Vol.2, No.5, 444-449 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.25054
Application of microspectral luminescent analysis to
study the intracellular metabolism in single cells and
cell systems
Natalia A. Karnaukhova*, Larisa A. Sergievich, Valery N. Karnaukhov
Institute of Cell Biophysics, Russian Academy of Science, Pushchino, Russian Federation; *Corresponding Author:
Received 4 December 2009; revised 27 January 2010; accepted 15 March 2010.
ABSTRACT
Spectral luminescent analysis of single cells
and cellular systems enable us to reveal the
initial changes of intracellular metabolism that
can followed by human diseases or failure in
biocenosis. Two cytodiagnostic systems of de-
vices and techniques have been developed: 1)
Microspectrofluorimeters registering the fluo-
rescent spectra of individual cells or intracellu-
lar organelles used for fundamental investiga-
tions of cell reactions and for discovering and
studying new dimensionless fluorescent char-
acteristic parameters reflecting the biochemical
or physiological properties of the cells; 2) Dou-
ble- and multi-wave microfluorimeters for rapid
registration of fluorescent characteristic para-
meters for many cells to obtain statistical in-
formation about cell population. These techni-
ques are useful especially in medical and eco-
logical investigations.
Keywords: Intracellular Metabolism; Spectral
Luminescent Analysis; Microspectrofluorimeters;
Double and Multi-Wave Microfluorimeters;
Cytodiagnostics; Low-Frequency Variable Magnetic
Fields; Solar Activity
1. INTRODUCTION
All cells have a general physical-chemical basis of func-
tioning in spite of different morphological structures and
functions (synthesis of nucleic acids and proteins, ener-
getic, etc.). Therefore, the success in the solution of this
problem is essentially dependent on finding specific
features which could form the basis of the algorithm for
recognition of cells by their chemical composition. Be-
sides, it is necessary to provide possibility for the mor-
phological analysis of “suspicious” cells. The character-
istic features common for cells of different kinds were
defined on the base of fundamental studies of intracellu-
lar regulation of metabolism with microspectral lumi-
nescent analysis. It formed a new trend in automation of
cytodiagnostics. To study plant, animal and microorgan-
ism cells both self-luminescence of some intracellular
compounds (NADH, flavoproteins, for example) and the
secondary luminescence induced by interaction of fluo-
rochromes with the cells are used. The advance in fluo-
rescent cytodiagnostics is only possible with best meth-
ods and tools available for microspectral analysis which
present a combination of a fluorescent microscope with a
spectroanalysing instrument supplied with electronic
registration and control units. Such a system satisfying
all needs of cytodiagnostics would consist of the two
types of instruments. The interconnection between them
is determined by their functional peculiarities which will
be considered below with some characteristic parameters
using in medicine and ecology [1-3].
2. DEVICES AND TECHNIQUES FOR
LUMINESCENT CYTODIAGNOSTICS
2.1. Microspectrofluorimeter-
Microspectrophotometer
This instrument is used to study luminescent spectral
characteristics of cell. The general diagram of micro-
spectrofluorimeters is given in Figure 1. This scheme
can be varied depending on concrete task. Different dis-
persing elements are used as monochromator: prisms,
diffraction gratings and interfering light filters of vary-
ing wave length [1]. Microspectrofluorimeter MSF-1
enables registration of luminescence spectra of cells and
intracellular compartments up to 0.5 µm in diameter in
the range from 400 to 800 nm. The luminescence spectra
of individual cells are insufficient to follow any process,
and information on the behavior of the whole cell popu-
lation under study is required. It is convenient for this
purpose to describe the luminescence spectrum of each
cell with one parameter only. Thus, microspectrofluo-

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445
rimeters registering the fluorescent spectra of individual
cells or intracellular organelles are used for fundamental
investigations of cell reactions and for discovering and
studying of the new dimensionless fluorescent charac-
teristic parameters, reflecting the biological or physio-
logical properties of the cells.
2.2. Microfluorimeter DMF-2
Using microspectrofluorimeter the luminescence spectra
of various types of cells have been studied and the char-
acteristic parameters of the cells have been defined,
there is no need to register the total spectrum for each
cell. It is enough to measure the luminescence intensity
of the cells in two characteristic spectral regions. Micro-
fluorimeter DMF-2 (Radical) interfaced to a PC/AT
compatible computer is used to measure fluorescence
intensities at two separate wavelengths (Figure 2). It is
based on a fluorescent microscope with a double-cha-
nnel fluorescent sensor assembly. The first channel is
tuned to record one fluorescence intensity and the sec-
ond another fluorescence intensity of the same object.
