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VO ~. 0 NO. 1 SCIENCE IN CHINA (Series C) February 1997

Spontaneous and light-induced photon emissionfrom intact brains of chick embryos

ZHANGJinzhu (ChangJiin-J u, E%%), YU Wendou (3- %4- ),S UN Tong ($ I\B(Insti tute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China)

and Fritz-Albert Popp(International Institute of Biophysics, Kaiserslautern 67661, Germany)

Received April 1, 1996

Abstract Photon emission (P E) and light-induced photon emission(LPE) of intact brains isolated from chickembryos have been measured by using the single photon counting device. Experimental results showed that the intensi-ty level of photon emission was detected to be higher from intact brain than from the medium in which the brain wasimmerged during measuring, and the emission intensity was related to the developmental stages, the healthy situationof the measured embryos, and the freshness of isolated brains as well. After white light i llumination, a short-life de-layed emission from intact brains was observed, and its relaxation behavior followed a hyperbolic rather thanan expo-nential law. According to the hypothesis of biophoton emission originating from a delocalized coherent electromagneticfield and Frohlich's idea of coherent long-range interactions in biological systems, discussions were made on the signifi-cance of photon emission in studying cell communication, biological regulation, living system's relevance to its environ-ment, and also on the relations between the detected photon emission and the coherent electromagnetic field. The de-tected photon emission should be comprehended in the manner of the interactions between the intrinsic fields within theliving systems and their environmental external fields.

Keywords: photon emission light-induced photon emission, chick embryos, intact brain, brain cells.

Biophoton emission is a common phenomenon in living systems. It concerns a weak electromagnetic radiation. The intensity of th e emission is from a few up to some hundred photons pecm2 per second, and the spectrum is whithin the optical range from 200 to 800 nm. PO^^ ] attributed it to a coherent field as the source of biophoton emission, which plays a role in intracellular and intercellular communication and biological regulation.

Recently the phenomenon of electromagnetic communication between cells was observed icultured HK cellsL2] nd in othe r living ~ ~ s t e m s [ ~ - ~ ]Some reports[3-51 have shown that photoemission technique is a good means to study the electromagnetic communication between cells. Iorder to explore the possibility of electromagnetic communication in the course of information processing in nerve system, the first step is to study biophoton emission from intact brains. However, up to now all researches on photon emission from brain were performed in brain cells or ibrain h ~ m o ~ e n a t e s ' ~ .1 , but not in intact brain. The phenomena concerned with white light-induced photon emission (LPE) from animal cells[9* o and the relationship of t he decay behavior othe L PE with the coherent field.in living systems[111have already been reported.


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44 SCIENCE IN CHINA SeriesC) Vol 4

Our previous studies showed that a specific pattern curve of emission from chick brain cellappeared in the embryos of more than 9 d, and intensities of the photon emission were related todevelopmental stage of the measuring embryoil2]. Here we report experimental results of photonemission and light-induced photon emission from intact brains. Our aim is to answer whetherbrains exist in a coherent field. The data will provide evidence for the possibility of cell communi-cation and regulation in brains based on the coherence theory.1 aterials and methods1.1 Preparation of intact brain and brain cell suspension

Fertilized white leghorn eggs used in this study, obtained from Kaiserslautern University,Germany, were incubated at 37 C for certain days in an incubator. Chick brains were separatedgently, washed in Hanks solution three times and finally immerged in DM EM medium for photon emission measurement. The whole preparation process was performed under aspect conditionand finished within 5-10 min. For comparison, in some experiments the measured brain was cuinto pieces by a scalpel and measured again; in some experiments the brain was fragmented andmixed with the medium in a test tube and then gently shaken to make cell suspension. Such celsuspension checked under a microscope, only contained a few cell aggregations. All processewere performed under aspect condition and finished within 10 min. The cell suspension was thenreplaced in the same cuvette and measured again under the same condition.1 . 2 Photon counting

Low-level spontaneous photon emissions and light-induced photon emissions were recorded bythe specific single photon counting system made in Popp s lab[13]. Th is device was equipped witha cooled EM1 9558 Q photomultiplier. The cathode had a diameter of 44 mm and was sensitivin the range from 200 to 800 nm. Intensities of photon emission were measured as the numbers ophotons integrated over a time interval by a computer. The temperature of the sample compartment could be controlled. In our experiments it was 37C

