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Absence of lines in Raman spectra of living cells
Volume 111, number 7
ABSENCE
PHYSICSLETTERS
OF LINES IN RAMAN SPECTRA
30 September1985
OF LIVING CELLS
Luciano FURIA and Om P. GANDHI Department
of Electrical Engineering,
University of Utah, Salt Lake City, UT 84112, USA
Received10 June 1985;acceptedfor publication20 June 1985
A previous paper reporting the absence of biologically criticized. A reply to these criticisms is presented.
In the past few years some investigators have claimed the presence of lines in Raman spectra of metabolizing cells while no lines could be detected in spectra of resting cells [l-3] . Consequently other investigators have linked these alleged lines to internal vibrations of Davydov solitons [4,5] , to coherent oscillations as postulated by Frijhlich [6] and to nonlinearoptics phenomena [7,8 ] . In our paper [9 ] we presented experimental data showing that no biologically related lines were found in Raman spectra of Bacillus megaterium suspensions under nonflowing conditions in the 20-300 and 800-l 800 cm-l regions and that under flowing conditions artifacts could be responsible for random lines appearing in the spectra of both cell suspensions and medium alone. For these experiments we closely followed the experimental protocol given by Webb in ref. [3]. Del Giudice et al. [lo] have raised several questions about both the correctness of the conclusions and the validity of experimental data presented in our previous paper [9] . In particular our conclusions have been attributed more to an alleged “...preconceived philosophy...” and to “... motive behind the two recent reports...” rather than to a logical connection to the experimental data. We find these allegations out of place in a scientific forum, especially when these are totally unsubstantiated. After a very careful rereading of our paper we have not been able to find any suggestion of an alleged preconception. Furthermore Del Giudice et al. attributed to us a criticism of the work of Webb [l-3] that was, instead, pointed out by Cooper and Amer [ 111. Cooper and Amer 376
related
lines in Raman
spectra
of Bacillus
megaterium
has been
suggested that the Raman spectra of bacterial cells, prepared according to Webb [3] were likely to be featureless due to the relatively low cell concentrations. In the present reply we hope to offer some clarifications and leave the conclusions to peers. Some of the questions asked in ref. [lo] are, however, already explicitly answered in our original paper [9 ] . In the following, citations from our first paper [9] are in quotes: (a) We did not use a nonnutrient medium. The legend of fig. 2 of ref. [9] contains a printing error: “untrient” instead of nutrient. We really wonder, however, how the word “untrient” can be read as nonnutrient. (b) The experimental conditions of 35’C and room temperature, were used because the latter is the one used by Webb in ref. [3] and the former is an optimal growth temperature for B. megaterium. (c) In addition to several experiments under nonflowing conditibns, we used also a flow-through system, since this was also the recommendation of Drissler [ 121 to avoid excessive laser heating of the cells. We pointed out in our paper [9] that in the experiments reported by Webb [l-3] “...the spectra were recorded under nonflowing conditions”. (d) Del Giudice et al. [lo] ask for clarification regarding the time elapsed between resuspension and recording of the spectra. In ref. [93 we clearly specified that “ ...no lines were ever detected in the 20300 cm-l region at any time between 10 min and 2 h 30 min after resuspension in DMM”. 0.375-9601/85/$03.30 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
Volume 111, number 7
30 September 1985
PHYSICS LETTERS
(e) All the apparent random Raman lines could be seen only in our spectra taken under flowing conditions. Such lines were narrow enough to be within one or two frequency steps (i.e. with a width of 4-8 cm-l) of the computer driven spectrometer. This is compatible with the hypothesis that either air bubbles or microscopic fluorescent contaminants may have caused these, by randomly passing through the scattering volume. (f) Under nonflowing conditions, the spectrum of the medium alone (Davis Minimal Medium 1 : 3 in HzO) was completely reproducible, as were the featureless spectra of B. megaterium suspensions taken at any time after resuspension. It may be useful to point out that all the spectra of a single experimental session were taken using either the same suspension for an extended period of time (up to 45 min) or by changing the suspension every 10 min with another sample taken from a culture kept in a water bath at the selected temperature. Furthermore, we have found that Kinoshita et al. [ 131 presented detailed calculations regarding possible artifacts that might have affected the intensity ratio of Stokes and anti-Stokes lines in Raman ‘speo tra of Chlorella pyrenoidosa [ 14,lS ] . These possible artifacts include the l/X4 dependence of the scattered light intensity, effective detection efficiency, resonance effect, laser-induced temperature increase and attenuation within the sample. These findings, along with our experimental data, show that several experimental artifacts can be introduced in the Raman spectroscopy of living cells and that to our knowledge no independent positive replication of the data pre-
sented in refs. [l-3,14,15] is available in the literature. In conclusion we do not think that the alleged presence of lines in Raman spectra of baterial cells can be claimed to be “...non questionable...” [lo] or that such “...revelations...” [lo] can be taken as a scientific proof of coherent phenomena occurring in living cells as suggested by Webb [3] , Frohlich [6] and Del Giudice et al. [7,8,10]. References
ill SJ. Webb and M.E. Stoneham, Int. J. Quantum Chem. Quantum Biol. Symp. 2 (1975) 339.
