IMAGE ANALYSIS COMBINED FLUORESCENCE

MICROSCOPY

Examples of ImageJ Software Application in Yeast Studies

Evgeny O. Puchkov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Institutskaya Str.,5, Pushchino, Russia

Keywords: Computer image analysis, Fluorescence microscopy, Yeast culture viability, Brownian motion, Intracellular

viscosity, Nucleic acid intercalator, Anticancer drug.

Abstract: For Saccharomyces cerevisiae yeast studies, three approaches have been developed. They are based on the

image analysis (ImageJ software) application for the fluorescence microscopy data treatment. The first is a

computer-aided fluorescence microscopy procedure for quantifying of the damaged cells in the ethanol-

producing yeast culture. It was shown to be applicable for the assessment of the culture viability. The

second is a means of characterizing Brownian motion of the insoluble polyphosphate complexes in the

vacuoles. Using this approach, the apparent viscosity in the vacuoles was measured. The third is a method

for locating intracellular sites/targets of the nucleic acid intercalators. This method may be of help in

designing of new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.

1 INTRODUCTION

The yeast Saccharomyces cerevisiae is an object of a

large research interest for at least two reasons. First,

it is widely used in food industry, including baking

and production of alcoholic beverages, ethanol and

food additives. Second, it serves as a model system

for studies of eukaryotic cells since many of the

basic cellular properties between yeast and humans

are highly conserved. This is also due to the

availability of the DNA sequence of the complete

genome and biochemical data, convenience for

molecular manipulations and the ease of handling.

The yeast cells are shown to be a good model in

many areas of cancer research and for new drug

discovery. Hence, the development of new

techniques and approaches for the studies of the

yeast S. cerevisiae is an important task.

Fluorescence microscopy is a very informative

method for single cell studies. Potentialities of the

method can be significantly improved by combining

it with the digital photography and computer image

analysis.

In this paper, three approaches based on the

ImageJ software (National Institute of Health, USA,

http://rsb.info.nih.gov/ij) application for the

fluorescence microscopy data treatment are

described. The first approach was developed for the

assessment of the yeast culture viability; the second

for to evaluate the apparent viscosity in the vacuoles

of the individual yeast cells; and the third for

locating intracellular sites/targets of the nucleic acid

intercalators.

2 ASSESSMENT OF THE YEAST

CULTURE VIABILITY

This study was undertaken to develop rapid

computer-aided fluorescence microscopy procedure

for the assessment of the ethanol-producing yeast

culture viability.

The fluorescence microscopy of rehydrated S.

cerevisiae cells from a dry commercial Fermiol

preparation (DSM Food Specialties Beverage

Ingredients, The Netherlands) stained by a

combination of ethidium bromide (E) and 4,6-

diamidino-2-phenylindole dilactate (DAPI) revealed

two types of cells. The cells of the first type showed

blue-green fluorescence (DAPI), whereas the cells of

the second type showed bright orange-red

fluorescence (E). In special experiments, it was

shown that the first type of fluorescence appearance

was a characteristic of the intact cells and the second

type was a characteristic of the damaged cells.

258

O. Puchkov E..

IMAGE ANALYSIS COMBINED FLUORESCENCE MICROSCOPY - Examples of ImageJ Software Application in Yeast Studies.

DOI: 10.5220/0003120902580261

In Proceedings of the International Conference on Bioinformatics Models, Methods and Algorithms (BIOINFORMATICS-2011), pages 258-261

ISBN: 978-989-8425-36-2

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

Using ImageJ “RGB Split” option, the initial

fluorescent images of the stained cells were

separated into three images containing red, green

and blue components of the fluorescence. This

procedure enabled to establish that, in the ethidium

stainable cells, along with the red component, there

was well expressed green component of the DAPI

fluorescence, too. This was an indication of the fact

that DAPI stained all the cells. So, RGB split of the

fluorescent image provided a means for assessing a

fraction of the E stainable/damaged or E

unstainable/undamaged cells. To this end, after an

appropriate threshold adjustment, “Analyse

Particles” option could be used for automated

counting of the fluorescing cells (“particles”) in the

“red” and in the “green” parts of the RGB-splitted

image.

