JPS63502782A - micro spectrofluorometer - 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] micro spectrofluorometer This is a continuation-in-part of U.S. Application No. 828.766 dated February 11, 1986. respond.

background In scientific research, materials are often used to evaluate the response of fluorescent probes to radiation (radiation). Its characteristics can be expressed by the answer. In some methods, the sample is exposed to different waves. The sample is irradiated alternately with long light beams, and the fluorescence of the sample is recorded at different irradiation wavelengths. Ru. For example, calcium ions drive various cellular processes to a high degree of spatial and temporal precision. It is thought to be controlled by degrees. Calcium within a single biological cell, using a microscope and the fluorescent calcium-sensitive dye Fura-2. In particular, measurements have been made with a high degree of spatial resolution. dye added The sample is alternately irradiated with 340i and 380 nanometer light. free Fluorescent dyes emit an odor at approximately 500 nanometers in response to excitation at 380 nanometers. dyes associated with calcium ions emit the most fluorescence, whereas dyes associated with calcium ions emit 34 In response to 0 nanometer excitation, the largest number of Generates a lot of fluorescence. Therefore, the concentration of cartarum can be calculated from the following formula: Wear.

(Ca”)i = Kd ((R-Rmin)/(Rmax-R)) β here K (1rl, effective dissociation constant for Fura-2-calcium reaction, R is 340 and the ratio of fluorescence intensity at 500 nm to excitation at 380 nm, Rm in is the limit value of R when the calcium concentration is zero, and RmaX is the limit value of R when calcium concentration is completely saturated. value when summed, β is the optical constant of the system, without calcium Relative quantum efficiency values at 380 nm for dyes and calcium-saturated dyes. Ru.

A photoelectron coupled to a microscope to detect fluorescence from the microscopic portion of the sample The use of multiplier tubes provides excellent spatial and temporal separation for spectrofluorophotometers. It has been demonstrated that it provides high resolution. The microscope further has an alternating light source It is necessary to change it so that One type of dual frequency light source uses an interference grating by separating the light into monochromatic rays, and then alternating this monochromatic ray using a rotating mirror. It is designed to be oriented toward the sample. Interference gratings are optically insufficient and Its inefficiency is further increased by the low shock coefficient of the system. like this The system is characterized by the temporal resolution of typical biological or chemical reactions, or Provides fast switching speeds needed to avoid problems caused by artificial motion and similar I can't. Additionally, light sources are difficult to match and maintain, and are very expensive. The microscope is adapted as a micro-spectrofluorometer. filter assembly The body is positioned in an optical path that includes an illumination source, a sample on a microscope, and a detector. I'm reading. The filter assembly includes multiple bandpass filters with different passbands. The passband is circumferentially around the filter axis offset from the optical path. Separated. The filter is driven in the rotation of the filter axis and has multiple filters. Moving the filter elements sequentially into the optical path and thus the waves of light passing through the filter assembly The length can be changed. A detector such as a photomultiplier tube with a photon counter detects light from the microscopic portion of the sample. Light detection depends on the position of the rotating filter Synchronized. The filter assembly is positioned between the illumination source and the sample and The wavelength of the wavelength can be changed sequentially. Separately, this is between the sample and the detector. can be positioned to detect different frequencies emitted by the sample. Ru.

In a preferred system, the filter is driven by a variable speed continuous drive motor. It will be done. Locate and rotate filter by detecting reference marks associated with filter elements It is designed to display speed. Filter discs pass light with different passbands. Contains fan-shaped filter sections separated by opaque sections. You can do what you want. The opaque area is precisely aligned with the light path to maximize the impact coefficient. The width is such that it blocks the The photon count for each filter is in the opaque region. It begins and ends when the minute passes through the optical path. Pass through each filter The light detected each time is normalized to the speed of the filter and the intensity of the broadband light source. It will be done.

