JP5033531B2 - Spectrofluorometer - Google Patents

Description

  The present invention relates to a spectrofluorometer. In particular, it is suitable for a spectrofluorometer having a function of easily and efficiently performing measurement and calculation for obtaining a fluorescence quantum yield calculation result.

  The calculation of the fluorescence quantum yield is obtained by measuring the spectrum necessary for the operation by scanning the wavelength, performing normalization at a specific wavelength from the data, four arithmetic operations, area calculation in a specific wavelength range, etc. Although the quantum yield was calculated from the ratio between the amount of absorption and the amount of fluorescence, these data processings were performed one by one, and finally calculated using spreadsheet software.

Further, since each calculation wavelength range is determined and calculated by a person based on the shape of the spectrum, an operation such as displaying the spectrum on an appropriate scale and tracing the data is necessary.

  For example, Patent Document 1 is a related art related to the above.

JP 2003-65956 A

  In the prior art, as a process of calculating the fluorescence quantum yield, normalization processing from a maximum of nine spectra, correction coefficient calculation processing, wavelength range designation processing in scattered light data, area calculation processing of scattered light data, fluorescence intensity data Since the fluorescence quantum yield must be calculated by performing many calculation procedures such as wavelength range designation processing and fluorescence intensity data area calculation processing, there is a problem that the calculation is very laborious.

  In addition, in the wavelength range designation process in the scattered light data for calculating the fluorescence quantum yield and the wavelength range designation process in the fluorescence intensity data, the energy levels of the scattered light data and the fluorescence intensity data of the sample itself are greatly different, so that the scattering is performed. When the scale is adjusted to the light peak, the fluorescence peak is displayed very small, making it difficult to discriminate, and it is difficult to accurately specify the wavelength range used for each calculation.

  Further, the spectrum for specifying the wavelength range in the fluorescence intensity data when a filter for cutting higher-order light of the scattered light is used is divided by the data (= 0) cut by the filter because the filter coefficient is applied. Contains data. Since the data divided by 0 is usually an overflow value, the spectrum is displayed on a large scale where the upper limit of the scale is not possible as normal fluorescence intensity, such as 10,000. Therefore, when the spectrum is drawn with a normal autoscale, There is a problem that the peak spectrum of the fluorescence intensity data to be designated as a range becomes very small, and it is difficult to designate the wavelength range of the fluorescence intensity data accurately.

  In addition, the fluorescence quantum yield calculation is usually calculated from a two-dimensional fluorescence spectrum focusing on a specific excitation wavelength. However, in the synthesized new phosphor, the optimum excitation wavelength is not known, and the fluorescence accompanying the change in the excitation wavelength Quantum yield needs to be obtained, but not using samples at individual excitation wavelengths, and integrating sphere correction spectra when using samples, fluorescence spectra of samples when not using samples, and when using samples Therefore, there is a problem that it takes a lot of labor and time to measure each spectrum by changing the excitation wavelength.

  Although the wavelength range in the scattered light data used for the fluorescence quantum yield calculation and the wavelength range in the fluorescence intensity data are specified by visual inspection every time, these calculations are performed as the data for calculating the fluorescence quantum yield increases. There is a problem that the work of specifying the range becomes a great effort.

  One feature of the present invention is that the correction data and sample data necessary for the calculation of the fluorescence quantum yield are read, and the wavelength range of the scattered light data and the wavelength range of the fluorescence intensity data are specified on the fluorescence quantum yield calculation screen. By providing the spectrofluorometer with a function (or means) that can obtain the calculation result of the fluorescence quantum yield, the work required to calculate the data required for the fluorescence quantum yield calculation one by one Even if it is not performed, it is possible to calculate the fluorescence quantum yield by executing the calculation button after specifying the wavelength range.

  In addition, among the data used to calculate the fluorescence quantum yield, diffusion element measurement data, data measured using an integrating sphere without setting a sample, and data measured using an integrating sphere with a sample set are The data indicating the characteristics of the device, and the baseline measurement data and filter measurement data are data indicating the characteristics of the filter and do not need to be measured again each time. A function that can be used. The screen for calculating the fluorescence quantum yield is provided with a function that makes it easy to select samples for which the fluorescence quantum yield is desired one after another.

