JPH05346399A - Spectrofluorometer - Google Patents

Abstract

PURPOSE:To exclude complicatedness due to subjectivity and trial and error and to automatically set shortest wavelength moving sequence and time resolving power by setting the sequence of a wavelength set wherein a timewise one cycle for measuring the data of all of wavelength sets becomes shortest on the basis of a function determining a time required in wavelength movement. CONSTITUTION:A function determining the wavelength moving time of a spectrofluorometer calculated from a present wavelength value and an objective wavelength value is inputted to a computer. Next, for example, a measuring person inputs sets (A-D) of four wavelength values of exciting/fluorescence wavelengths EX1/EM1-EX4/EM4. The matrix showing the moving times related to all of cases moving between two points in the sets A-D is formed from the function and the wavelength values and the times starting from an A-point to pass between B-D and again returning to the A-point are calculated with respect to the combinations of B-D to calculate the min. value and measuring sequence is determined. The min. time (time resolving time) required in one cycle is determined on the basis of the measuring required time of one cycle measured according to the measuring sequence.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is mainly applied to medicine, chemistry,
In the fields of pharmacy and the like, a spectrofluorometer used for multi-wavelength time-change measurement for measuring the time-dependent change in fluorescence intensity of a group of multiple wavelengths for the purpose of tracking chemical changes or reactions of organisms to drugs , The improvement of time resolution and operability for tracking the reaction.

[0002]

2. Description of the Related Art An example of multi-wavelength time change measurement is as a combination of excitation / emission wavelengths, (1) 340/510 (nm), (2) 380/510, (3) 500.
Four sets of / 530 and (4) 440/530 are designated, the fluorescence intensity is measured in this order, and the time change of the fluorescence intensity in each wavelength set is displayed in a graph. The wavelength switching time is 1 second between each wavelength, and the number of wavelength pairs is 4, so one cycle is 4 seconds. The method for determining the order of wavelength switching is not described.

[0003]

In the prior art, the order of measurement of each wavelength set was set by the intuition of the measurer or by trial and error. If the measurement order of the set of four wavelengths shown in the above conventional technique is intuitively set in the order of the smallest excitation wavelength (1)-(2)-(4)-(3), the same measurement is performed. When it is repeatedly executed by using the device, the wavelength shift amount of (3)-(1) returning from wavelength (3) to wavelength (1) becomes large, and it takes about 2 seconds as the movement time between this pair of wavelengths. Therefore, the time resolution that the wavelength switching time between each wavelength pair is 1 second cannot be achieved.

When performing multi-wavelength time change measurement, the measurer must set the measurement cycle, which has a lower limit value. When this lower limit value is called the time resolution, the time resolution also changes depending on the wavelength value set by the measurer, even if the same device is used.Therefore, when tracking a fast response, the measurer also measures by trial and error. Had to set the cycle of.

The present invention eliminates the subjectivity of the measurer and the complexity due to trial and error in setting the measurement sequence and period, and automatically sets the shortest wavelength shift sequence and the time resolution simply by inputting a set of wavelengths. Is to be set. Furthermore, it also meets the demand that the movement time between each wavelength group is to be suppressed within a certain time, or that the movement time is to be as uniform as possible.

[0006]

[Means for Solving the Problems] Although it is clear that there is an optimum permutation in the order of measurement, in order to find the optimum permutation by hand calculation, the amount of calculation increases greatly as the number of wavelength groups increases. I will do it.

Also, spectrofluorometers typically utilize gears to rotate the optical dispersive element. Therefore, when moving in the direction in which the wavelength value decreases in order to eliminate the wavelength selectivity error due to the play of the gear, it moves once to a wavelength value that is smaller than the target wavelength value by a certain amount, and then to the target wavelength value. The gears are moved to increase (called backlash). In other words, even if it simply makes a round trip between two sets of wavelengths, the traveling time differs between the forward and backward paths, and therefore a reduction in the calculation amount cannot be expected.

Further, if there are upper and lower limit constraints on the moving time, such as setting the time required for moving between wavelengths to be as equal as possible, the calculation becomes more complicated. After all, the relationship between the number of wavelength pairs and the total number of these permutations is: total number of permutations = (number of wavelength pairs-1)! The number of wavelengths is increased to 6 in this example, 6 in 5, 24 in 5, 6 in 120, so the number of wavelengths increases enormously. Is very difficult to set up.

In the present invention, for the shortest measurement order, for all the measurement orders (permutation of wavelength groups), the sum of the time required for wavelength shift between wavelength groups is calculated by a computer, and the minimum value is calculated. It is realized by asking.

In order to perform calculation for all the order of wavelength shifts between arbitrary wavelength sets inputted, and to cope with different wavelength shift speeds depending on the device, the computer is required to:
A function for calculating the time required for wavelength shift is stored from the current wavelength value and the target wavelength value. In addition, this function considers backlash and divides the calculation results depending on the magnitude relationship between the current wavelength value and the target wavelength value.