A comparison of the viability rates of cultures,

determined by the plate count method, and the

relative numbers of intact cells, determined in the

same cultures by the developed procedure, showed a

good correlation between these parameters, thereby

indicating a possibility of using this procedure for

the assessment of the viability of rehydrated Fermiol

preparations. It was noted that fluorescence

microscopy underestimates the viability of yeast

populations by 10–15%, compared to cultural

methods. This disagreement can probably be

explained by the fact that some damaged cells may

recover during the subsequent cultivation of

rehydrated cells.

The main advantage of the proposed approach is

that it allows more rapid assessment of cell viability

as compared to not only the cultural methods, but to

the “manual” microscopy as well. Also, this

approach opens a way to the automation of the

analysis. For more details see (Puchkov, 2006).

3 MEASUREMENT

OF VISCOSITY IN VACUOLES

In the S. cerevisiae cells, at some cultivation

conditions, appear vividly moving particles, <1 µm

in size, known as ‘dancing bodies’. They were

shown to be insoluble polyphosphate complexes

(IPCs) localized in the vacuoles. Upon staining of

the cells by DAPI, IPCs acquire a bright yellow

fluorescent colour, while the nuclei and

mitochondria fluoresce blue.

The aim of this study was to quantitatively

characterize, by fluorescence microscopy combined

with computer image analysis, the movement of

IPCs in the vacuoles of S. cerevisiae VKM Y-2549

cells and to evaluate the apparent viscosity in the

vacuoles, using the obtained data.

The immobilized cells were photographed in a

Speed Burst regime of the Sony DSC-V3 digital

camera. It gave a series of eight frames at intervals

of 0.43 s and an interval between series of 2–3 s.

Using ImageJ, in a frame of a series, a fluorescing

particle was selected as a region of interest (ROI) by

“Oval Selection” option. “ROI Manager” option was

switched on to get the same ROI area in other

frames, although position of the ROI was changed

according to the position of the particle in a new

frame. The locations of the IPCs in the two

dimensional space (X and Y locations) were

evaluated using “Center of Mass” option.

The results of this analysis indicated that IPC

movements were chaotic or, as it is often referred to,

were random walks, or Brownian motion.

The Brownian motion of particles obeys the

Einstein–Smoluchowski equation, which for two-

dimensional (2D) movement is as follows:

> = 4κTt/3πηD, (1)

where <s

> is the average of the square of

displacement, κ is the Boltzmann constant, T is the

thermodynamic temperature, t is the elapsed time, η

is the viscosity and D is the diameter of the particle.

To evaluate the apparent viscosity in the yeast

vacuoles via Brownian motion of the IPCs, the

average displacement in 2D space and the diameters

of the moving particles need to be estimated

[equation (1)]. As IPCs move in three dimensions, a

criterion for selecting the 2D displacements in the

photorecords, which are in the focusing plane or at

least close to it, must be found. To learn how this

could be done, experiments were performed on

suspensions of fluoresceinisothyocyanate-labeled

latex microspheres of 2.1 μm and 3.1μm diameter in

water. In this model, two parameters were known,

the diameters of the microspheres and the viscosity

of distilled water in which they were moving.

Using photorecords similar to those of IPC, two

parameters of Brownian motion of fluorescing

microspheres were estimated for each series of the

eight speed regime shots – the consecutive two-

dimensional locations and the mean fluorescence

intensities (“Mean Gray Value” option) at these

locations. The normalized fluorescence intensity

served as a quantitative measure of the microsphere

shift from the two-dimensional motion in each eight

frames series of the speed regime shots. It was tested

whether the fluorescence decrease of no more than

15% may be used as a criterion for taking

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displacements of the microspheres close enough to

the two-dimensional space for viscosity assessment

by formula (1). Upon selecting “appropriate”

displacements by this criterion from photorecords of

the series of speed regime shots, computation of the

apparent water viscosity using formula (1) has been

made. The viscosity values of water estimated by

these measurements agreed with 30% accuracy with

the value (0,89 сР at 25 °C, the temperature of

measurements) obtained by other methods. This

result indicated that the developed approach may be

used for viscosity assessment.

