Shifted excitation Raman difference spectroscopy (SERDS) was applied for an effective fluorescence removal in the Raman spectra of meat, fat, connective tissue, and bone from pork and beef. As excitation light sources, microsystem diode lasers emitting at 783 nm, 671 nm, and 488 nm each incorporating two slightly shifted excitation wavelengths with a spectral difference of about 10 cm−1 necessary for SERDS operation were used. The moderate fluorescence interference for 783 nm excitation as well as the increased background level at 671 nm was efficiently rejected using SERDS resulting in a straight horizontal baseline. This allows for identification of all characteristic Raman signals including weak bands which are clearly visible and overlapping signals that are resolved in the SERDS spectra. At 488 nm excitation, the spectra contain an overwhelming fluorescence interference masking nearly all Raman signals of the probed tissue samples. However, the essentially background-free SERDS spectra enable determining the majority of characteristic Raman bands of the samples under investigation. Furthermore, 488 nm excitation reveals prominent carotenoid signals enhanced due to resonance Raman scattering which are present in the beef samples but absent in pork tissue enabling a rapid meat species differentiation.
Due to its fingerprinting characteristics, Raman spectroscopy is well suited for the investigation of biological material, for example, for rapid and nondestructive identification purposes. Here, excitation wavelengths in the visible or near-infrared range are preferable to avoid strong absorption of water leading to sample heating [
There exist certain methods to remove the fluorescence background from the Raman spectra to overcome the fluorescence issue. In that way, mathematical approaches as polynomial [
This has been demonstrated in the NIR spectral region by da Silva Martins et al. [
This paper presents SERDS investigations using microsystem diode lasers with two slightly shifted emission lines at 783 nm, 671 nm, and 488 nm as excitation light sources. The lasers emitting in the near-infrared and red spectral region were integrated into compact Raman measurement heads which are connected to laboratory (783 nm) as well as miniature spectrometers (671 nm), while the 488 nm light source is part of a laboratory setup. To test the performance of the SERDS technique, we chose meat, fat tissue, connective tissue, and bones from pork and beef as sample material to realize a variety of optical properties and fluorescence background levels.
To realize a SERDS measurement head for near-infrared excitation, an in-house developed and tested Raman optical bench [
The SERDS measurement head is coupled to a PI 320 spectrometer (Princeton instruments) equipped with a 600 lines/mm grating. An attached back-illuminated deep-depletion CCD detector (DU4020-BR-DD, Andor Technology) thermoelectrically cooled down to −60°C serves for detection of the Raman spectra. CCD readout and data storage is performed by means of a computer running Andor Solis software (Andor Technology). With applied laser powers of 50 mW (782.65 nm) and 110 mW (783.15 nm) at sample position, Raman spectra of the biological samples were recorded with an integration time of 10 s.
For the 671 nm measurement head, the same concept of a Raman optical bench with attached microsystem diode laser is applied. Here, the excitation light source contains two separate laser cavities each comprising a volume Bragg grating as frequency selective element [
The laboratory setup applied for Raman spectroscopic investigations at 488 nm is depicted in Figure
Scheme of experimental setup with (1) 488 nm microsystem diode laser, (2) BG 38 color glass filter, (3) bandpass filter, (4) dielectric mirror, (5) Raman edge filter, (6) lens, (7) sample, (8) pinhole, (9) spectrometer, (10) CCD, and (11) computer.
To obtain meat, fat, connective tissue, and bones from pork and beef, fresh meat slices including these components were bought in a local supermarket and investigated at the day of purchase. The biological tissue of interest was cut from the slices during sample preparation using a knife and transferred into Petri dishes to prevent it from drying. Until investigation, all sample material was stored in a laboratory refrigerator (Spezial-468, Philipp Kirsch, Germany) at 5°C. During the Raman measurement, each type of tissue was probed at 15 different positions and 10 spectra were averaged each. From the two slightly shifted Raman spectra of each measurement spot, SERDS spectra were calculated applying an in-house developed reconstruction procedure [
Figure
Raman bands identified in the SERDS spectra of meat from beef and pork and their vibrational assignment, Phe: Phenylalanine, Trp: Tryptophan, Tyr: Tyrosine.