  In addition, in this fluorescence quantum yield calculation screen, in order to make it easy to specify the calculation range, the spectrum for the range specification of the scattered light data and the spectrum for the range specification of the fluorescence intensity data are drawn on separate graphs, By setting the screen configuration so that each scale can be changed, it is possible to display spectra with different levels of scattered light data and fluorescence intensity data by setting the optimal vertical axis, and setting an appropriate calculation range. Can do.

  Next, in the graph for specifying the wavelength range of the fluorescence intensity data after the filter correction processing of the spectrum using the filter for cutting the higher-order light of the scattered light, a sample using the filter for cutting the higher-order light of the scattered light was used. By using the auto-scaling value of the raw spectrum data and displaying it, the fluorescence intensity peak data to be noticed that could not be seen in the auto-scaling spectrum after the filter correction process is displayed on the optimal scale. The wavelength range of the appropriate fluorescence intensity data can be specified.

  Also, data measured using an integrating sphere without using a sample, and an integrating sphere corrected spectrum calculated from data measured using an integrating sphere using a sample, data measured without using a sample, and a sample The data measured by using the three-dimensional measurement is acquired at once, the fluorescence quantum yield at each excitation wavelength is acquired, and drawn on a two-dimensional graph of X axis: excitation wavelength, Y axis: fluorescence quantum yield. This makes it possible to find the optimum excitation wavelength.

  Next, regarding the designation of the wavelength range of the scattered light used for the calculation of the fluorescence quantum yield, the scattered light has the same fluorescence wavelength as the excitation wavelength ExWL and is twice the slit width MaxSlit of the larger one of the excitation side slit ExSlit and the fluorescence side slit EmSlit. It is possible to designate ExWL ± (MaxSlit × 2) as the wavelength range of the scattered light from the characteristics detected in the wavelength range, and for the designation of the wavelength range of the fluorescence intensity data, the long wavelength ExWL + of the wavelength range of the scattered light By specifying the wavelength range from (MaxSlit × 2) to the end wavelength, or specifying the wavelength range from the start wavelength to the end wavelength of the detection peak, by specifying the start wavelength and end wavelength of the specific peak in advance It is not necessary to perform an operation for setting the wavelength range on the screen.

  In addition, the fluorescence quantum yield can be acquired only by an operation of executing the calculation button by automatically specifying the wavelength range of the scattered light data and the fluorescence intensity data by this method.

In the fluorescence quantum yield calculation, by incorporating a function that holds the state of reading the spectrum indicating the characteristics of the device once, only the data of the sample for which the fluorescence quantum yield is desired is read, and the wavelength range of the scattered light data It is possible to calculate the fluorescence quantum yield simply by specifying the wavelength range of the fluorescence intensity data, and the work efficiency of the calculation is greatly improved.

  In addition, since the spectrum that specifies the wavelength range of the scattered light data and the wavelength range of the fluorescence intensity data is displayed on an optimal scale, the wavelength range can be specified appropriately.

  In addition, the spectrum for specifying the wavelength range in the fluorescence intensity data when using a filter for cutting the higher-order light of the scattered light is the peak of the fluorescence intensity data for which you want to specify the wavelength range when the spectrum is drawn with the normal autoscale. The spectrum became very small, and it was difficult to specify the wavelength range of fluorescence intensity data accurately, but by using the autoscale value of the raw spectrum before correction with the filter correction factor, fluorescence The scale is optimized for the peak spectrum of the intensity data, and the wavelength range in the fluorescence intensity data can be specified appropriately.

  In addition, by utilizing 3D measurement, data measured using an integrating sphere without setting a sample, data measured using an integrating sphere with a sample set, data measured without using a sample, 3D fluorescence spectrum of sample-free and sample-free samples by maintaining the integrating sphere correction spectrum over the entire excitation / fluorescence wavelength by reducing the time and effort of measuring the data measured using the sample and changing the excitation wavelength. The fluorescence quantum yield calculation result at each excitation wavelength can be acquired simply by measuring, and the fluorescence quantum yield characteristics with respect to the excitation wavelength can be seen by displaying this in a two-dimensional graph.

  In addition, the wavelength range of the scattered light data and fluorescence intensity data used for the fluorescence quantum yield calculation is not set manually but automatically. The quantum yield can be automatically acquired without specifying the wavelength range, which is effective for calculation after acquiring a large amount of data.