An ordinary microscope with a photomultiplier tube is installed between an ordinary light source and the microscope, and between the microscope The attachment quickly connects the photodetector and the micro-spectroscopic fluorometry It can be converted into a meter. The fitting has an optical conduit between the light inlet and the light outlet. There is. The inlet connector at the inlet end of the optical conduit is connected to a conventional microscope light source or microscope. is combined with The exit connector at the exit end of the optical conduit is Combined with a vent.

The filter assembly is eccentrically mounted relative to the optical conduit. This is the filter housing and a filter disc within the housing. The filter disc is an optical conduit a plurality of circumferentially spaced filters in rotation of the filter axis offset from It has a ruta. The attachment rotates the filter around the filter axis. Contains a drive motor for driving. To adapt the attachment to a wide range of microscopes The inlet and outlet connectors are typically pinned between the light source and the microscope. shall be equivalent to a plug-in connector.

The foregoing and other objects, features and advantages of the invention will be realized as illustrated in the accompanying drawings. This will become apparent from the following detailed description of preferred embodiments of the invention. next species Like parts in the various drawings are represented by like reference characters. The drawing is In order to clarify the principle of the present invention, it does not necessarily have a common scale. This is shown with emphasis.

FIG. 1 is a perspective view of a micro spectrofluorometer in which the present invention can be implemented; Figure 2 is an enlarged view of the filter attachment in the stem of Figure 1; Figure 3 is a perspective view of the filter attachment removed from the system in Figure 1, and is shown after Figure 2. Figure 4 is a perspective view of the filter attachment in Figure 3, as seen from the side, with the filter housing Figure 5 shows a typical microscope mounted with the engraved cap removed. an end view of one member of a built-in optical connector; Figure 6 shows the partially sectional side of the plug-in optical connector that connects the accessory to the microscope. side view, FIG. 7 is a developed sectional view of the filter assembly, FIG. 8 is a sectional view of the filter assembly, Figure 9 shows synchronization marks on the filter assembly; Figure 10 shows micro-spectral fluorescence. Optical and electrical block diagram of the photometer, Figure 11 is a set of time charts for the spectrofluorophotometer; Figure 12 shows the filter assembly mounted in the optical path between the sample and the detector. perspective view, FIG. 13 is a rear view of the filter assembly in the system of FIG. 12.

A microscope 18 modified according to the invention is shown in FIG. There are 2 sample slides It is represented by 0. The sample is illuminated from above by a light source 22.

Instead of doing this, the sample is irradiated by an ordinary xenon arc lamp 24. This lamp is typically coupled directly to the microscope at 25. death However, according to the present invention, filter assembly 26 is disposed between light source 24 and sample 20. is positioned within the optical path.

Using either light source, the specimen can be viewed through the binocular viewer 28. , and the video output can be displayed by a television camera 30. from the sample A photomultiplier tube 32 is coupled to quantitatively display the fluorescence of the sample. Accept parts. In an ordinary microscope, the crosshair assembly is attached to the microscope housing. It extends within the housing from the closure member 34 to the surface of the sample. In the case of the present invention, these ten A variable field of view diaphragm hole is not used in place of the character lines, and this diaphragm hole is used for viewing with a photomultiplier tube. Define the portion of the sample to be detected.

An enlarged view of the filter fitting is shown in FIG.

This is the female part 3 of the connector for coupling the fitting to the xenon arc lamp 24. 6. This attachment also has a connector for connecting it to the microscope 18. It has a male portion 38. The light beam from the light source 24 passes through the optical conduit 40 between the coifters. conducted through. Filter housing 42 is eccentrically mounted relative to the optical conduit. It also houses a rotating filter disk, which will be described later. This filter disk is block 4 6 and is rotated by a motor 44 mounted on the optical conduit. light source A guide 48 is provided for a manual shutter for blocking from the filter assembly. It is.

The filter attachment removed from the microscope is shown in FIGS. 3 and 4. this In these figures, the attachment is seen from the rear of FIG. In Figure 4 The end plate 50 of the filter housing is shown removed. rigid filter The disc 52 is mounted within the housing so as to rotate about its central axis.