Vibrational assignment | 783 nm excitation | 671 nm excitation | 488 nm excitation | Literature value |
---|---|---|---|---|
[References] | ||||
Quartz | 492 | 492 | 489 | |
Quartz | 603 | 602 | 602 | |
Myoglobin | — | — | 671 | 671–678 [ |
Adenine | 720 | 717 | — | 720 [ |
Trp | 757 | 753 | 752 | 750–760 [ |
Quartz | 782 | 783 | 780 | |
Tyr |
827 | 825 | 826 | 827–834 [ |
Tyr |
855 | 853 | 852 | 850–860 [ |
Trp |
880 | 875 | — | 877–881 [ |
|
901 | 902 | — | 900–901 [ |
|
936 | 935 | 937 | 934–944 [ |
Carotenoid | — | — | 960 | 960–965 [ |
Phe |
1002 | 1002 | 1001 | 1000–1006 [ |
Carotenoid | — | — | 1002 | 1000–1014 [ |
C–C ring bend (Phe) | 1033 | 1032 | — | 1030–1033 [ |
|
1047 | 1049 | — | 1040–1120 [ |
|
1081 | 1077 | 1076 | 1040–1120 [ |
|
1126 | 1125 | 1125 | 1127–1130 [ |
|
1156 | 1154 | — | 1156 [ |
Carotenoid | — | — | 1154 | 1151–1172 [ |
Tyr | 1173 | 1171 | — | 1172–1175 [ |
Carotenoid | — | — | 1188 | 1191–1193 [ |
Tyr, Phe | 1206 | 1203 | — | 1205–1209 [ |
Carotenoid | — | — | 1210 | 1213–1215 [ |
Amide III ( |
1242 | 1234 | — | 1225–1250 [ |
Amide III ( |
1266 | 1266 | — | 1265–1278 [ |
Amide III ( |
1304 | 1304 | — | 1301–1309 [ |
Trp; |
1341 | 1339 | — | 1339–1342 [ |
Myoglobin | — | — | 1351 | 1353–1371 [ |
|
1395 | 1394 | 1397 | 1395 [ |
|
1447 | 1446 | 1447 | 1447–1451 [ |
CH2 and CH3 bending | 1460 | 1461 | 1465 | 1460–1483 [ |
Carotenoid | — | — | 1520 | 1511–1535 [ |
Trp |
1550 | 1549 | — | 1553-1554 [ |
Trp, Phe, Tyr |
1603 | 1610 | 1601 | 1605–1618 [ |
Amide I ( |
1648 | 1647 | 1653 | 1645–1658 [ |
Comparison of Raman (upper traces) and SERDS spectra (lower traces) of meat from pork (red curves) and beef (green curves) excited at (a) 783 nm, (b) 671 nm, and (c) 488 nm. Each curve represents the average from 15 measurement positions. Resonance-enhanced carotenoid signals in (c) are indicated by asterisks.
Applying an excitation wavelength of 671 nm results in a strongly increased fluorescence interference which becomes obvious in particular for beef as illustrated in Figure
Figure
The recorded spectra for fat from pork and beef are presented in Figure
Raman bands identified in the SERDS spectra of fat tissue from beef and pork and their vibrational assignment.
Vibrational assignment | 783 nm excitation | 671 nm excitation | 488 nm excitation | Literature value |
---|---|---|---|---|
[References] | ||||
Quartz | 492 | 494 | 490 | |
Quartz | 605 | 606 | 607 | |
=C–H in-plane deformation | 726 | 723 | — | 727 [ |
C–C stretch, CH3 rock, C–O stretch | 842 | 843 | 843 | 800–920 [ |
C–C stretch, CH3 rock, C–O stretch | 868 | 869 | 872 | 800–920 [ |
C–C stretch, CH3 rock, C–O stretch | 890 | 886 | 887 | 800–920 [ |
C–C stretch, CH3 rock, C–O stretch | 921 | 919 | 920 | 800–920 [ |
Carotenoid | — | — | 960 | 960–965 [ |
=C–H out of plane bend |
970 | 969 | 970 | 970–972 [ |
Carotenoid | — | — | 1003 | 1000–1014 [ |
C–C aliphatic out-of-phase stretch | 1061 | 1060 | 1060 | 1060–1068 [ |
C–C aliphatic stretch | 1084 | 1080 | — | 1076–1090 [ |
C–C aliphatic in phase stretch | 1126 | 1125 | 1125 | 1119–1129 [ |
Carotenoid | — | — | 1153 | 1151–1172 [ |
Carotenoid | — | — | 1188 | 1191–1193 [ |
Carotenoid | — | — | 1211 | 1213–1215 [ |
=C–H symmetric rock |
1262 | 1262 | 1263 | 1263–1266 [ |
>CH2 in phase twist | 1297 | 1297 | 1294 | 1295–1305 [ |
CH3 symmetric deformation | 1368 | 1368 | 1366 | 1368 [ |
>CH2 symmetric deformation | 1436 | 1436 | 1436 | 1436–1443 [ |
CH3 antisymmetric deformation | 1457 | 1459 | 1461 | 1455–1460 [ |
Carotenoid | — | — | 1521 | 1511–1535 [ |
C=C |
1651 | 1650 | 1653 | 1650–1670 [ |
Carbonyl C=O stretch | 1740 | 1740 | 1738 | 1730–1750 [ |
Comparison of Raman (upper traces) and SERDS spectra (lower traces) of fat from pork (red curves) and beef (green curves) excited at (a) 783 nm, (b) 671 nm, and (c) 488 nm. Each curve represents the average from 15 measurement positions.