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

  First, with reference to FIG. 20, the basic structure of the spectrofluorometer of the present embodiment will be described.

  In FIG. 20, the light beam emitted from the light source 2001 enters the excitation light side spectrograph 2002. The set wavelength of the excitation light side spectrometer 2002 is controlled by a pulse motor 2003. The operation of the pulse motor 2003 is controlled by an instruction issued from the data processing device (arithmetic control means) 2004 to the pulse motor 2003 via the interface 2005. The instruction from the data processing device 2004 is transmitted in the data processing device 2004. Issued by a program stored in memory.

  The monochromatic light extracted by the excitation light side spectroscope 2002 irradiates the measurement sample 2008 in the sample cell 7 via the beam splitter 2013, and the fluorescence emitted from the measurement sample 2008 enters the fluorescence side spectroscope 2009.

  The fluorescence side spectroscope 2009 is driven by a pulse motor 2010, and its operation is controlled by the data processing device 2004 in the same manner as the pulse motor 2003. The fluorescence of the wavelength selected by the fluorescence side spectroscope 2009 enters the detector 2011 and is converted into an electrical signal.

  The operation settings of the excitation light side spectroscope 2002 and the fluorescence side spectroscope 2009 are set by an analyst through an operation panel 2006 having a display device such as a CRT or a liquid crystal. The excitation light side spectroscope 2002 and the fluorescence side spectroscope 2009 include a plurality of slits (not shown) having various widths at the light incident position and the light emitting position, respectively. The slit width is arbitrarily changed according to the measurement.

  The electric signal converted by the detector 2011 is converted into a digital signal by an analog-digital converter 2012.

  On the other hand, a part of the monochromatic light extracted from the excitation light side spectroscope 2002 enters the monitor detector 2014 via the beam splitter 2013 and is converted into an electric signal in order to monitor the light amount of the light source. The electrical signal converted by the monitor detector 2014 is also converted into a digital signal by the analog-digital converter 2012.

  The digital signal (S) from the detector 2011 and the digital signal (M) from the monitor detector 2014 are sent to the data processing device 2004, and a ratio (S / M) between them is calculated. The fluorescence intensity at the wavelength is stored in the internal memory of the data processing device 2004. It is also possible to omit the monitor detector 2014 and send light amount data directly from the excitation light side spectrograph 2002 as a digital signal to the data processing device 2004.

  Next, processing in the data processing apparatus 2004 will be described.

  The measurement data obtained by the above apparatus is three-dimensional matrix data composed of three parameters of excitation light wavelength, fluorescence wavelength, and fluorescence intensity. At the time of measurement, first, at an arbitrary excitation light wavelength, a fluorescence spectrum is obtained by driving the fluorescence side spectrometer 2009 at a predetermined sampling interval to scan the fluorescence wavelength and detect the fluorescence intensity. Then, the excitation light side spectroscope 2 is driven for the sampling interval and set to the next wavelength, and similarly, the fluorescence side spectroscope 2009 is driven to obtain a fluorescence spectrum. By repeating this, three-dimensional measurement data can be obtained.

  The data processing apparatus 2004 stores the measurement result (three-dimensional measurement data array) and measurement conditions (excitation light side spectrometer, fluorescence side spectrometer slit width, sampling interval, etc.) each time measurement is repeated, and analysis. To be output at the request of the user.

  FIG. 1 shows the entire system of the spectrofluorometer of this embodiment.

  The spectrofluorometer system includes a main body analysis unit 101 and an operation / data processing GUI unit 102. The main body analysis unit 101 receives an instruction corresponding to the analysis procedure 103 from the operation / data processing GUI unit 102 via the communication unit 104, and issues an execution request to the control unit 105 according to the received content. The control unit 105 is programmed with a sequence corresponding to each execution request, and executes photometer condition setting 106, two-dimensional spectrum data measurement 107, three-dimensional spectrum data measurement 108, and the like. The measurement result is acquired / displayed / saved 109 in the operation / data processing GUI unit 102. The fluorescence quantum yield calculation 110 is incorporated in the operation / data processing GUI unit, reads the measurement result data acquired / displayed / saved 109, and calculates the fluorescence quantum yield. Further, the main body analysis unit 101 is equipped with a sample chamber 111 in which a sample to be measured is placed, and measurement using a square cell or an integrating sphere is possible. The main optical system 112 constituting the main body analysis unit 101 includes a light source, excitation wavelength, fluorescence wavelength, excitation side slit, and fluorescence side slit.