In consideration of the calculation in the case where the time constraint condition is set, the calculation of the wavelength shift time of one period is performed based on the set function of the stored shift time and the input wavelength and the current excitation. / Make a matrix that has the fluorescence wavelength position (corresponding to one wavelength group) and the target wavelength position (corresponding to another wavelength group) as rows and columns, and have each element as the movement time, and It was selected according to each permutation, and the sum of them was calculated and output as one cycle time. When a constraint condition is added, the time of each permutation is calculated after performing an operation of replacing an element that does not satisfy the constraint with a numerical value that is larger by several digits in this matrix. The calculation result of permutations that do not satisfy the constraint is several orders of magnitude larger than that of those that satisfy the constraint, so that permutations outside the constraint can be eliminated.

After the order of measurement is determined, the time for one cycle is roughly estimated. However, when the spectrofluorometer is controlled by an external computer, the time required for communication is added, Further, due to the difference in the integration time set for the stability of the data, the time of one cycle obtained by calculation does not match the time of one cycle actually measured in the given order.

Therefore, after the measurement order is determined, the measurement start time is stored, the measurement for one cycle is executed immediately, and the time of one cycle obtained from the difference between the measurement end time and the measurement start time is calculated. As a guide, the minimum time (time resolution) required for one cycle is determined.

[0014]

By storing the function of the wavelength shift time, it is possible to determine the shortest measurement order according to the difference between the measurement wavelength and the device, and it is also possible to deal with the case where a constraint condition is added to the measurement order.

After the measurement order is determined, one cycle is actually measured to measure the time, so that one cycle (time resolution) including the communication time can be determined.

From the results of the above operations, the measurer only needs to set the measurement wavelength value and the constraint condition, and in setting the measurement condition, the optimum measurement order and time resolution can be obtained in a short time without subjectivity and complexity. The decision can be made and the measurement can be performed.

[0017]

Embodiments of the present invention will be described below with reference to the drawings.

In FIG. 1, the white light emitted from the Xe lamp 1 is converted into monochromatic light by the excitation-side spectroscope 2 and applied to the sample container 3. The fluorescence emitted from the sample is the fluorescence side spectroscope 4
After the wavelength is selected by the detector, its intensity is measured by the detector 5. The signal output from the detector 5 is amplified by the signal amplifier 8 and then sent to the computer 9 for data processing. The wavelength of the excitation side spectroscope 2 is controlled by the computer 9 via the excitation side wavelength drive system 6, and similarly, the wavelength of the fluorescence side spectroscope 4 is controlled by the computer 9 via the fluorescence side wavelength drive system 7. ing. Here, the order of measurement of the wavelength set on the computer 9,
Determine the minimum period, set the measurement conditions based on the result, and execute the measurement.

FIG. 2 shows an example of the obtained four-wavelength time change measurement result. Each graph shows excitation / emission wavelength, EX
1 / EM1, EX2 / EM2, EX3 / EM3, EX4
/ EM4 represents the change over time in the fluorescence intensity for each set of wavelength values of EM4. The set of wavelength values is A, B, C,
When D is taken, the actual measurement is a graph in which the measurement results for the same wavelength group are repeated by repeating the measurement for each wavelength group as ABCD-AB-.

An execution example for determining the measurement order will be described with reference to the flowchart of FIG.

1) Input a function for determining the wavelength shift time of the spectrofluorometer into a computer. Here, assuming that the function when moving in the wavelength increasing direction is t = FT (fw, tw), FT (fw, tw) = (tw-fw) / 500 (tw-
When fw <40) FT (fw, tw) = (tw-fw-22.4) / 1000
+0.04 (when tw-fw ≧ 40) Assuming that the function when moving in the wavelength decreasing direction is t = BT (fw, tw), BT (fw, tw) = FT (tw-10, fw) +0.
12 where fw is the current wavelength value and tw is the target wavelength value.

For FT (fw, tw), fw-tw
When <40, wavelength is moved at a constant speed of 500 nm / s, and when fw-tw ≧ 40, 0.02 seconds (total 0.04 seconds) of slow-up and slow immediately after the start and end of wavelength movement, respectively. There is down time, and the distance traveled during this period is 22.4n.
m 2 and others mean moving at a constant speed of 1000 nm / s.

For BT (ft, tw), fw is always
Since> tw, the term of tw is the first argument of FT. The reason why 10 is subtracted from tw is that it is temporarily moved to a wavelength smaller by 10 nm than the target wavelength due to backlash. Then, 0.12 seconds is added as the travel time from that point to the true target wavelength.

Generally, both excitation and fluorescence wavelengths are moved, but since the present apparatus moves both excitation and fluorescence wavelengths one by one, the sum of the respective wavelength movement times corresponds to the movement time between points. Although this function differs depending on the device, once the same device is stored, there is no need to store it again.

2) The operator inputs each wavelength set.

Here, (excitation wavelength, fluorescence wavelength) is A (300,380), B (300,450), C (4
20,550) and D (500,550).

