively, it may be replaced with an optical trap. Or the fiber light 1317 itself The light is transmitted away from the planar waveguide 1316 as a light trap. light beam 13 26B and the surface of the plane plate waveguide 1316. Both of the solution fluorescence excited by the light beam 1326A emitting the It is transmitted to filter 1321 over optical path 1327B. The filter 1321 is Only the emitted fluorescent wavelengths are selected to pass.

FIG. 13 shows another embodiment, in which the contents of the housing 1331 of the embodiment of FIG. The contents of the housing 136o are used instead. This example uses a slow optical beam. For example, it is suitable for blocking frequencies of several Hertz or a fraction of 1 Hertz. This is a case like 1. Chopping device shown in the direction of arrow 14 in Figure 13 The location is shown in FIG.

The plane plate 182 is attached to the shaft 181, and the shaft 181 is connected to the deceleration clutch 18. It is attached to 3. A plane mirror 171 is attached to a plate 182.

The plate 182 to which the mirror 171 is attached is located at the position shown by the solid line in FIG. If not, it is rotatably supported between the positions indicated by broken lines. The stopper 172 is Limit the movement of mirror 171 so that plate 182 is adjacent to it, and repeatedly and precisely It acts like a fixed point to fix the position of. Mirror 173 is similar to mirror 17. 1, and -i, in the embodiments of FIGS. 8 and 12. Same as mentioned above.

Illustrated list of elements 40 Lightweight rod 41 Fluorescence detector metal housing 42 Optical fiber spring transparent exterior 43 Optical fiber transparent cladding 46 Opening in the housing 47 Chamber in the housing 65 Exterior 66 Liquid medium in the chamber 171 Mirror rotating frame 172 Rubber buffer stop collar (movement stopper) 173 Mirror 181 Electric motor shaft 182 Counterweight 183 Mirror overtorque prevention friction clutch 184 Low torque reversible electric motor 1301 Light source 1302 Excitation beam lens 1303 Excitation beam filter 1304 Typical optical fiber opaque device 305 Pumping fiber light - Evanef Cent Wave 1306 Optical fiber transparent clanging 1307 Fluorescent enclosure cover 1308 Fluorescent enclosure 1309 Fluorescent enclosure cover retainer bezel 1310 Excitation beam prism 1311 0-ring 1312 Reactor container wall 1313 Particulate matter 1314 Reaction medium 1315 Evanescent wave guide coating 1316 Flat plate evanescent Light wave guide 1317 Light emitting fiber light - Evanescent wave 1318 Light emitting beam light chopper 1319 Light emitting fiber light - direct wave 1320 Pumped fiber light - Direct wave 1321 Emission beam filter 1322 Luminous beam lens 1323 1 pre-detection system 1324 Prism, excitation fiber light, and light emitting fiber light - Evanescent 7 cent wave liquid separator between 1325 Excitation beam optical chopper 1326 Excitation beam 1327 Luminous beam 1328 Optical chopper disk (typical shape) 1329 Optical chopper mirror 1330 Optical chopper motor shaft 1331 Light source and detector housing 1332 Fluorescence/absorbance detector enclosure 1333 Grommet 1334 Fluorescence detector retainer flange 1335 Waveguide plate position adjustment insertion surface ( Various sizes) 1336 Waveguide mounting sleeve 1337 Waveguide end of tube 1338 Front end side of waveguide 1339 Evanescent wave 1340 Plate with opening 1341 Light emitting beam prism 1342 Optical chopper disk aperture 1343 AC/DC converter and linearizer 1344 Ronfin ratio pump 1345 Analog/digital converter 1346 Digital display panel or screen Gunarprose, S1347 Digital storage device 1348 Chopper Synchronizer 1349 Dichroic mirror 1350 Reference detector 1351 Cell fluorescence modified to medium fluorescence 1352 From bulk fluorescence detection device Digitized signal 1353 strip chart recorder 1354 Retainer sleeve 1355 Optical fiber mounting block 1356 Waveguide edge seal 1358 Emission-excitation beam lens 1360 Light source and detector housing 1361 0-ring groove 1362 Mounting port 44〒Kamishi^121〒Issue amount logo RL〒Shikyochudou vomiting view 2〒＼ま　IQ／ l name of 91 division/71 'E'F + Eyo F　ii’t? Song Dynasty on Mujaku 10' 0) July 14, 1988 Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1. Display of patent application PCT/US 8710 OO8B 2. Name of the invention Detection of Evanescent Wave Background Fluorescence/Absorbance 3, Patent Applicant Address: Swi, Philadelphia, Pennsylvania 19103, United States 2706 Chesnut Street 1919 Name Levin Berman Double Nationality United States 4. Agent Postal code 102 Telephone (03) 238-0031 Address Tokyo 6-1-18 Kojimachi, Chiyoda-ku, Miyako August 14, 1987 6. List of attached documents (1) Copy and translation of the written amendment) According to my invention described in 1. , the scope of the claims for which protection is desired by letters patent is as follows: As written.