This filter disc has two sector-shaped filter sections 54 and 56, which They are separated by opaque portions 58 and 60 within the body structure. Fura-2 In a system for measuring calcium ion concentration in samples using dyes one filter has a passband centered at 340 nanometers, and Others have passbands centered at 380 nanometers. used The special filter has a full bandwidth of 10 nanometers at half the maximum conduction. There is.

When the disk is rotated by the motor 44, the two filters will move through the conduit 40. It is moved sequentially into the optical path it passes through. As described below, opaque portions 58 and 60 are the width of the optical conduit is such that it just blocks the light beam in the conduit when the light beam is at the center of the conduit; There is.

A pair of reflective marks are placed on the filter assembly, and these marks Each time the disk rotates, a light emitting diode mounted on the end plate 50 of the housing and Sensed by photoelectric detector cubicle 63. The signal from the photoelectric detector is as described below. Synchronize photon counting with filter rotation.

The filter attachment can be easily mounted between an ordinary microscope and its ordinary light source. 5 and 6, the plug-in optical connector 36 and and 38 are provided. Figures 5 and 6 are typically used in actual microscopes. The female portion 62 of the connector and the male portion 38 of the attachment are shown. xenon arcra Connector 64 on the pump is substantially similar to connector 38, and has no attachments. The connector 36 is functionally similar to the connector on the microscope housing. .

As shown in FIG. 5, the female portion of the connector has two fixing pins 66 and 68. , the pins extend radially inwardly from the rim 70 of the connector socket. third Pin 72I/'X, biased by a spring and pushed outwardly by pin head 74. It is supposed to be pulled out and obtained.

Pin 72t - When pulled out of the socket in this way, the truncated circle of the connector on attachment T6 The male conical portion can be slid into position behind the pins 66 and 6. next When the pin 72 is released, it presses against the conical surface and forces the male part into the socket. Begin to push it firmly into place.

Therefore, the pin can be locked in place by operating the head 74. Wear.

Details of the filter assembly are shown in FIGS. 7 and 8. The drive shaft 78 is a drive motor. The motor 44 extends into the housing of 42. The filter disc carrier 80 is It is positioned on the drive shaft 70. The outer diameter of the carrier 8° and the filter disk 52 A gap of approximately 1 mil is provided between the inner diameter of the bore 82 and the filter It is designed to be able to slide on 80 hubs. The carrier 80 hub is a filter Projecting through the disk, a brass retainer 84 slides over the hub. This retainer 8 4 is fixed to the carrier 8o by a set screw 86. The set screw is still attached along the length. The split carrier 80 is fastened onto the drive shaft 78.

Filter disc 52 is a brass plate with internal and external threads that is threaded onto retainer 86. The sleeve 90 presses well against the two flange 88 of the carrier 80. There is. This sleeve 90 is also fixed by a set screw. Retainer 84 and three Each of the tabs 90 has a pair of holes in the front of the tube and is tightened with two pin wrenches. It looks like this. Finally, end plate 92 is positioned over the assembly and attached to the set screw. Therefore, it is maintained.

This end plate 92UM9 is blackened as shown in Figure 9 and carries a reflective synchronization mark 61. ing.

Figure 10 shows the block diagram of the entire system for obtaining micro spectrofluorometer readings. Show the diagram. Light from light source 24 passes through rotating filter disk 52 to the specimen on microscope 18. oriented to irradiate the material. The light emitted from the sample within one day of the microscope is photoelectric. It is directed to the daughter multiplier tube 32. This photomultiplier tube includes a bandpass filter 94, The filter is 500nm when measuring calcium concentration with Fura-2. The meter has a center frequency. The filter is the problem light. However, light at the excitation wavelength reflected from the sample is blocked. Note that on top of the photomultiplier tube A shutter 96 is arranged.