This allows measurement of the fluorescent characteris-
tics of definite single cells or their compartments, the
structure of which is controlled by microscopy. The
fluorescence of the cells is excited by the emission of a
DRSh-250-2 mercury arc lamp with chosen wavelength.
The size of the photometered area corresponds to the cell
size. A special program “Microfluor” made it possible to
obtain the distribution histograms of the fluorescence
intensities in the different regions of the spectrum as
well as the distribution histograms of the characteristic
parameters for 200 cells within 15-20 min with the
points plotted onto the phase plane, and to perform sta-
tistical analysis of the data [4,5]. These techniques are
useful especially in medical and ecological investiga-
tions.
3. APPLICATIONS OF DEVICES AND
METHODS FOR LUMINESCENT
CYTODIAGNOSTICS
3.1. Synthetic Activity of Cells
To investigate synthetic activity of cells the characteris-
tic parameter α have been offered. Fluorochrome ac-
ridine orange (AO) is widely used to investigate the nu-
cleic acids in living as well as fixed cells. As an example
of information obtained with the help of MSF-1 it can
consider fluorescence spectra of AO stained blood lym-
phocytes of rabbit at different stages of their immune
response to exogenous protein ovalbumin [6]. The spec-
tra consist of two emission bands. AO interacts with
DNA and RNA by intercalation or electrostatic attraction
respectively. DNA (double-helical nucleic acids) inter-
calated AO fluoresces green (530 nm), RNA (single-
helical nucleic acids) electrostatically bound AO fluo-
resces red (640 nm).
Using the fluorescent microscope, investigator visu-
ally observes the cells with colors from green through
yellow, orange to red in depend of ratio in emission
bands only in two spectral diapasons-in green (I530) and
red (I640). And if the color in cell fluorescence can’t be
Figure 1. General scheme of MSF-1. 1-luminescent
microscope; 2-probe nozzle; 3-system of monochro-
mator: a-objective, b-mirror, c-diffraction grating; 4-
mercury arc lamp (DRSh-250-2); 5-power supply unit;
6-photomultiplier; 7-high voltage power unit; 8-am-
plifier; 9-register (X,Y-recorder).
Figure 2. General scheme of two-channel (double-
wave) microfluorimeter “Radical DMF-2”. 1-lumine-
scent microscope LUMAM; 2-probe nozzle; 3-di-
chroic mirror-analyzer; 4-two-channel (double- wave)
registration system;5-mercury arc lamp (DRSh-250-
2); 6-power supply unit for mercury arc lamp; 7-
power supply unit for photomultipliers; 8-ADC; 9-
computer.

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446
used for analytical purpose, the description of cell’s
color as a ratio of fluorescence intensities.
][
][
2
1
1
530
640
NA
NA
A
I
I
 (1)
where А1 is the proportionality coefficient, [NA1] and
[NA2] are the concentrations of single-helical (NA1) and
double-helical (NA2) nucleic acids, permits to analyze
quantitatively the processes in the cells.
Taking into account that in differentiated nonproli-
firating cells (blood lymphocytes) the main quantity of
single-helical nucleic acids is RNA, double-helical nu-
cleic acids is DNA, expression (1) should be refined.
][
][
1
530
640
DNA
RNA
A
I
I
 (2)
It has been shown that under definite conditions of
cell staining with AO parameter
 reflects the amount of
RNA per unit DNA and, hence, characterizes cell syn-
thetic activity [1-3,6]. Series of α-distribution histograms
is indicative of changes in synthetic activity of all popu-
lation of immunocompetent cells at different stages of
processes in immune system (Figure 3).
The above example demonstrates only one way of de-
termining the parameter characterizing the ratio between
the nucleic acids in the cell and the synthetic potentiality
of the cell. Being, presumably, of greatest interest to
medicine and biology [6-10], it is not the only possible
method. Using the microspectral analysis of cells and a
large set of luminescent dyes-labels, other useful char-
acteristic parameters can be found. We further elaborate
on our method for monitoring the synthetic activity of
lymphocytes, testing the two-dye, three-color assay with
regard to the temporal organization of the immune re-
sponse. Within the same methodological framework, a
good probe for protein is 1-anilino-8-naphthalene sul-
fonate (ANS); we have already shown that consecutive
staining of fixed cells with AO and ANS adds a third
fluorescence peak in the blue region (470 nm) character-
istic of protein-bound ANS [11].