PI SJ. Webb and M.E. Stoneham, Phys. Lett. 60A (1977) 267. 131 SJ. Webb, Phys. Rep. 60 (1980) 201. [41 A.C. Scott, Phys. Lett. 86A (1981) 60. iSI P.S. Lomdahl, L. MacNeil, A.C. Scott, M.E. Stoneham and SJ. Webb, Phys. Lett. 92A (1982) 207. [61 H. Frijhlich, Adv. Electron. Electron Phys. 53 (1980) 85. 171 E. Del Giudice, S. Doglia and M. Milani, Phys. Lett. 85A (1981) 402. PI E. Del Giudice, S. Doglia, M. Milani and SJ. Webb, Phys. Lett. 91A (1982) 257. PI L. Furia and OP. Gandhi, Phys. Lett. 102A (1984) 380. VOI E. Del Giudice, S. Dog@, M. Milani, C.W. Smith and SJ. Webb, Phys. Lett. 107A (1985) 98, [Ill M.S. Cooper and NM. Amer, Phys. Lett. 98A (1983) 138. 1121 F. Drissler, COB. Phen. 3 (1981) 147. 1131 S. Kinoshita, K. Hirata and T. Ku&da, J. Phys. Sot. Japan 49 (1980) 314. [I41 F. Drissler and R.M. MacFarlane, Phys. Lett. 69A (1978) 65. 1151 F. Drissler, Phys. Lett. 77A (1980) 207.
377
ABSENCE
PHYSICSLETTERS
OF LINES IN RAMAN SPECTRA
30 September1985
OF LIVING CELLS
Luciano FURIA and Om P. GANDHI Department
of Electrical Engineering,
University of Utah, Salt Lake City, UT 84112, USA
Received10 June 1985;acceptedfor publication20 June 1985
A previous paper reporting the absence of biologically criticized. A reply to these criticisms is presented.
In the past few years some investigators have claimed the presence of lines in Raman spectra of metabolizing cells while no lines could be detected in spectra of resting cells [l-3] . Consequently other investigators have linked these alleged lines to internal vibrations of Davydov solitons [4,5] , to coherent oscillations as postulated by Frijhlich [6] and to nonlinearoptics phenomena [7,8 ] . In our paper [9 ] we presented experimental data showing that no biologically related lines were found in Raman spectra of Bacillus megaterium suspensions under nonflowing conditions in the 20-300 and 800-l 800 cm-l regions and that under flowing conditions artifacts could be responsible for random lines appearing in the spectra of both cell suspensions and medium alone. For these experiments we closely followed the experimental protocol given by Webb in ref. [3]. Del Giudice et al. [lo] have raised several questions about both the correctness of the conclusions and the validity of experimental data presented in our previous paper [9] . In particular our conclusions have been attributed more to an alleged “...preconceived philosophy...” and to “... motive behind the two recent reports...” rather than to a logical connection to the experimental data. We find these allegations out of place in a scientific forum, especially when these are totally unsubstantiated. After a very careful rereading of our paper we have not been able to find any suggestion of an alleged preconception. Furthermore Del Giudice et al. attributed to us a criticism of the work of Webb [l-3] that was, instead, pointed out by Cooper and Amer [ 111. Cooper and Amer 376
related
lines in Raman
spectra
of Bacillus
megaterium
has been
suggested that the Raman spectra of bacterial cells, prepared according to Webb [3] were likely to be featureless due to the relatively low cell concentrations. In the present reply we hope to offer some clarifications and leave the conclusions to peers. Some of the questions asked in ref. [lo] are, however, already explicitly answered in our original paper [9 ] . In the following, citations from our first paper [9] are in quotes: (a) We did not use a nonnutrient medium. The legend of fig. 2 of ref. [9] contains a printing error: “untrient” instead of nutrient. We really wonder, however, how the word “untrient” can be read as nonnutrient. (b) The experimental conditions of 35’C and room temperature, were used because the latter is the one used by Webb in ref. [3] and the former is an optimal growth temperature for B. megaterium. (c) In