For LPE measurements a 150 W-tungsten halogen lamp(7158 Philips) was used. Th e lamwas situated at the right angle to the axis between the measuring sample and the photomultiplierThe illumination was carried out continuously for s (per 20 s ) or 10 s (pe r 200-1 000 s ) . During the illumination period the shutter between the sample measuring chamber and the photomultiplier was closed and opened alternatively automatically after turning off the lamp. Each samplwas illuminated for 4-10 times in our experiments.2 Results2 . 1 Photon emission from intact brains of chick embryos

Measurements of photon emission from embryonic chick brain immerged in DMEM medium


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No. 1 PHOTO N EMISSION FROM INTACT BRAINS O F CHICK EMBRYOS 45

Table shows the relationship of PE intensities with the developmental stages. The averageintensities shown in this table are statistical values of two samples. For each sample 000-2 000measurements were made, and for each measurement the photons were registered continuously fo5 s . The intact brain emits higher intensities of photons than DMEM medium. The average PEintensities increased with th e increasing developmental stages till the 1 4th day, but decreasedfrom later embryos.

Table 1 Relationship between P E intensities from em bryo s brains and their stagesSample n = 2 ) Average intensity /photon. sDMEM medium 2 9 . 9 k 1 . 6E8-E9 intact bra in 3 6 .0 k 2 .0E13-E l4 intact b ra in 5 6 .0 + 8 .3E16-E l8 int ac t b ra in 41 .2 k2 .2

PE intensity from unhealthy brain or brains treated at high temperature 42-43C) for ashort period or treated at low temperature was lower than those from healthy one at the same age

Figure shows the result from the intact brains of a 14-d healthy chick embryo and unhealthy embryo suffered from the storage in a refrigerator for certain days before incubation. Suchembryo was not active when being dissected. Similar results were also obtained from 16-d and 18d embryos.

Fig. 1 Comparison between PEs from the intact brain of healthy (A ) and unhealthy (B) embryos. Each brain was immerge in5 mL DMEM medium and measured at th e same temperature of 379 : . The figure shows the r sults from 14-d embryos.

Besides the healthy situation, photon emissions from the intact brain of chick embryos wer


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46 SCIENCE IN CHINA Series C) Vol. 40

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oo

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Fig. 2 . Relationship between the PE intensities and freshness of the brain. a ) The result from twobrains of 17-d embryo; b) the result of the same brain kept in DMEM medium for 16 h at room tem-perature photon emission was measured in chamber of the double measuring chamber system).

2 2 Comparison between photon emissions from intact brain and the brain cell suspensionBrain cell suspension was prepared from the measured intact brain without any treatments by

chemicals or trypsin, and then replaced into the same cuvette and measured again under the sameconditions. Th e results showed that besides the specific pattern of PE curve from suspended brain


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No. 1 PHOTON EMISSION FROM INTACT BRAINS O F CHICK EMBRYOS 47

neutrophil population undergoing respiratory burstLs1.When an intact brain was cut into 10-20fragments, PE intensity from the fragments was also higher than from their intact brain. However, for unhealthy embryos, the patterns of photon emission and PE intensities from the intacbrains could not be distinguished from those of brain cell suspension.

2 . 3 Light-induced photon emission from intact brain and brain cell suspension

1400

200

1000

b)I I Iloo0 2 3 4000 5000 6 7000 8 9 1r s

Fig. 3 . Comparison between photon emission from Intact brain a) and its cell suspension b ) . T hecurves show ch nges of emission intensities with the running time.


