Application of microspectral luminescent analysis to study the intracellular metabolism in single cells and cell systems

doi:10.4236/ns.2010.25054

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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: [email protected]

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-

N. A. Karnaukhova et al. / Natural Science 2 (2010) 444-449

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.

N. A. Karnaukhova et al. / Natural Science 2 (2010) 444-449

446

used for analytical purpose, the description of cell’s

color as a ratio of fluorescence intensities.

][

530

640

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.

][

530

640

DNA

RNA

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][

][

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 .

N. A. Karnaukhova et al. / Natural Science 2 (2010) 444-449

447

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

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

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

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

A



(7)

572

680

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

448

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.

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