It was also found by analysis of the fluorescence

intensity profile (“Line selection” across “Center of

Mass” and “Plot Profile” options) of the

microspheres that the level of the 85% fluorescence

intensity corresponds to their outer borders. That

gave the means of estimating the size of the

fluorescing IPC particles by their fluorescence

intensity profiles.

Using methodology developed on the latex

microspheres, Brownian motions of the IPCs in four

cells were analyzed. Displacements for each IPC in

the two-dimensional space in the series of the eight

speed regime shots were estimated. Outer borders of

IPC were determined by estimating 85% level of

fluorescence intensity in the IPC fluorescence

profile. That gave the dimensions of IPC. Since the

shape of IPC is not known, this parameter was

assumed to be corresponding to the diameter of the

microspheres.

In four yeast cells, the 2D displacements and

sizes of the IPCs were evaluated. Apparent viscosity

values in the vacuoles of the cells computed by the

Einstein-Smoluchowski equation using the obtained

data, were found to be of 2.16 ± 0.60; 2.52 ± 0.63;

3.32 ± 0.9; 11.3 ± 1.7 cP. The first three viscosity

values correspond to 30 – 40% glycerol solutions.

The viscosity value of 11.3 ± 1.7 сР was supposed to

be an overestimation caused by the peculiarities of

the vacuole structure and/or volume in this particular

cell. This conclusion was supported by the particular

quality of the Brownian motion trajectories set in

this cell as compared to the other three cells. For

more details see (Puchkov, 2010).

4 INTRACELLULAR LOCATION

OF INTERCALATORS

The aim of this study was to test if intact (not fixed)

yeast cells of S. cerevisiae can be used as a model

for locating intracellular sites/targets of the nucleic

acid intercalators (NAI). To this end, intracellular

distributions of three fluorescing NAI – the

anthracycline anticancer drug doxorubicin (DR)

(trade name adriamycin; also known as

hydroxydaunorubicin) along with nucleic acid dyes

ethidium (E) and 4',6-diamidino-2-phenylindole

(DAPI), were investigated using fluorescence

microscopy combined with computer image

analysis.

Upon incubating for at least 20 h, all the cells of

S. cerevisiae VKM Y-2549 appeared to be stainable

by the NAI. In the cells stained by DAPI, there were

clearly visible fluorescing blue spots of the nuclei

and small dots of the mitochondria. As compared to

DAPI, intracellular fluorescence distribution of DR

and E did not have the clear “spot and dot”

appearance. Although nuclear region could be

distinguished, even distribution of the DR and E

fluorescence was observed in the regions where

mitochondria should be located. There was

heterogeneity in the expression of staining by DR

and E. The observed difference in this feature of the

individual cells may be a reflection of a specific

combination of the cytoplasmic membrane diffusion

barrier properties and of the presence/activity of

drug export permeases.

Combined application of DR or E with DAPI

visualized the location of the nuclear and of the

mitochondrial DNA. However, visual analysis could

not answer the question whether DR and E were

located in the sites of the DAPI-stainable DNA.

To investigate the potential location of DR and E

in the nuclei and in the mitochondria marked by

DAPI, distribution of the red, green and blue

components of the fluorescence (“pseudospectral

analysis”) in these regions was assessed

quantitatively using ImageJ “Measure RGB“ option

of the “Analyze“ plugin after appropriate ROI

selection.