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48 SCIEN CE IN CHIN A (Series C). Vol. 40

4 shows LPE from the intact brain a ) of a 12-d embryo and its cell suspension b ) by illumination for s at a time interval of 20 s . Comparing curves a) and (b.) we can see that the LPE intensities were lower from brain than from its cell suspension see Embryo No. 1 in +,able 2 .

lo

Fi g.4 . Changes of th e LP E intensities from the intact brain ( a ) of a 12-d embryo and its cell suspen-sion ( b ) w ith th e running time. Each light illumination was carried out at the time interval of 20 s andlasted 1 s. T he photons were collected at a time interval of 100 rns

Table 2 Light-induced photon emission from intact brain and the brain cell suspension (El21Intact brain Brain cell suspension

( n = 1 0 ) ( n = 1 0 )


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No. 1 PHOTON EMISSION FROM INTACT BRAINS OF CHICK EMBRYOS 49

Figure 5 shows the delayed photon emission changing with the running time during the re-laxation after white light illumination. Curves 1 and 2 represent respectively the average results o10 times of illumination of ( a ) and of ( b ) shown in fig. 4 The circles represent the predicatedtheoretical hyperbolic values. The results suggested that the decay kinetics of photon emissionfrom intact brain and brain cell suspension did not obey exponential law.

Fig. 5 Changes of t he LPE intensitlea ..om bra~n 1 ) and from its cell suspension (2) with the run-ning time. The curves show the average result of 10 times of illuminations shown in fig. 4 Accordingto the hyperbolic law, the theoretical values are shown by the circle lines.

According to the following relation of hyperbolic decay law' ]

the average LPE results from the intact brains and their cell suspension of two 12-d embryos wercalculated and shown in table 2 . Each measurement was taken over 200 s and each illuminatioperiod lasted 1 s. Table 2 shows the statistic results of 10 times of illumination for each sampleAlthough the values of I. from the intact brains and brain cell suspension of the same embryo werdifferent due to some reasons, to, i. e. the period when th e intensity decayed to I ,) 1/2 1 0were almost the same (table 2 ) .


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50 SCIENCE IN CHINA (Series C Vol. 4

iscussionBy using the high sensitive single photon counting system, PE can be detected from the em-

bryonic chick intact brain immerging in the DMEM medium. The intensity of the emission is dependent on the developmental stages, the healthy situation of the embryos and the freshness ofbrain. LPE from intact brain and brain cell suspension is a kind of short-life delayed emission.The decay kinetics of LPE satisfies a hyperbolic law rather than an exponential law. The intensityand the life of the LPE are also dependent on embryonic healthy situation. There is no obvioulinear relationship between the LPE intensity and the illuminating time even under the conditionthat other factors remain stable.

Photon emission caused by lipid peroxidation of brain cell membranes was reported earlier['. I . It may contribute to explaining partially the higher PE intensities from brain cell suspension than from intact brain, because cell membrane can be easily oxidized in cell suspension. However, we cannot exclude the consideration based on the coherence theory. PE intensity from intacbrain was not simply the sum of each brain cell due to the coherent interaction of the cells. A verysimilar phenomenon was also found in normal cells from which the PE intensity decreased withthe increasing cell den~ it ie s ~] In our experiments, the relaxation behavior of LPE explains preliminarily that chick embryo brain and its cells are existing in a coherent electromagnetic field[ ].

Delocalized coherent field in biological systems exists in a steady state, i. e. it stabilizearound a threshold between a chaotic and an ordered state. ~ r o h l i s h l ~ ~ ~ndicated theoretically thabased on the dipolar properties of cell membrane and of certain bonds recurring (such as H bondsin biomacromolecules and possibly on existence of non-localized electrons, biological systems werexpected to have a branch of longitudinal electric modes in a frequency of 10~~-10 '~s- ' . If theris enough energy available, which is almost always the case in biological systems, the supplied energy is not completely thermalized but stored in highly ordered fashion. This order expresses itself in long-range phase correlations, i. e. coherence. Because the coherent field in biological systems remains in a threshold between coherent state and chaotic state, a steady state far away fromthermal equilibrium, it is very difficult to measure this field directly. For investigation of the internal coherent field in biological systems, it may be a practical method to influence the internafield with some external fields, for instance with electromagnetic fields. Biophoton emission anaysis is a powerful measuring method since it can be performed at different levels of living cells, tissues, and even at intact whole body level. Biophoton analysis provides the information concerninsub-molecular and microscopical processes and states in living systems by macroscopical measurement, therefore it is known as the optical window . Photon emission from living systems wereflects their living states and functions. For example, in our experiments, comparing with thmeasurements of photon emission as indicated above, we found that there were more cells in MG phase in 14-d embryos than in other stages of embryos by cell sorting analysis for brain cesuspension in different developmental stages of chick embryos.