  FIG. 2 shows a screen example for calculating the fluorescence quantum yield.

  The fluorescence quantum yield calculation screen 201 has a spectrum data reading function 202 measured without using a sample and a spectrum reading function 203 measured using the sample. The spectrum data read here is shown in FIG. The correction is calculated according to the calculation formula, and is displayed on the spectrum display unit 204 for designating the wavelength range of the scattered light data and the spectrum display unit 205 for designating the wavelength range of the fluorescence intensity data. What is important here is that the same spectrum is displayed on each of the spectrum display units 204 and 205, but the scale can be changed independently. Therefore, in two wavelength regions where the levels of the scattered light 206 and the fluorescence intensity 207 are different, respectively. The optimum wavelength axis (X axis) and fluorescence intensity axis (Y axis) scale can be set, and the wavelength range and the vertical axis can be set appropriately according to the shape of the spectrum (FIG. 2). The example in (1) describes a graph with an already optimized scale. Next, the wavelength range of the scattered light data is designated by the cursor 216 using the wavelength range designation function 208 of the scattered light data, and the wavelength range of the fluorescence intensity is designated by using the wavelength range designation function 209 of the fluorescence intensity data. When the cursor 217 is designated, the wavelength range to be calculated is determined, and the calculation button 211 is executed, the fluorescence quantum yield calculation is executed according to the calculation formula of FIG. 3c, and the fluorescence quantum yield calculation result 212 is displayed.

  The correction coefficient necessary for the calculation is set by a correction coefficient setting function 210 (integral sphere correction spectrum setting and filter correction coefficient setting are possible). In addition, since the contents set by this function are held independently from the data readings 202 and 203, there is no need to read the correction coefficient again even if the spectrum is read again.

  In addition, when the fluorescence quantum yield is calculated using data measured using a filter for cutting higher-order light of scattered light, a data readout function 214 without using a sample with the filter attached, Data is read using a data reading function 215 measured using a sample with the filter attached. The spectrum data read here is corrected and calculated in accordance with the calculation formula of FIG. 3b, and is displayed on the spectrum display unit 205 for designating the wavelength range of the fluorescence intensity data. As in the case where no filter is used, the wavelength range to be calculated is calculated. When it is confirmed and the calculation button 211 is executed, the fluorescence quantum yield calculation is executed according to the calculation formula of FIG. 3d, and the fluorescence quantum yield calculation result 212 is displayed.

  In addition, by executing a report print button 213, it has a function of outputting a calculation result and used data as a report.

  Next, FIG. 3 shows the flow of calculation of the fluorescence quantum yield.

  For the calculation of the fluorescence quantum yield, A: excitation light quantity 301 obtained from a spectrum measured without using a sample, B: fluorescence background 302 obtained from a spectrum measured without using a sample, and a sample It requires C: the amount of reflected light 303 acquired from the spectrum measured using it, and D: the amount of fluorescence 304 acquired from the spectrum measured using the sample.

  The spectra for obtaining A, B, C, and D need to be corrected as shown in FIGS. 3a and 3b because the entire apparatus including the sample chamber needs to be corrected.

  In FIG. 3a, a spectrum (λ) 305 measured without using a sample is multiplied by an integrating sphere correction coefficient (λ) 306 without using a sample to calculate a corrected spectrum (λ) 307, and A flow of calculating a corrected spectrum (λ) 310 by multiplying a spectrum (λ) 308 measured using a sample by an integrating sphere correction coefficient (λ) 309 when the sample is used is shown. The integrating sphere correction coefficient (λ) 306 when the sample is not used and the integrating sphere correction coefficient (λ) 309 when the sample is used are measured using a diffusing element, a reference reflector, a standard reflector, and an integrating sphere, respectively. Calculated from the obtained spectrum and normalized by data of an arbitrary wavelength. The integrating sphere correction coefficients (λ) 306 and 309 are set and held in the correction coefficient setting 210 of FIG. Here, it is conceivable that various methods are used as the calculation formula itself. However, it is important that the calculation is performed manually by the operator in the past, and the correction coefficient of FIG. It is important that the settings 210 are calculated and maintained by the program.