The output of the photomultiplier tube is amplified by a preamplifier 98 and output by a discriminator 100. It is designed to eliminate pulses caused by noise. photomultiplier tube The power supply device 104, amplifier 98, discriminator 100 and counter 102 are Ortic It is a common PMT device as manufactured by the company. The counter 10 2 has dual channels and exposes the sample to 340 nm and 380 nm light. Therefore, when the photons are alternately irradiated, the photons can be counted alternately. This card The counter 102 is configured by a microprocessor 106 to convert one channel to another. channel and photons are being counted by one channel. First, the counts obtained by other channels are processed by the microprocessor. is read and stored. The microprocessor then calculates the normal The sample is exposed through each of the 34Q nm and 380 nm filters. Calcium concentration is calculated based on photon counts as they are released. Sara The microprocessor senses the intensity of the light source 24 through the fiber beam. A detector 108 normalizes variations in the light source intensity.

The microprocessor detects the reflective mark 61 detected by the photon detector 63. The switching of counter 102 between channels based on the time adjustment derived from control changes. When switching the counter 102 between these two channels The interval adjustment is shown in FIG. As mentioned above, the opaque portion 58 of the filter disk and 60 complete the ray passing through the conduit when the section is centered relative to the conduit 40. It has a width that completely blocks it. Once one opaque area is determined, the photoelectric A cell multiplier detects the lowest intensity of fluorescence. Counter when opaque area is determined By switching 102, each counter channel can effectively 50 inch impact coefficient with the flat part of the highest strength across the area, or Counting can be done nearby. therefore accepted by the photomultiplier tube Counting is performed for maximum efficiency near 100% of the photons received.

For this purpose, a first pulse generator is used to switch the counter between the two channels. As shown in Figure 1B (centered on the minimum value of the PMT output).

The switching and the rotation of the filter disk are synchronized by detecting the two reflective marks 61. This occurs by allowing a pulse such as that shown in Figure 1C to be obtained. 1st The rising edge of the pulse starts the timing interval as shown in FIG. 11D. next This timing interval ends when the rising edge of the pulse is sensed as the second mark.

iEl　1　In the timing interval represented by the pulse in figure D, the megahertz clock The output of the lock is counted. The velocity of the filter disc is as long as the two marks are sensed. is expressed by the number of pulses counted in time T1. Filter failure Since the position of the synchronization mark relative to the transparent part is known, next The time Td at which the counter should be switched and the next time following this when the counter should be switched again. The interval Tf can be easily confirmed. Is the rotational speed measured every cycle? Thus, the system responds immediately to variations in the rotational speed of the filter disc. On the other hand The temporal resolution of the system can be controlled by changing the rotation speed. At this time, the microprocessor 106 automatically responds every cycle and registers the counter. Traps are provided to maintain synchronization between switching and rotation of the filter.

The microprocessor controls the filter when controlling the switching of photon counting versus rotational speed. The photon count by the composite counter channel is always 1 because it responds to the rotation of the router. Close to 00%. Therefore, the system always operates at maximum light collection efficiency and All light received is thus counted.

A special application of this system is to detect ion fluorescence or molecular sensitivity at two excitation wavelengths. This is the case when measuring fluorescent molecules and calculating the concentration of ions or molecules. and For example, calcium ions react with Fura-2 through the following reversible reaction.

Fx HX 10F d where x12 represents a calcium ion and F represents Fura-2 fluorescent dye . The reaction constant Kd is defined by the following equation.

In other words, the concentration of Kd-bound calcium and Fura-2 It is equal to the product of the concentration of free calcium ions and free Fura-2. Free Fur a-2 generates its primary fluorescence in response to an excitation wavelength of 580 nm.

On the other hand, the bound Fura-2 mainly responds to the excitation wavelength of 540 nm. Ru. Both bonded and unbonded Fura-2 have a wavelength of approximately 500 nm. It emits fluorescence in its length. Therefore, the sample can be exposed to two excitation frequencies at different times. of free Fura-2 and bound Fura-2 by excitation by The relative concentration can be measured and the concentration of calcium ions calculated. Karushi The concentration of ion can be calculated from the following formula.