470530
470
4.0][
][
II
I
DNA
protein
A 
 (3)
The investigations conducted on blood lymphocytes
showed that parameter α is sensitive to the action of the
stimulatory and damaging effects of environmental fac-
tors, including electromagnetic fields and solar activity
[12,13].
Under the action of low-frequency variable magnetic
fields with the specified parameters, the synthetic activ-
ity (parameter α) increased by 22 to 35% (Figure 4).
These results and the data obtained by another methods
showed that the variable magnetic fields enhances the
synthetic activity of lymphocytes, improves the type of
adaptive response, and thereby increases the level of the
immune resistance of the organism.
Figure 3. Distributions histograms of the parameter 
for rabbit blood lymphocytes in process of immune re-
sponse in organism onto albumin introduction: (а) be-
fore immunization; (b) on stage of the maximum activ-
ity; and (c) on the later stage. Along the ordinate is the
number of cells. Along the abscissa are the  parameter
values.
Figure 4. Diagrams of the changes in the average values of the
parameters α for lymphocytes in the blood of rats as a function
of frequency. * p < 0.05, ** p < 0.01 .

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Correlation was revealed between solar activity pa-
rameters (sunspot number and the 10.7 cm solar radio
flux) and the synthetic activity of blood lymphocytes in
different species of animals during the same periods
(January-April) of 1993, 1994, 2000, and 2002 (Table
1).
In the others seasons (April-June, August-December),
the negative correlation between the studied indices was
weaker and was not reliable. The data suggest seasonal
regularities in the connection between the studied proc-
esses.
Sign reversal of the correlation coefficient was ob-
served in the second maximum of solar cycle 23. The
change of sign may depend on a change in the ratio be-
tween phases of the oscillatory processes studied. How-
ever, the mechanism of the phenomenon observed re-
mains unknown.
It was also shown that the correlation decreased under
stronger internal (disease) or external (chronic gamma-
irradiation) influences (Table 2).
3.2. Energetics of Animal Cells
Also the self-luminescence of living cells can be used
for cytodiagnostics. For instance, it was shown [1,14,15]
that the state of the mitochondrial energetic apparatus of
a cell can be quantitatively characterized by the ratio of
the intensities of luminescence of oxidized flavoproteins
(530 nm) and reduced pyridinenucleotides (470 nm):
470
470530 5.0
I
II 
  (4)
In this case the application of the microspectral analy-
sis enabled one to find that in one cell (neurons of
stretch-receptor) there are two mitochondrial pools re-
sponsible for the energy supply of different functional
mechanisms of the cell-membrane transport and syn-
thetic processes (Figure 5). It was shown that these two
mitochondrial pools are controlled by different mecha-
nisms. Therefore, in one and the same cell, at one and
the same instant the mitochondria belonging to different
Table 1. Correlation coefficients between the 10.7 cm solar
radio flux and the synthetic activity of blood lymphocytes in
different years.
1993 1994 2000 2002
-0.81 ** ± 0.08 -0.63 * ± 0.17 -0.63 * ± 0.15 0.71 * ± 0.09
* p < 0.05, ** p < 0.01
Table 2. Correlation coefficients between the 10.7 cm solar
radio flux and the synthetic activity of blood lymphocytes in
health, in pathology, and after gamma-irradiation.
Control
(healthy state)
Pathology
(cholelithiasis)
Gamma - irradiation (14.4
cGy/day to 15 Gy)
-0.63 * ± 0.17 -0.24 ± 0.26 -0.45 ± 0.22
* p < 0.05
pools may be in different states of activity. It is only the
method of spectral analysis that enables one to obtain
such information about the state and organization of in-
tracellular organelles in a functioning cell [1].
Taking into consideration the changes in the energetic
apparatus of cells upon malignization found by Warburg
it can be supposed that the characteristic parameter can
also be used for automatic detection of cancerous ma-
lignant cells in preparation [16].
470
530
1I
I
B
A
 (5)
3.3. Ecology and Environment Protection.
In solving the problems of environment protection, the
relationship between autotrophic and heterotrophic en-
ergy provision reflects the wellbeing of an autotrophic
organism [2]. The energetic state of high plant cells can
be determined from the ratio luminescence band intensi-
ties of chlorophyll (680 nm) and oxidized flavoproteins
(530 nm).