Next, FIG. 3b shows a correction calculation in the case where the fluorescence quantum yield is calculated using data measured using a filter for high-order light cut of scattered light. The spectrum (λ) 312 measured without using the sample with the filter set is multiplied by the integrating sphere correction coefficient (λ) 307 and the filter correction coefficient (λ) 313 when the sample is not used to obtain the corrected spectrum (λ ) 314, and the integral sphere correction coefficient (λ) 310 and the filter correction coefficient (λ) 313 when the sample is used are added to the spectrum (λ) 315 measured using the sample with the filter set. A flow of multiplying and calculating a correction spectrum (λ) 316 is shown.
The integrating sphere correction coefficients (λ) 307 and 310 are the same as those shown in FIG. The filter correction coefficient (λ) 313 is a spectrum obtained by normalizing a ratio with a spectrum measured by setting a filter, with a state where no filter is set as a baseline. This filter correction coefficient (λ) 313 is also set and held in the correction coefficient setting 210 of FIG. As for the filter correction coefficient, various calculation methods can be used. However, it is important that the operator manually calculates such a filter correction coefficient. It is important that the correction coefficient setting 210 of FIG.

  Next, FIG. 3C calculates A: excitation light quantity 301, B: fluorescence quantity background 302, C: reflected light quantity 303, and D: fluorescence quantity 304 in FIG. 3 necessary for calculating the fluorescence quantum yield. The method is shown.

  From the corrected spectrum (λ) 308 of the spectrum measured without using the sample calculated in FIG. 3A, the wavelength range of the scattered light data is specified 317, and the area value A318 obtained by integrating the data within the wavelength range is calculated. This area A318 is A: excitation light quantity 301 in FIG. Further, the wavelength range of the fluorescence intensity data is designated 319, and the area value B320 obtained by integrating the data within the wavelength range is calculated. This area B319 is the background 302 of B: fluorescence amount in FIG. Next, the wavelength range of the scattered light data is specified 317 from the corrected spectrum (λ) 311 of the spectrum measured using the sample calculated in FIG. 3a, and the area value C321 obtained by integrating the data in the wavelength range is calculated. . This area C321 is C: reflected light amount 303 in FIG. Also, the wavelength range of the fluorescence intensity data is designated 319, and an area value D322 obtained by integrating the data within the wavelength range is calculated. This area D322 is D: fluorescence amount 304 in FIG. Here, the operation 317 for designating the wavelength range of the scattered light data corresponds to the wavelength range designation function 208 of the scattered light data in FIG. 2, and the operation 319 for designating the wavelength range of the fluorescence intensity data is shown in FIG. This corresponds to the wavelength range designation function 209 for fluorescence intensity data.

  Next, FIG. 3d shows A: excitation light quantity 301 and B: fluorescence quantity in FIG. 3 required for calculating the fluorescence quantum yield using data measured using a filter for cutting higher-order light of scattered light. A method of calculating the background 302, C: reflected light amount 303, and D: fluorescence amount 304 is shown.

  The wavelength range of the fluorescence intensity data is designated 319 from the corrected spectrum (λ) 314 of the spectrum measured without using the sample with the filter calculated in FIG. 3B, and the area value B ′ obtained by integrating the data within the wavelength range 324 is calculated. This area B ′ 324 is the background 302 of B: fluorescence amount in FIG. Next, from the corrected spectrum (λ) 316 of the spectrum measured using the sample with the filter calculated in FIG. 3b, the wavelength range of the fluorescence intensity data is designated 319, and the area within which the data within the wavelength range is integrated A value D'325 is calculated. This area D ′ 325 is D: fluorescence amount 304 in FIG. 3. Here, the operation 319 for specifying the wavelength range of the fluorescence intensity data corresponds to the wavelength range specifying function 209 of the fluorescence intensity data in FIG.

  As described above, A: excitation light amount 301, B: fluorescence amount background 302, C: reflected light amount 303, D: fluorescence amount 304 shown in FIG. 3 are obtained, and calculation of fluorescence quantum yield 323 in FIG. Alternatively, the fluorescence quantum yield calculation 326 of FIG. 3d can be performed to calculate the fluorescence quantum yield. Here, the operation of obtaining the calculation result of the fluorescence quantum yield corresponds to the calculation 211 in FIG.

  In this embodiment, whether to use a spectrum measured using a filter is selected in the correction coefficient setting 210 of FIG.