(X) = Kd(R-Rmin)/(Rmax-R)βIU sensed fluorescence , the microprocessor determines both the rotation speed of the filter and the intensity of the light source. It has been normalized. Subscripts represent excitation wavelengths. The difference in strength is between the sample and , the difference from a blank sample such as a saline solution sample. Rmin and RmaX are Measured from a sample containing only Fura-2 and no calcium ions. completely saturated by providing a sufficiently high concentration of R and a sufficiently large concentration of calcium ions. This is the R measured from Fura-2. Excitation at 380 nm with β Ratio of fluorescence due to only saturated Fura-2 and fluorescence due to saturated Fura-2 It is.

Therefore, in one experimental method, a sample of a saline solution is placed in a microscope sample holder. at each excitation wavelength and illuminated through a rotating filter disk. The blank strength measurement is then performed. Next, it does not contain calcium ions. Fura-2 sample and Fura-2 sample saturated with calcium ions and separately positioned above the sample holder and excited each sample through the rotating filter disc. Measurements are taken separately by raising the Finally, place the sample in question on the sample holder. Place and excite through the rotating filter wheel to complete the measurement. Data is All stored by microprocessor and calculate calcium ion concentration used when

Many studies have focused on dynamic changes in calcium ion concentration over a short period of time. It is important to follow. For example, if you want to monitor calcium levels in muscle cells, If this is the case, the cells can be electrically or chemically stimulated to and to be able to measure the calcium ion concentration while this contraction is occurring. do. Since this process takes milliseconds, measurements at both excitation wavelengths are also milliseconds long. It is important that this is done in the second degree. Filter discs are recommended for this purpose. is rotated at speeds up to 12,000 revolutions per minute. This reaction can be carried out using a microscope. By using It can be monitored with fast response speed.

12 and 13 show alternative positions of filter fitting 26. FIG. This attachment is As before, optical connectors 36 and 38't are used to connect the microscope and detector 3. It is connected between 2. Light source 24 is coupled directly to the microscope. This arrangement is used to detect different emission wavelengths from a molecule for a given excitation wavelength. be able to. One example of the use of this system is Indo, which also binds calcium. This is a case where -i is detected.

The system of FIG. 10 is unchanged except for the position of the filter wheel 52. photons Detection by the counting photomultiplier tube 32 is performed by a continuously rotating filter as described above. It is synchronized with Photon counting depends on the speed of the rotating filter and the intensity of the radiation source. and can be normalized.

While the invention has been illustrated and described with particular reference to its preferred embodiments, it will be appreciated by those skilled in the art The spirit of the invention as defined by the claims of the present invention as easily understood by the practitioner and its shape and details can be transformed into species without leaving the range. Should.

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Claims (1)