530
680
I
I
B
A
X (6)
This makes it possible to detect regions with polluted
atmosphere, to assess the state of the forests as well as to
predict the harvest of cultivated plants [2,17]. For the
same purpose, the following parameters can also be
used.
645
680
I
I
B
A
 (7)
572
680
I
I
B
A
 (8)
Those characterize the state of lichens symbiotic
Figure 5. Luminescence spectra of reduced pyridi-
nenucleotides (NADH, 470 nm) and oxidized flavo-
proteins (FPo, 530 nm) in neuron of stretch-receptor.
Ordinate-fluorescence intensity in rel. units. Ab-
scissa-the wave length in nm.

N. A. Karnaukhova et al. / Natural Science 2 (2010) 444-449
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blue-green algae which are extremely sensitive to at-
mosphere pollution in places of their habitat. In the both
cases, the ratio of luminescence band intensity of chlo-
rophyll (680 nm) to that of phycocyanine (645 nm) or
phycoerythrin (572 nm) is used [2]. It is clearly seen that
the autotrophic cells having intense chlorophyll band in
the luminescent spectrum (Figure 6, curve 1) later start
to switch from autotrophic (algal) to heterotrophic (bac-
terial) energy provision (Figure 6, curves 2, 3).
It is important that in the tropical Atlantic the majori-
ties of the Cyanophyceae contain no chlorophyll and are
actually heterotrophic cyanobacteria. Their typical fluo-
rescence spectra (Figure 7, curve 1) have only phyco-
erythrin (572 nm) and phycocyanine (645 nm) bands,
though there are occasional cells that have not com-
pletely lost chlorophyll (Figure 7, curves 2, 3).
Figure 6. Luminescence spectra of single cells of
blue-green microorganisms in different states: 1-
autotrophic (algal); 2, 3-mixed (autotrophic and
heterotrophic) energy provision.
Figure 7. Luminescence spectra of blue-green mi-
croorganisms in the tropical Atlantic: 1-a most typi-
cal spectrum, 2, 3-deviant spectra.
However, most interesting seems the application of
the parameters φ and ψ in the cases when it is necessary
to predict the onset of “blooming” of blue-green algae in
water storage basins or lakes [18,19] for determining the
optimal instant of treating algae population with bacte-
riophages (cyanophages) in order to prevent “blooming”
which causes disastrous effect. Of particular importance
may be the application of the parameters φ and ψ for
predicting the initiation of the disease “ciguatera”. It is
the poisoning of people with usually harmless fishes and
mollusks living in regions of intense “blooming” of
blue-green algae in tropic zones of the oceans [19].
4. CONCLUSIONS
These few examples indicate that the methods of lumi-
nescent cytodiagnostics have promising applications in
different fields of biology, medicine, environment pro-
tection and biotechnology. The realization of these per-
spectives necessitates the development of special equip-
ment. Two cytodiagnostic systems of devices and tech-
niques have been used for the purpose:
1) Microspectrofluorimeters-microspectrophotometers
registering the fluorescent spectra of individual cells or
intracellular organelles are used for fundamental inves-
tigations of cell reactions and for discovering and study-
ing of the new dimensionless fluorescent characteristic
parameters, reflecting the biochemical or physiological
properties of the cells.
2) Double-and multi-wave microfluorimeters are used
for the rapid registration of many cells to obtain statisti-
cal information about cell population. These techniques
are useful especially for medical and ecological investi-
gations.
Thus, this study confirms that parameters derived
from luminescent spectral analysis of fluorochromed
cells (such as α and β) or self-luminescence of living
cells (such as ξ, χ, φ, ψ) are valid as dynamic indices of
intracellular metabolic activity in single cells and cell
systems. There are broad possibilities of further devel-
oping this approach and equipment into developing in-
creasingly differentiated fluorescence techniques.
REFERENCES
[1] Karnaukhov, V.N. (1978) Luminescent spectral analysis
of cell. Nauka, Moscow.
[2] Karnaukhov, V.N. (2001) Spectral analysis in cell-level
monitoring of environmental state. Nauka, Moscow.
[3] Karnaukhov, V.N. (1978) Luminescent analysis of cell.