  When calculating the fluorescence quantum yield using a filter for cutting higher-order light of scattered light, the spectrum that specifies the wavelength range of the fluorescence intensity data is corrected by the filter correction coefficient, and therefore usually as shown in FIG. In addition, since the division by 0 occurs due to the completely filtered wavelength value = 0, the result is an overflowed value. Therefore, when auto-scaling with this spectrum, the peak spectrum of the fluorescence intensity data cannot be determined.

  Here, since the raw spectrum data before being corrected by the filter correction coefficient is filtered raw data, the scattered light 501 is cut and becomes a spectrum in which only the fluorescence intensity spectrum 502 exists as shown in FIG. When the spectrum (205 in FIG. 2) specifying the wavelength range of the fluorescence intensity data is displayed using the autoscale value 503 of this spectrum, a scale optimized for the peak of the fluorescence intensity data as shown in FIG. (The same scale as the autoscale value 503), and the wavelength range of the fluorescence intensity data can be appropriately designated. Note that a portion 601 overflowing on the left side of FIG. 6 is data obtained by dividing 0 by filter correction.

  In FIG. 2, the spectrum display unit 205 for designating the wavelength range of the fluorescence intensity data employs this embodiment so that the filter-corrected spectrum can be displayed at an optimum scale.

  FIG. 7 shows a three-dimensional spectrum measured without using a sample. The vertical axis 701 is the excitation wavelength and the horizontal axis 702 is the fluorescence wavelength. As described in FIG. 20, the excitation wavelength is fixed and the fluorescence wavelength is scanned. The spectrum obtained by repeating the fluorescence spectrum acquired in this manner while changing the excitation wavelength is displayed with contour lines at the same measured value level.

  FIG. 9 is a three-dimensional spectrum measured in the same manner using a sample. The cut line 703 in FIG. 7 and the cut line 901 in FIG. 9 indicate that the fluorescence spectrum is cut out at an arbitrary excitation wavelength. FIGS. 8 and 10 show the fluorescence spectra cut out from FIGS. 7 and 9, respectively. Show. A striped pattern 704 existing in FIG. 7 indicates scattered light, and a peak corresponding to a portion 705 overlapping the cut line is a peak 801 in FIG.

  The two-dimensional spectrum shown in FIG. 8 cut out from the three-dimensional spectrum measured without using the sample and the two-dimensional spectrum shown in FIG. 10 cut out from the three-dimensional spectrum measured using the sample are converted into the fluorescence quantum yield of FIG. It can be read into the rate calculation function and the fluorescence quantum yield can be calculated.

  FIG. 11 shows the wavelength range designation of the scattered light data, and the range is designated by the cursor 1101 and the cursor 1102. This corresponds to the specification 208 of the scattered light wavelength range in FIG. FIG. 12 shows the wavelength range designation of the fluorescence intensity data. Similarly, the range is designated by the cursor 1201 and the cursor 1202. This corresponds to the fluorescent wavelength range designation 209 in FIG.

  Next, FIG. 13 corresponds to the integrating sphere correction set in the correction coefficient setting 210 in FIG. 2, and shows a method of measuring a three-dimensional spectrum and calculating a three-dimensional integrating sphere correction spectrum. . This corresponds to the integrating sphere correction coefficient (λ) 307 when the sample of FIG. 3a is not used and the integrating sphere correction coefficient (λ) 310 when the sample is used.

A three-dimensional spectrum of the diffusing element: A (ex, em) is a value A ( ex , λ) at an arbitrary wavelength λ1.
From the spectrum 1301 normalized in 1) and the spectrum 1302 obtained by normalizing the integrating sphere three-dimensional spectrum B (ex, em) measured at a desired wavelength λ1 without using a sample with a value B ( ex , λ1). Integrated sphere corrected 3D spectrum measured without using a sample:
F1 is calculated 1303, and the three-dimensional spectrum of the diffusing element: A (ex, em) is set to an arbitrary wavelength λ.
The spectrum 1301 normalized by the value A ( ex , λ1) at 1 and the integrating sphere three-dimensional spectrum measured using the sample: C (ex, em) is the value C ( ex , λ1) at an arbitrary wavelength λ1.
The integrated sphere corrected three-dimensional spectrum F2 measured using the sample is calculated 1304 from the spectrum 1303 normalized in (1).