The obtained data indicated that, as it was

expected, Red/Green ratio of DR and E were

significantly higher than that of DAPI. In contrast to

E, DR fluorescence had appreciably higher the

Red/Green ratio in the nuclei than in the

mitochondrial region. These data indicated that at

least a fraction of DR molecules bond in the nuclei

had more expressed red component of their

fluorescence spectra than those in the mitochondrial

region. Interaction of DR with nucleic acids is

known to increase the “red” maximum in its

fluorescence spectrum. So, the less expression of the

red fluorescence in the mitochondria may be

explained assuming that, along with binding to

DNA, some molecules of DR are bound to the

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membranes. Binding to the membranes may also be

a plausible reason for visible even fluorescence

distribution in the mitochondrial region in the cells

stained by DR as compared to “dot”-like staining of

the mitochondrial DNA by DAPI. There may be at

least two binding sites for DR and E in the

mitochondria: DNA and the membranes. Ability of

E to interact with the mitochondrial membranes was

demonstrated by others. Potential ability of DR to

interact with biomembranes was shown on artificial

membranes.

Upon combined addition of DR+DAPI

simultaneously, in both the nuclei and in the

mitochondria, the Red/Green fluorescence ratio was

higher in comparison with the application of DAPI

alone, but it was lower as compared to exposing the

cells to DR only. If the cells were first incubated

with DR, and DAPI was added later, the Red/Green

ratio in the nuclei was higher than in case of

simultaneous addition of the NAI, but still lower

than after the addition of DR alone. Similar results

were obtained for the pair E+DAPI with the

difference that the order of the NAI addition did not

significantly influence the final result. At the same

experimental conditions, there were no appreciable

changes in the Blue/Green ratio. So, quantitative

image analysis revealed appearance of the red

fluorescence component in the regions of DAPI

stained DNA upon combined application of DAPI

with DR and E. This is an indication of a

colocalization of DAPI with DR and E in the nuclei

and in the mitochondria.

In the sites of the colocalization of DR and E

with DAPI, the red component of their fluorescence

was less as compared to the application of DR and E

alone. These data can be interpreted as a competition

of DAPI with DR and E for the same DNA binding

sites. It should be mentioned here that, although NAI

can intercalate between base pairs, they may also be

bound to some other parts of DNA, e.g. in the minor

groove. The detailed mechanism of the competition

of DAPI, DR and E for binding sites in DNA is not

known as of yet. For more details see (Puchkov,

2011, in the press).

5 CONCLUSIONS

1. Three computer image analysis algorithms of

the ImageJ software for the fluorescence microscopy

data treatment have been developed.

2. The first, based on the use of “RGB-split” and

“Analyze Particles” options, was shown to be

successfully applied in a new method for rapid yeast

viability assessment. This method could be of

potential use for rapid viability evaluation of other

microbial cultures, too.

3. The second enables the determination of the size

and of the center of mass locations of the fluorescing

particles, <1 µm in size, by their space fluorescence

intensity distribution assessment. Using this

algorithm, the apparent viscosity in the vacuoles of

the individual yeast cells was evaluated by Brownian

motion measurements. The methodology developed

in this work may be of use for some other studies of

moving microscopic fluorescing particles.

4. The third algorithm, the “pseudospectral

analysis”, was designed for locating intracellular

sites/targets of fluorochromes in the yeast cells. The

intact (not fixed) yeast cells of S. cerevisiae can be

used as a model for intracellular locating of the

nucleic acid intercalators by fluorescence

microscopy combined with this computer image

analysis algorithm. The model and the approach

presented herein may be of help in the new DNA-

targeted drug discovery and in preliminary studies of

their interaction with eukaryotic cells.

ACKNOWLEDGEMENTS

Participation of Miss Millicent McCarren in the

experiments and discussions of the studies described

in the 4

Section of the paper is highly appreciated.

REFERENCES

Puchkov, E. O., 2006. ‘Viability assessment of

ethanol-producing yeast by computer-aided

fluorescence microscopy’, Microbiology, vol. 75, no.

2, pp. 154-160.

Puchkov, E. O., 2010. ‘Brownian motion of polyphosphate

complexes in yeast vacuoles: characterization by

fluorescence microscopy with image analysis’, Yeast,

vol. 27, no. 6, pp. 309-315.

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Yeast Studies

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