Nauka, Moscow. http://www.edu.ru/db/ portal/e-library/
00000048/00000048.htm
[4] Karnaukhov, V.N., Yashin, V.A., Karnaukhova, N.A.,
Kazantsev, A.P. and Karnaukhov, A.V. (1999) Dou-
ble-wave microfluorimeter “Radical DMF-2”. Book of
Abstracts II Congress of Biophysicists of Russia, 2,

N. A. Karnaukhova et al. / Natural Science 2 (2010) 444-449
449
449
594-595.
[5] Karnaukhova, N.A., Sergievich, L.A., Kuzhevskij, B.M.
Sigaeva, E.A., Nechaev, O.Y., Karnaukhov, V.A. and
Karnaukhov, V.N. (2007) A study of the correlation be-
tween the functional activity of blood lymphocytes in
different animals and intensity of neutrons near the earth
surface. Biophysics, 52(4), 699-704.
[6] Karnaukhova, N.A. (1984) Luminescence parameters of
blood nuclear cells in process of immune reaction in or-
ganism. Biophysics, 29(2), 276-279.
[7] Karnaukhova, N.A. (1991) Changes in fluorescent spec-
tra of acridine orange stained blood cells from patient
suffering from lymphosarcoma and leukemias in the
course of chemotherapy. Experimental Oncology, 13(1),
50-53.
[8] Gordon, R.Y., Bocharova, L.S., Kruman, I.I., Popov, V.I.,
Kazantsev, A.P., Khutzian, S.S. and Karnaukhov, V.N.
(1997) Acridine orange as an indicator of ribosome state
in cell. Cytometry, 29(3), 215-221.
[9] Karnaukhova, N.A., Sergiyevich, L.A., Aksenova, G.E.
and Karnaukhov, V.N. (1999) Synthetic activity of rat
blood lymphocytes under acute and continuous gamma
irradiation-fluorescent microspectral study. Radiation
and Environmental Biophysics, 38(1), 49-56.
[10] Karnaukhova, N.A., Lubet, P.Е., Katania, R., Karnauk-
hov, А.V., Sergievich, L.A. and Karnaukhov, V.N. (2003)
Microspectral studies on neuroendocrine regulation of
gametogenesis in mollusk. Biophysics, 48(5), 869-873.
[11] Karnaukhova, N.A., Sergiyevich, L.A. and Karnaukhov,
V.N. (2008) Dinamics of ribosomal activity and protein
production in peripheral blood lymphocytes during an
immune response. Biophysics, 53(4), 632-637.
[12] Karnaukhova, N.A., Sergievich, L.A., Kvakina, E.B.,
Barsukova, L.P., Mar’yanovskaya, G.Y. and Kuz’-menko,
T.S. (2000) Study into the changes in the functional state
of the synthesis apparatus of blood lymphocytes under
the action of weak low-frequency magnetic fields. Bio-
physics, 45(4), 697-703.
[13] Karnaukhova, N.A., Sergievich, L.A., Karnaukhov, V.A.
and Karnaukhov, V.N. (2004) Changes in the synthetic
activity of lymphocytes under the action of physical fac-
tors related to Solar activity variations. Biophysics,
49(suppl.1), 552-559.
[14] Karnaukhov, V.N., Lebedev, O.E. and Pavlenko, V.K.
(1976) About two mitochondrial pools in a stretch-re-
ceptor neuron. Tsitologia, 18(10), 1189-1193.
[15] Rudenko, Y.N., Bigdai, E.V. and Samoilov, V.O. (2007)
Kinetics of Са2+, NADH and oxidized flavoproteids in
the frog olfactory living under the effect of odorants.
Biophysics, 52(1), 88-94.
[16] Thorell, B. (1981) Flow cytometric analysis of cellular
endogenous fluorescence. Cytometry, 2(1), 39-43.
[17] Roshchina, V.V. (2003) Autofluorescence of plant se-
creting cells as a biosensor and bioindicator reaction.
Journal of Fluorescence, 13(5), 403-420.
[18] Karnaukhov, V.N., Martsenuk P.P. and Yashin, V.A.
(1980) Luminescence spectral characteristics of physio-
logical state of cells of blue-green algae. Fisiologia Rast,
27(1), 11-17.
[19] Karnaukhov, V.N. and Yashin, V.A. (2003) Spectral stud-
ies on single cells of sea microplankton: History and
prospects. Biophysics, 48(5), 940-949.