Compared to meat, fat has a higher Raman scattering intensity and thus despite of the high fluorescence level even in the Raman spectra excited at 488 nm some strong lipid signals can be recognized (see Figure
Connective tissue possesses a complex Raman spectrum comprising a large number of collagen signals [
Raman bands identified in the SERDS spectra of connective tissue from beef and pork and their vibrational assignment, Hyp: Hydroxyproline, Pro: Proline, Phe: Phenylalanine, Trp: Tryptophan, Tyr: Tyrosine.
Vibrational assignment | 783 nm excitation | 671 nm excitation | 488 nm excitation | Literature value |
---|---|---|---|---|
[References] | ||||
Quartz | 492 | 494 | 489 | |
Quartz | 602 | 602 | 602 | |
CH2 rocking | 721 | 723 | — | 730 [ |
Trp ring breathing | 757 | 757 | 753 | 756 [ |
CH2 rocking | 782 | 781 | 783 | 779 [ |
|
814 | 813 | 817 | 812–821 [ |
|
853 | 853 | 852 | 852–860 [ |
|
875 | 875 | 873 | 873–884 [ |
|
918 | 919 | 919 | 920–925 [ |
|
939 | 938 | 937 | 936–938 [ |
Carotenoid | — | — | 963 | 960–965 [ |
|
970 | 969 | — | 957–961 [ |
Phe | 1002 | 1002 | 1002 | 1003–1006 [ |
Carotenoid | — | — | 1002 | 1000–1014 [ |
Phe, Pro | 1031 | 1032 | — | 1032–1037 [ |
Bend of carboxyl OH | 1060 | 1058 | — | 1062–1065 [ |
|
1100 | 1099 | — | 1093–1101 [ |
|
1126 | 1125 | 1124 | 1122–1125 [ |
Carotenoid | — | — | 1154 | 1151–1172 [ |
|
1159 | 1162 | — | 1163 [ |
Carotenoid | — | — | 1188 | 1191–1193 [ |
Hyp, Tyr | 1203 | 1202 | — | 1202-1211 [ |
Carotenoid | — | — | 1211 | 1213–1215 [ |
Amide III | 1240 | 1240 | 1243 | 1239–1248 [ |
Amide III | 1271 | 1273 | 1270 | 1263–1273 [ |
CH2 twisting | 1318 | 1317 | 1314 | 1314–1319 [ |
CH2 wagging | 1342 | 1341 | 1340 | 1340–1343 [ |
CH2 deformation | 1378 | 1377 | 1374 | 1379 [ |
|
1424 | 1424 | 1423 | 1416–1427 [ |
|
1450 | 1448 | 1448 | 1447–1452 [ |
|
1461 | 1464 | 1468 | 1461–1464 [ |
Carotenoid | — | — | 1521 | 1511–1535 [ |
Phe, Tyr | 1603 | 1599 | 1598 | 1603–1606 [ |
Amide I | 1630 | 1629 | — | 1636–1642 [ |
Amide I | 1662 | 1660 | 1658 | 1650–1680 [ |
Comparison of Raman (upper traces) and SERDS spectra (lower traces) of connective tissue from pork (red curves) and beef (green curves) excited at (a) 783 nm, (b) 671 nm, and (c) 488 nm. Each curve represents the average from 15 measurement positions.
As in the case of meat, the Raman spectra of connective tissue excited at 488 nm presented in Figure
The Raman and SERDS spectra of bone from pork and beef are displayed in Figure
Raman bands identified in the SERDS spectra of bone from beef and pork and their vibrational assignment, Hyp: Hydroxyproline, Pro: Proline, Phe: Phenylalanine, Tyr: Tyrosine.