  Next, as shown in FIG. 14, a three-dimensional spectrum 1401 measured without using a sample (three-dimensional spectrum in FIG. 7) is measured using an integrating sphere corrected three-dimensional spectrum without using the sample: F1 (the sample in FIG. 13 is changed). 3D spectrum 1303 of the integrating sphere correction coefficient when not used is multiplied and corrected 3D spectrum 1402 measured without using the sample (when replaced with the 2D spectrum of FIG. 3A, the sample of FIG. 3A is used without measurement) (This corresponds to the corrected spectrum (λ) 308 of the spectrum.).

  In addition, as shown in FIG. 15, a three-dimensional spectrum 1501 (three-dimensional spectrum in FIG. 9) measured using the sample is used for integrating sphere correction three-dimensional spectrum measured using the sample: F2 (using the sample in FIG. 13). The three-dimensional spectrum 1304 of the integrating sphere correction coefficient in this case, and the corrected three-dimensional spectrum 1502 measured using the sample (the spectrum measured using the sample of FIG. 3a when replaced with the two-dimensional spectrum of FIG. 3a) (Corresponding to the correction spectrum (λ) 311).

  In the corrected three-dimensional spectrum 1402 measured without using a sample, the A: excitation light quantity 1403 is calculated by designating the wavelength range as shown in FIG. 11 for the two-dimensional spectrum cut out at each excitation wavelength Exn, and FIG. B: Fluorescence amount background 1404 is calculated by specifying the range as described above, and in the corrected three-dimensional spectrum 1502 measured using the sample, the two-dimensional spectrum cut out at each excitation wavelength Exn is shown in FIG. As shown in FIG. 12, C: the amount of reflected light 1503 is calculated, D: the amount of fluorescence 1504 is calculated as shown in FIG. 12, and the fluorescence quantum yield QY is calculated from A, B, C, and D. As shown in FIG. 16, the fluorescence quantum yield QY with respect to Exn is expressed two-dimensionally to obtain the magnitude of the fluorescence quantum yield with respect to the change in the excitation wavelength of the sample. Kill.

  In FIG. 17, as a method for automatically determining the wavelength range of the scattered light data, the wavelength range of excitation wavelength 1701 ± slit width 1702 (excitation side slit, fluorescence side slit larger slit width × 2) nm is used as the scattered light data. A designation method for determining the wavelength range is shown.

FIG. 18 shows a designation method for determining the wavelength range 1802 from the long wavelength 1801 to the long wavelength side of the scattered light data as the wavelength range of the fluorescence intensity data.

  Further, FIG. 19 shows from the peak start wavelength (valley wavelength on the short wavelength side from the peak wavelength) to the end wavelength (from the peak wavelength) detected by detecting the peak on the long wavelength side 1902 from the long wavelength 1901 of the scattered light data. A designation method for determining a wavelength range 1903 to 1904 (valley wavelength on the long wavelength side) as a wavelength range of fluorescence intensity data is shown. In this case, the sum of the integrated values of all peaks is used.

  If these designation methods are applied to the three-dimensional spectra of FIGS. 14 and 15, the fluorescence quantum yield can be automatically acquired.