addition to several experiments under nonflowing conditibns, we used also a flow-through system, since this was also the recommendation of Drissler [ 121 to avoid excessive laser heating of the cells. We pointed out in our paper [9] that in the experiments reported by Webb [l-3] “...the spectra were recorded under nonflowing conditions”. (d) Del Giudice et al. [lo] ask for clarification regarding the time elapsed between resuspension and recording of the spectra. In ref. [93 we clearly specified that “ ...no lines were ever detected in the 20300 cm-l region at any time between 10 min and 2 h 30 min after resuspension in DMM”. 0.375-9601/85/$03.30 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
Volume 111, number 7
30 September 1985
PHYSICS LETTERS
(e) All the apparent random Raman lines could be seen only in our spectra taken under flowing conditions. Such lines were narrow enough to be within one or two frequency steps (i.e. with a width of 4-8 cm-l) of the computer driven spectrometer. This is compatible with the hypothesis that either air bubbles or microscopic fluorescent contaminants may have caused these, by randomly passing through the scattering volume. (f) Under nonflowing conditions, the spectrum of the medium alone (Davis Minimal Medium 1 : 3 in HzO) was completely reproducible, as were the featureless spectra of B. megaterium suspensions taken at any time after resuspension. It may be useful to point out that all the spectra of a single experimental session were taken using either the same suspension for an extended period of time (up to 45 min) or by changing the suspension every 10 min with another sample taken from a culture kept in a water bath at the selected temperature. Furthermore, we have found that Kinoshita et al. [ 131 presented detailed calculations regarding possible artifacts that might have affected the intensity ratio of Stokes and anti-Stokes lines in Raman ‘speo tra of Chlorella pyrenoidosa [ 14,lS ] . These possible artifacts include the l/X4 dependence of the scattered light intensity, effective detection efficiency, resonance effect, laser-induced temperature increase and attenuation within the sample. These findings, along with our experimental data, show that several experimental artifacts can be introduced in the Raman spectroscopy of living cells and that to our knowledge no independent positive replication of the data pre-
sented in refs. [l-3,14,15] is available in the literature. In conclusion we do not think that the alleged presence of lines in Raman spectra of baterial cells can be claimed to be “...non questionable...” [lo] or that such “...revelations...” [lo] can be taken as a scientific proof of coherent phenomena occurring in living cells as suggested by Webb [3] , Frohlich [6] and Del Giudice et al. [7,8,10]. References
ill SJ. Webb and M.E. Stoneham, Int. J. Quantum Chem. Quantum Biol. Symp. 2 (1975) 339.
PI SJ. Webb and M.E. Stoneham, Phys. Lett. 60A (1977) 267. 131 SJ. Webb, Phys. Rep. 60 (1980) 201. [41 A.C. Scott, Phys. Lett. 86A (1981) 60. iSI P.S. Lomdahl, L. MacNeil, A.C. Scott, M.E. Stoneham and SJ. Webb, Phys. Lett. 92A (1982) 207. [61 H. Frijhlich, Adv. Electron. Electron Phys. 53 (1980) 85. 171 E. Del Giudice, S. Doglia and M. Milani, Phys. Lett. 85A (1981) 402. PI E. Del Giudice, S. Doglia, M. Milani and SJ. Webb, Phys. Lett. 91A (1982) 257. PI L. Furia and OP. Gandhi, Phys. Lett. 102A (1984) 380. VOI E. Del Giudice, S. Dog@, M. Milani, C.W. Smith and SJ. Webb, Phys. Lett. 107A (1985) 98, [Ill M.S. Cooper and NM. Amer, Phys. Lett. 98A (1983) 138. 1121 F. Drissler, COB. Phen. 3 (1981) 147. 1131 S. Kinoshita, K. Hirata and T. Ku&da, J. Phys. Sot. Japan 49 (1980) 314. [I41 F. Drissler and R.M. MacFarlane, Phys. Lett. 69A (1978) 65. 1151 F. Drissler, Phys. Lett. 77A (1980) 207.
377