1. (a) a continuous phase consisting of a component that emits fluorescence at an optically detectable wavelength; (b) a discontinuous phase consisting of a component that also emits fluorescence at the optically detectable wavelength; In an optical analysis method for a slurry consisting of: (i) exciting the light emission of the component; At the same time, the slurry is optically excited with an evanescent wave having a wavelength of (fi) detecting the intensity of the fluorescence generated therefrom; (ffi) said detected intensity of fluorescence is related to the concentration of said component in said continuous phase; Consequently, the detected intensity of the fluorescence is in the discontinuous phase in which the fluorescence also emits. (a) emits fluorescence at an optically detectable wavelength; a continuous phase consisting of components; (b) A discontinuous component consisting of a component that also emits fluorescent light at the optically detectable wavelength. phase and A method for optically analyzing a slurry comprising: (i) exciting the fluorescence of the component; simultaneously optically exciting the slurry with an evanescent wave having a wavelength of and (ii) the intensity of the fluorescence resulting therefrom at different but close times. a step of detecting; (ij) simultaneously irradiating the system with non-evanescent waves; and (iv) therein. detecting the intensity of fluorescence or absorbance resulting from the (v) determining the difference between the two intensity values; and (vi) determining the difference between the two intensity values; and (vi) determining the difference between the two intensity values. The difference is related to the concentration of said components in the continuous phase, so that said differences also emit fluorescence. a step that is independent of the concentration of the component in the continuous phase; A method consisting of

3. The continuous phase consists of the aqueous solution in the bioreactor, and the discontinuous phase consists of living cells. The method according to claim 2, comprising:

4. The method according to claim 3, wherein the fluorescence of NADH and NADPH is measured.

5. The excitation beam is approximately 366 nm and the fluorescence is measured at approximately 460 nm. The method described in item 4.

6. Excitation or illumination by light at an optically detectable wavelength emitted by the device. Disconnection of a system with a continuous phase that also emits or absorbs fluorescence when irradiated. In an apparatus for determining the fluorescence or absorbance of a subsequent phase, (a) means for exciting a continuous phase having an evanescent wave at said wavelength; (g) means for measuring the intensity of fluorescence or absorbance resulting from excitation of the continuous phase; (C) means for irradiating the system with non-evanescent waves at said wavelength; (means for measuring the intensity of fluorescence or absorbance resulting from irradiation of the target system; (e) measuring the magnitude of the intensity produced by one of the aforementioned measuring means; Compare the magnitude of the intensity generated by others and display the differences between them. means for determining the difference value so that said difference value is related to the concentration of said component in said discontinuous phase; , and the value is independent of the concentration of the component in the continuous phase that also emits fluorescence. A device.

7.Furthermore, (a) before operating the measuring means (d). activating and deactivating said irradiating means (C) before activating the measuring means (b). and the means to As a result, the measuring means (d) is activated only during the irradiation of the system by the irradiating means (C). , and the measurement means (b) excite the continuous phase with the evanescent wave excitation means (a). 7. The device of claim 6, which operates only during startup.