Vibrational assignment | 783 nm excitation | 671 nm excitation | 488 nm excitation | Literature value |
---|---|---|---|---|
[References] | ||||
|
432 | 432 | — | 422–454 [ |
|
455 | 451 | — | 422–454 [ |
Quartz | 494 | 494 | 489 | |
|
583 | 583 | 581 | 578–617 [ |
|
615 | 612 | 612 | 578–617 [ |
CH2 rocking | 723 | 719 | — | 730 [ |
CH2 rocking | 784 | 781 | 780 | 779 [ |
|
815 | 813 | 812 | 815 [ |
|
853 | 851 | 850 | 855-856 [ |
|
880 | 884 | — | 871–876 [ |
|
918 | 912 | — | 920-921 [ |
|
958 | 958 | 957 | 957–963 [ |
Phe |
1004 | 1002 | 1001 | 1003-1004 [ |
Carotenoid | — | — | 1001 | 1000–1014 [ |
|
1031 | 1030 | 1026 | 1030–1032 [ |
|
1071 | 1070 | 1067 | 1065–1075 [ |
|
1103 | 1099 | 1101 | 1093–1101 [ |
|
1128 | 1125 | 1125 | 1122–1125 [ |
Carotenoid | — | — | 1155 | 1151–1172 [ |
|
1161 | 1160 | — | 1163 [ |
Hyp, Tyr | 1203 | 1200 | 1197 | 1202–1211 [ |
Amide III | 1243 | 1241 | 1241 | 1243–1320 [ |
Amide III | 1272 | 1269 | 1266 | 1243–1320 [ |
Amide III | 1320 | 1317 | — | 1243–1320 [ |
CH2 wagging | 1344 | 1341 | 1344 | 1340–1343 [ |
CH2 deformation | 1378 | 1375 | 1374 | 1379 [ |
|
1450 | 1448 | 1447 | 1447–1452 [ |
|
1464 | 1466 | — | 1461–1464 [ |
Carotenoid | — | — | 1518 | 1511–1535 [ |
Phe, Tyr | 1603 | 1599 | 1602 | 1603–1606 [ |
Amide I |
1630 | 1629 | 1629 | 1640–1670 [ |
Amide I |
1662 | 1658 | 1659 | 1640–1670 [ |
Amide I |
1683 | 1681 | 1679 | 1640–1670 [ |
Comparison of Raman (upper traces) and SERDS spectra (lower traces) of bone from pork (red curves) and beef (green curves) excited at (a) 783 nm, (b) 671 nm, and (c) 488 nm. Each curve represents the average from 15 measurement positions.
Using an excitation wavelength of 671 nm, the fluorescence interference is even more pronounced (see Figure
The bone Raman and SERDS spectra excited at 488 nm are presented in Figure
In this paper, we applied shifted excitation Raman difference spectroscopy (SERDS) at different excitation wavelengths to demonstrate the potential for an efficient rejection of the fluorescence background in the Raman spectra of selected meat components. Microsystem diode lasers emitting at 783 nm, 671 nm, and 488 nm were used as excitation light sources. Each laser device was able to emit at two slightly different wavelengths with a spectral separation of about 10 cm−1 necessary to perform SERDS. To realize a variety of biological samples, meat, fat, connective tissue, and bone from pork and beef were selected for our investigations.
With an excitation wavelength of 783 nm, the fluorescence interference was moderate. However, the SERDS technique effectively removed the signal background resulting in a straight horizontal baseline. Furthermore, weak bands in the Raman spectra become clearly visible in the SERDS spectra, and also overlapping signals are separated and resolved applying the SERDS technique.
An excitation at 671 nm results in an increased fluorescence background which becomes obvious in particular for the beef samples. Despite of this fluorescence issue, SERDS allows for efficient background removal enabling the identification of essentially the same spectral patterns of the samples under investigation as for 783 nm excitation.
As expected, the Raman spectra excited at 488 nm exhibit an overwhelming signal background masking nearly all Raman signals of the probed tissue except in the case of fat. Application of SERDS allows determining not all, but a majority of the signals which were present in the spectra excited at 783 nm and 671 nm. In addition, the tissue samples from beef reveal prominent signals of carotenoids which are enhanced by means of the resonance Raman effect. These bands are not present in samples from pork and thus allow for a rapid meat species distinction.
The presented study demonstrated the ability of the SERDS technique for an effective fluorescence background removal of selected biological material. Furthermore, weak bands are enhanced, and overlapping signals are resolved allowing for improvement of spectral quality. In that way, SERDS has a great potential for numerous other analytical Raman applications which were limited by the fluorescence issue up to now, for example, investigation of various natural compounds, medical diagnostics, and forensic usage.
The authors wish to thank Bernd Sumpf and Martin Maiwald, Ferdinand-Braun-Institut, Leibniz-Institut für Höchstfrequenztechnik Berlin, for the development of the microsystem diode laser sources suitable for SERDS emitting at 783 nm, 671 nm, and 488 nm. K. Sowoidnich is grateful for the financial support by the state of Berlin in the frame of an Elsa-Neumann scholarship.