The figure which shows an example of the spectrofluorometer of the Example of this invention. The figure which shows an example of the fluorescence quantum yield calculation screen of the Example of this invention. The fluorescence quantum yield calculation of the Example of this invention. The figure which shows an example of the correction | amendment spectrum calculation method when not using the filter of the Example of this invention. The figure which shows an example of the correction | amendment spectrum calculation method in the case of using the filter of the Example of this invention. The figure which shows an example of the fluorescence quantum yield calculation method when not using the filter of the Example of this invention. The figure which shows an example of the fluorescence quantum yield calculation method in the case of using the filter of the Example of this invention. The figure which shows an example of the graph display which carried out the auto scale of the spectrum after the filter correction | amendment of the spectrum which set the filter of the Example of this invention and measured using the sample. The figure which shows an example of the raw spectrum which set the filter of the Example of this invention, and was measured using the sample. The figure which shows an example of the graph display which autoscaled the spectrum after filter correction | amendment by the autoscale value of the raw spectrum which set the filter of the Example of this invention and was measured using the sample. The figure which shows an example of the three-dimensional spectrum when not using the sample of the Example of this invention. The figure which shows an example of the two-dimensional spectrum cut out with the specific excitation wavelength from the three-dimensional spectrum when not using a sample. The figure which shows an example of the three-dimensional spectrum at the time of using the sample of the Example of this invention. The figure which shows an example of the two-dimensional spectrum cut out with the specific excitation wavelength from the three-dimensional spectrum at the time of using a sample. The figure which shows an example of designation | designated of the wavelength range of the scattered light data from the two-dimensional spectrum of the Example of this invention. The figure which shows an example which also designates the wavelength range of the fluorescence intensity data from the two-dimensional spectrum of the Example of this invention. The figure which shows an example of the calculation method of the integrating sphere correction coefficient using the three-dimensional spectrum of the Example of this invention. The figure which shows an example of the explanatory drawing which repeatedly cuts out the two-dimensional spectrum of a specific excitation wavelength from the three-dimensional spectrum measured without using the sample of the Example of this invention, and performs a fluorescence quantum yield calculation. The figure which shows an example of the explanatory drawing which repeatedly cuts out the two-dimensional spectrum of a specific excitation wavelength from the three-dimensional spectrum measured using the sample of the Example of this invention, and performs fluorescence quantum yield calculation. The figure which shows an example of the excitation wavelength characteristic graph of the fluorescence quantum yield which expressed two-dimensionally the fluorescence quantum yield with respect to the excitation wavelength calculated | required in FIG. 14, FIG. The figure which shows an example of the automatic designation | designated method which judges the wavelength range of the excitation wavelength +/- (excitation side slit, the larger slit width of a fluorescence side slit x2) nm of the Example of this invention as a wavelength range of scattered light data. The figure which shows an example of the automatic designation | designated method which judges the wavelength range longer wavelength side of the scattered light data of the Example of this invention as the wavelength range of fluorescence intensity data. The figure which shows an example of the automatic designation | designated method which judges the peak wavelength of the long wavelength side from the long wavelength of the scattered light data of the Example of this invention as the wavelength range of fluorescence intensity data. It is a schematic block diagram of an example of the spectrofluorometer to which this invention is applied.

Explanation of symbols

2001 Light source 2002 Excitation light side spectroscope 2003 Pulse motor 2004 Data processing device 2005 Interface

Claims (6)

A light source;
An excitation light side spectroscope that converts the light emitted from the light source into monochromatic light;
A fluorescence-side spectrometer that irradiates the sample with light emitted from the spectrometer and separates the light emitted from the sample; a detector that detects a signal from the fluorescence-side spectrometer;
A processing device for processing the signal from the detector;
A display device for displaying data processed by the processing device;
It has an arithmetic unit that calculates the fluorescence quantum yield,
The display device is a signal obtained without installing a sample, and is a signal obtained by installing a spectrum and a sample from which the higher order light of the scattered light has been removed by the filter, and the higher order light of the scattered light is obtained by the filter. Display the scattered light spectrum and fluorescence spectrum of the sample obtained based on the removed spectrum, the display range of the scattered light spectrum and the fluorescence spectrum can be independently changed,
The arithmetic device calculates a fluorescence quantum yield based on a wavelength range of the scattered light spectrum and a wavelength range of the fluorescence spectrum specified on the display screen.
A spectrofluorometer characterized by. The spectrophotometer of claim 1,
A spectrofluorophotometer characterized in that the excitation light side spectroscope irradiates a sample with different monochromatic light and obtains a three-dimensional fluorescence spectrum using a spectrum obtained by irradiation with each monochromatic light. The spectrofluorometer according to claim 2, wherein
A spectrofluorometer capable of calculating a fluorescence quantum yield by cutting out a fluorescence spectrum of an arbitrary excitation wavelength from the three-dimensional fluorescence spectrum. The spectrofluorometer according to claim 1, wherein
A spectrofluorophotometer characterized in that light emitted from the sample is measured using an integrating sphere, and wavelength characteristics of the integrating sphere are corrected. The spectrofluorometer according to claim 1, wherein
A spectrofluorimeter characterized by two-dimensionally displaying a fluorescence quantum yield at each wavelength of light irradiated on a sample. The spectrofluorometer according to claim 1, wherein
A spectrofluorometer comprising: a determining unit that determines a wavelength range of an absorption region and a fluorescence region based on a peak wavelength of a detected fluorescence spectrum.

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