[Claims] 1. Regarding the microscope, a device for positioning the sample; a radiation source for irradiating the sample; a detector for detecting radiation from the microscopic observation part of the sample; A filter assembly located in the optical path between the source and the sample and deflected from the optical path. have different passbands spaced circumferentially around the filter axis. Multiple bandpass filters and rotation of the filter around the filter axis and sequentially move a plurality of filter elements into the optical path and through the filter assembly. a filter assembly having a drive adapted to change the wavelength of the radiation; A microscope having a device for synchronizing radiation detection with the position of a rotating filter. 2. The microscope according to claim 1, wherein the detector is a photomultiplier tube and a photon multiplier tube. A microscope that has a device for counting. 3. The microscope according to claim 1, wherein the drive device is a variable speed continuous drive motor. - A more sophisticated microscope. 4. The microscope according to claim 1, wherein the filter assembly further includes a rotary filter. A synchronization mark that is fixed relative to the router and A microscope having a device for detecting arcs. 5. The microscope according to claim 4 further includes two synchronization marks and an electronic The rotation speed of the filter is determined by the processor device and the time it detects two marks. A microscope that has a processor device that calculates . 6. A microscope according to claim 1, wherein an opaque segment is provided between the filters. , and when each opaque segment is centered on the ray, the opaque segment A microscope whose width is just such that it blocks this ray. 7. The microscope according to claim 1, wherein the filter assembly comprises a fan-shaped opaque segment. Consisting of disks with sector-shaped filter segments separated by microscope. 8. A microscope according to claim 7, wherein a filter segment and an opaque A microscope in which the segments are formed within an integral filter disk. 9. In the microscope according to claim 1, the filter assembly is arranged between the filters. An opaque segment that is wide enough to block broadband radiation sources. segment, the microscope further converts into an electronic processor device, and the corresponding film cumulative measurement of radiation detected from a sample by irradiating the sample through a the opaque segment has a processor device configured to control the When the line is interrupted, the processor terminates the cumulative measurements for one filter. The microscope is adapted to initiate cumulative measurements on other filters. 10. A microscope according to claim 9, in which cumulative measurements are performed using dual channel light. is performed by the child counter and the counter is activated when the opaque segment blocks the radiation. A microscope in which the scanner can be switched between channels. 11. The microscope according to claim 9, further comprising: a rotation speed of the filter assembly; The processor normalizes the cumulative measurement of filter speed. A microscope that has equipment for 12. The microscope according to claim 11, further comprising: an intensity of the broadband radiation source; and a processor that makes cumulative measurements of broadband radiation intensity. A microscope that has a device for normalization. 13. In the microscope according to claim 1, the filter assembly is detachable from the microscope. A microscope is a microscope accessory that can be mounted freely. 14. The microscope according to claim 13, wherein the filter assembly attachment is complementary to to the radiation source and microscope housing via a pinned plug-in connector. A linked microscope. 15. The microscope according to claim 13, wherein the filter assembly attachment is complementary. Connected to the microscope housing and detector by a pinned plug-in connector Microscope. 16. The microscope according to claim 1, in which the filter assembly connects the source and the sample. A microscope positioned in the optical path between. 17. In the microscope according to claim 1, the filter assembly includes a sample and a detector. a microscope positioned in the optical path between the 18. In a microscope, a device for positioning the sample; a radiation source for irradiating the sample; Photomultiplier tube and photon meter for detecting radiation from the microscopic portion of the sample a filter assembly in the optical path between the source and the detector; Different channels spaced circumferentially around the filter axis offset from the optical path It consists of a plurality of bandpass filters with overband, and the filters just block radiation. a filter assembly separated by an opaque portion of sufficient width to cut the filter; Use a high-speed continuous drive motor to rotate the filter around the filter axis. move a plurality of filter elements into the optical path to reduce the amount of light passing through the filter assembly. The motor that changes the wavelength, the position and rotation speed of the rotating filter a device for sensing; An electronic processor device for controlling photon counting of radiation detected from a sample. When the opaque segment blocks the radiation source, the processor uses one filter. terminating photon counting for the filter, starting photon counting for the other filter, and Prohitsa now normalizes photon counts to filter speed. A microscope having a setsa device. 19. In microscope accessories, an optical conduit between the light inlet and the light outlet; Inlet connector located at the entrance end of the optical conduit to couple the conduit to the microscope system The connector that has become Exit connector located at the exit end of the optical conduit to connect the conduit to the microscope system The connector that has become A filter assembly mounted eccentrically with respect to the optical conduit and a filter housing. and a filter disc within the filter housing, the filter disc being an optical guide. A plurality of filters spaced circumferentially around a filter axis offset from the tube. a filter assembly having a filter; and a drive device for rotating the filter around the filter axis. Microscope accessories. 