3) From the function of 1) and the wavelength value of 2), A,
A matrix representing the travel time for all cases of moving between two points of B, C, and D is created. The results are as follows. For example, the element 0.22 in the second row and the first column indicates that the wavelength shift time from the point B to the point A is 0.22 seconds.

4) The time from the point A as a starting point to the points B to D and returning to the point A is calculated for all combinations of B to D, and the minimum value is obtained. In all cases AB-
C-D-A, A-B-D-C-A, A-C-B-D-
A, A-C-D-B-A, A-D-B-C-A, A-D
There are 6 types of CBA.

As a result of the calculation, the shortest order is ABCD.
In the case of -A, the time was 1.15 seconds. In this way it is determined that the shortest sequence of measurements is ABCD. Also, assume that there is a request such as setting the expected value of each wavelength movement time to be less than 0.6 seconds. In this case, all the elements of the matrix created in 3) are checked, and those of 0.6 or more are replaced with 10, for example. The number of elements related to movement of CA, DA, and DB is 10. If the same calculation as 4) is executed here, in the permutation that follows the routes of 0.6 seconds or more of the above three types,
Since the sum of all traveling times exceeds 10, the calculation result becomes extremely large compared to the permutation that follows these three types of routes,
It will not be detected as the minimum value. If there is no route satisfying the set constraint condition, the detected minimum value exceeds 10. Therefore, it can be determined that there is no required route. In the case of this request, 1.38 seconds of A-D-C-B-A is judged to be the shortest.

5) After setting the parameters of the apparatus so that the measurement is performed in the order determined in 4), one cycle of measurement is performed and the required time is measured. Here, three wavelengths of three points A to C were quantitatively measured at an integration time of 0.1 seconds. The shortest order is ABCA and the travel time is 0.9.
Although it was calculated to be 5 seconds, the time actually required to measure one cycle was 2.08 seconds. This means that it is not possible to set the measurement period (time resolution) to 2 seconds in order to repeatedly perform this quantitative measurement to perform time change measurement of 3 wavelengths, and the shortest period is about 2.1 seconds. is doing. In this way, the time resolution can be set to be about 2.1 seconds.

The present invention is not limited to the multiwavelength time change measurement,
Applications such as re-execution of multi-wavelength quantitative calculation in a short time are also possible.

Further, the application is not limited to the shortest route, and it is also possible to apply a constraint condition to the travel time of one cycle so that the travel time of one cycle is included in a certain time range.

Finding the optimal order in the present invention mathematically results in the traveling salesman problem. When finding the optimal path, it is possible to set it in a shorter time if the number of wavelengths is further increased, by not investigating all the permutations and introducing other mathematical methods (for example, linear programming, application of neural network, etc.) Becomes

[0034]

According to the present invention, the measurement order of multi-wavelength measurement is determined by relying on trial and error and the intuition of the measurer until now, but the measurer only inputs the wavelength value. It has become possible to automatically set the optimum measurement order and the minimum one cycle time (time resolution) in consideration of the time constraint of wavelength shift.

[Brief description of drawings]

FIG. 1 is a diagram showing an embodiment of the present invention.

FIG. 2 is a diagram illustrating an example of a multiwavelength time change measurement result.

FIG. 3 is a diagram illustrating a procedure for determining a measurement order.

[Explanation of symbols]

1 ... Xe lamp, 2 ... Excitation side spectroscope, 3 ... Sample container, 4
... Fluorescence side spectroscope, 5 ... Detector, 6 ... Excitation wavelength drive system, 7
... Fluorescent wavelength drive system, 8 ... Signal amplifier, 9 ... Computer.

Claims (3)

[Claims] 1. A light source, an excitation-side spectroscope that disperses light from the light source and irradiates a sample with monochromatic light, a fluorescence-side spectroscope that disperses fluorescence emitted from the sample, and light emitted from the fluorescence-side spectroscope. It is composed of a detector for detecting the wavelength, a wavelength shift of the above-mentioned two spectroscopes, a data acquisition from the detector, and a control unit for displaying and processing the data, each of which sets a plurality of sets of excitation wavelength and fluorescence wavelength. In order to measure the fluorescence intensity at the wavelength value of the set, the fluorescence intensity at each wavelength value on the excitation side and the fluorescence side is sequentially measured, and the fluorescence intensity at all the wavelength value sets is acquired, and these data are displayed. In a fluorescence spectrophotometer equipped with a function of performing processing, a permutation of wavelength pairs having the shortest one time period for measuring data of all wavelength pairs is characterized by being automatically set. Spectrofluorometer. 2. In the above spectrofluorophotometer, within one time period for measuring data of all wavelength groups, the wavelength shift time between each group is restricted by an upper limit, a lower limit, or both. A spectrofluorometer, wherein, when provided, a permutation of a set of measurement wavelengths that satisfies the constraint condition and has the shortest one cycle is automatically set. 3. The spectrofluorometer described above, wherein one cycle of measurement is performed in accordance with a given measurement order, and the time required for this measurement is measured to obtain a wavelength shift time and data acquisition. A spectrofluorometer, which is characterized by automatically determining the shortest period for actual measurement including the integration time.