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No. 1 PHOT ON EMISSION FROM INTAC T BRAINS O F CHICK EMBRYOS 5 1

the functions of the livings but also the influences of environments on the livings. However thereare still more interesting problems yet to be further studied in particular the relation of PE withthe environmental factors and electric stimulation the relationship between PE and the actions othe brain in situ including objective consciousness and the response to the external stimulationsand the nonlinear behavior of these relations.

eferences

Popp, F . A . Som e essential questions of biopho ton research and probable answers, in Recent Advances in Biophoton Research and Its Applications ( eds . P opp , F . A . , L i , K . H . , G u , Q. , Singapore-London: World Scientific Publishing CoL t d . , 1992, 1-46.Albrecht-Buehler, G . Rudim entary form of cellular vision , Proc N a t l . Ac a d . S c i U S A , 1 9 9 2 , 8 9 :8 2 8 8 .Popp, F. A., Chang, J J . , Gu, Q. e t a l . , Non-sub stantial biocom mun ication in term s of Dicke' s theory, in Bioelectrodynam ics and Biocommunication (eds . Ho, M. W . , Popp, F . A . , Warnke, U. , Singapore-London: World Scientific Publishing Co t d . , 1 9 9 2 , 2 9 3 - 3 1 8 .Zhang,J . Z . Popp, F. A. Yu, W . D . , Research on cell communication of P . elegans by means of photon emission, ChinesScience Bul le t in , 199 5 .40 : 7 6 .Shen, X Mei, W . P . Xu, X. Activation of neutrophils by a chemically separated optically coupled neutropil population undergoing respiratory b urst, Erper ient ia 1994 .50 63 .Galantsev, V. P . Kovaleako, S . G. Moltchanov, A. A. et a1 Lipid peroxidation, low-level chemiluminescence and regulation of secretion in th e mamm ary gland, Exper ient ia , 1993 , 49:8 7 0 .Reiber. H . , Martens, U . , Prall , F. et a l . , Relevan ce of en dogeno us ascorbate and tocophero l for brain cell vitality indicateby photon emission, J N euro chem 1 9 9 4 , 6 2 2 ) 0 8 .Cadenas, E . , Varsavsky, A. I. , Boveris, A. et a1 Oxyg en- or organic hydroperox ide-induced chem iluminesc ence of braiand liver homogenates, Biochem 1981 ,198 45 .Scholt, W . , Stasjkiewiez, U. Popp, F. A . et a1 Light-stimu lated u ltraweak photon reem ission of hu man amn ion cells anwish cells, Cell Biophys . 1 9 8 8 , 1 3 5 .Wijk, R. Aken, H. Mei, W. P . et al Light-induced photon emission by mammalian cells, 1 hotochem Photobiol BB i o l . , 1 9 9 3 , 1 8 : 7 5 .Popp, F . Li, K . H . Hyperbolic relaxation as a sufficient condition of a fully coherent ergodic field, Int J The or. Physic a . . 1 9 9 3 , 3 2 : 1 5 7 3 .Yu, W. D . , Zhang, J . Z . Deve lopme ntal of em bryonic chick brain cells by mea ns of ph oton em ission, Chinese Science Bu ll e t i n , 1 9 9 5 , 4 0 :1 9 9 2 .Mieg, C. Mei, W . P. Popp, F . A . Technical notes to biophoton emission, in Recent Advances in Biophoton Research anIts Applications (eds. Popp . A. Li, K . H . G u , Q. , Singapore-London: World Scientific Publishing Co Ltd. 1992197-205.Popp, F . A . , Gu, Q. Li, K . H . , Biophoton emission: experimental background and theoretical approaches, Modern PhysicLetters B, 1 9 9 4 . 8 : 1 2 6 9 .Frohlich, H . , Long-range coherence and energy storage in biological systems, Int Quentum Chem 19 68 ,2 : 64 1 .


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