20. The microscope accessory according to claim 19, wherein the drive device is a variable speed continuous A microscope attachment consisting of a drive motor. 21. The microscope accessory according to claim 19, further comprising: a filter assembly; Furthermore, a synchronization mark is fixed to the rotating filter, and the mark passes through a predetermined position. and a device for detecting this mark when the mark is detected. 22. The microscope accessory according to claim 21, further comprising two synchronous machines. mark and the electronic processor device between the detection points of the two marks. A microscope accessory having a device for calculating the rotational speed of a rotor. 25. The microscope accessory according to claim 19, further comprising an opaque material between the filters. has a bright segment and each opaque segment when the opaque segment is centered on the beam. A microscope attachment whose bright segment is adapted to exactly block the light rays passing through the optical conduit. Ingredients. 24. The microscope accessory according to claim 23, wherein the filter assembly is sector-shaped. From a disk with sector-shaped filter segments separated by opaque segments A familiar microscope accessory. 25. The microscope accessory according to claim 19, wherein the filter segment and and an opaque segment formed within an integral filter disk. 26. The microscope accessory according to claim 19, further comprising an inlet connector and A microscope attachment in which the outlet connector is a bayonet coupling member with complementary pins. 27. In filter accessories, an optical conduit between the optical inlet and the optical outlet; an inlet connector adapted to couple to the system and located at the outlet end of the optical conduit; An exit connector adapted to couple the conduit to the optical system and for the optical conduit. An eccentrically mounted filter assembly including a filter housing and a filter housing. a filter disc within the optical conduit housing, the filter disc being offset from the optical conduit; It has a plurality of filters spaced circumferentially around the filter axis. The routers are separated by opaque segments, and the opaque segments are placed on the rays. Each opaque segment, when centered, just blocks the light ray passing through the optical conduit. A filter assembly that looks like this, A variable speed continuous drive motor rotates the filter around the filter axis. The motor to rotate the filter and the synchronization mark fixed to the rotating filter are in the specified position. a filter attachment having a device for detecting the mark when passing through the device; . 28. radiation from a sample in response to excitation by radiation of a distinct wavelength In the method of measuring At the determining stage, the filter assembly is rotated circumferentially around the filter axis. consisting of a plurality of bandpass filters having different passbands spaced apart from each other; Continuously rotate the filter around the filter axis to rotate multiple filters. a step in which the sample is sequentially moved within the optical path; By starting and stopping photon counting in synchronization with the filter position, At the stage where the photons emitted from the sample are counted, the sample passes through a corresponding filter. The stage at which photons emitted when excited by light are sequentially counted How to have. 29. 29. The method of claim 28, wherein the filter blocks the broadband radiation source. separated by opaque segments that cut through the When blocked, the photon count switches between the corresponding filter and the associated channel. The duration of photon counting now depends on the rotation speed of the filter. How to be. 30. 29. The method according to claim 29, further comprising a rotating film for counting photons. The method includes a step of normalizing to the speed of the data. 31. 31. The method of claim 30, further comprising broadband radiation counting the photons. A method that has a step of normalizing to the line intensity. 32. In a method for measuring radiation of a clearly distinguishable wavelength from a sample, By positioning the filter assembly between the sample and the photon counting detector, the filter assembly is positioned between the sample and the photon counting detector. Filter assemblies have different passbands spaced circumferentially around the filter axis a stage consisting of a plurality of bandpass filters having a range of multiple filters are sequentially rotated between the sample and the detector. The photons emitted from the sample are moved into the optical path of the filter. counting by starting and ending photon counting in synchronization with position; The photons emitted from the sample through the corresponding filter are now counted sequentially. A method comprising steps. 33. 32. The method according to claim 32, wherein the filter is a radiation blocking element. Corresponds when separated by a transparent segment and the opaque part blocks radiation The photon counting is switched between the filter and the associated channel, and the photon counting A method in which the duration is determined by the rotation speed of the filter. 34. In the method according to claim 33, the photon counting is further performed on a rotating disk. A method comprising a step of normalizing for speed. 35. In the method according to claim 34, the photon counting is further performed by measuring the intensity of the radiation. A method that has a step of normalizing for degrees.