8.Furthermore, (-a light source of the wavelength, (c) Light is alternately guided from the light source to the excitation means (a) and the irradiation means (C). means and (i) means for generating a signal having a value related to the intensity of light of said wavelength detected; and, 0) means for guiding light from the system to the signal generating means (i); and (b) light deriving means. when the mouth is guiding light to the excitation means (a) and to the irradiation means (C); detecting when the light is being guided to the excitation means (a) and the measuring means (d); and whenever the light is guided to the irradiation means (a), said signal generating means means for directing a signal instead from (i) to said measuring means (b). The device according to scope item 7.

9. The excitation means (a) is an optical fiber at least partially covered with cladding. 9. The device of claim 8, comprising a fiber.

10. The apparatus according to claim 8, wherein the excitation means (a) comprises an optical plate.

11. Excitation or excitation by light at an optically detectable wavelength emitted by the device. system with a continuous phase that also emits fluorescence or absorbs light when irradiated In an apparatus for determining the fluorescence or absorbance of a discontinuous phase of (a) a light source of the wavelength; (b) a waveguide plate having a surface in contact with said system; and (b) a waveguide plate having a surface in contact with said system; at a sufficiently inclined angle to the surface to induce evanescent waves. directing light from the light source to the waveguide, resulting in excitation of a first state of the system; A means of being rubbed, (C) directing light from the light source to the waveguide at an angle substantially perpendicular to the plane; means for causing excitation of a second state of the resulting system; (d) alternately transmitting light from the light source to the deriving means (b) or the deriving means (C); a chopper means for guiding; (e) means for detecting the wavelength of light emitted by the system upon fluorescence; (0) Guides light from the waveguide plate to the detection means (e) at an angle substantially perpendicular to the plane. and means to (6) The detection means and the derivation means from the waveguide plate at an angle inclined to the plane. (f) Alternatively, the deriving means (chop which alternately guides light from the chestnut to the detecting means (e)) means and (:1) means for synchronizing the chomba means (d) with the chofba means (sha), and (j ) Compare the value obtained during the excitation of the first state with that obtained during the excitation of the second state and receiving a signal from the detection means (e) to produce an output related to the difference in said values. means for processing signals.

12. The means (b), (c), and (b) for guiding light are fiber-shaped optical members. 12. The apparatus of claim 11.

13. The means (C) and (f) for guiding light are each composed of a bundle of optical fibers. The device according to item 12.

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Claims (1)

[Claims] My invention which has been described for a while and which I wish to protect and claim under a letter patent teeth, 1. The process of exciting the continuous phase with evanescent waves and the resulting fluorescence a discontinuity that fluoresces or absorbs light at the relevant wavelength. The continuous phase of a slurry (muddy fluid) having a continuous phase (phase) in the presence of a A method of detecting fluorescence or absorbance. 2. The process of exciting the continuous phase with epanescent waves and the resulting fluorescence or detecting the absorbance and irradiating the system with non-evanescent waves; Detecting the resulting fluorescence or absorbance and measuring the difference between the two values. and a step of determining the Discontinuous phase of a system with a continuous phase that fluoresces or absorbs light at the wavelengths of interest A method for measuring the fluorescence or absorbance of 3. Means of exciting the continuous phase with evanescent waves and the resulting fluorescence or means for detecting absorbance and means for irradiating the system with non-evanescent waves. , means for detecting the resulting fluorescence or absorbance, and means for measuring the difference between the two values. Equipped with Disconnection of a system with a continuous phase that absorbs light at related wavelengths and emits fluorescence Device for measuring continuous phase fluorescence or absorbance. 4. Claims having means for performing each step continuously in real time The device according to paragraph 3. 5. The means to excite the continuous phase with evanescent waves is to remove part of the cladding. 4. A device according to claim 3, comprising an optical fiber. 6. Claim 3: The means for exciting the continuous phase with evanescent waves is an optical plate. The device described. 7. The continuous phase is a bioreactor and the discontinuous phase consists of living cells. The method described in item 2 of the scope of the request. 8. 8. The method according to claim 7, wherein the fluorescence of NADH and NADPH is measured. 9. The excitation beam is approximately 366 nm and the fluorescence is measured at approximately 460 nm. The method described in box 8.