Fluorescence and Fluorescence and fluorescence-lifetime fluorescence-lifetime imaging microscopy imaging microscopy

(FLIM) to characterize (FLIM) to characterize yeast strains by yeast strains by

autofluorescenceautofluorescence

H. BhattaH. Bhattaaa, E.M. Goldys, E.M. Goldysaa and J. Ma and J. Mabb

aaDepartment of PhysicsDepartment of Physics, , bbDepartment of StatisticsDepartment of Statistics

Macquarie University - Sydney, AustraliaMacquarie University - Sydney, Australia

Problem: Quantiative Problem: Quantiative indicators in the presence indicators in the presence

of heterogeneityof heterogeneity• Diversity within microbial populationDiversity within microbial population

– Size, fluorescence intensity, textureSize, fluorescence intensity, texture

Solution: Characterise populations

Evolution of yeast Evolution of yeast fluorescence with fluorescence with

culture ageculture age

A9

Y275

24 hrs 48 hrs 72 hrs

Capturing and Capturing and quantifying quantifying

heterogeneity (1)heterogeneity (1)• Image analysisImage analysis

– Extracting features (size, intensity, Extracting features (size, intensity, texture) using customized plugins for texture) using customized plugins for ImageJ*ImageJ*

*http://rsb.info.nih.gov/ij/*http://rsb.info.nih.gov/ij/

Cell # Area Intensity IDM Entropy1 250 12.55 0.556 8.572 153 13.69 0.586 8.213 88 9.43 0.762 6.204 230 11.70 0.666 7.835 130 10.42 0.693 7.176 177 10.33 0.625 7.267 241 11.86 0.625 8.098 88 9.20 0.711 6.159 138 10.13 0.623 7.37

10 173 10.24 0.670 7.3311 111 10.18 0.634 6.8412 189 11.17 0.708 7.4813 110 10.06 0.672 6.9214 82 9.60 0.731 6.4115 206 9.72 0.724 7.1316 155 9.76 0.614 7.0417 217 10.08 0.702 7.1818 129 9.39 0.686 6.7219 147 9.86 0.625 7.2320 201 10.33 0.608 7.53… … … … …96 176 13.99 0.480 8.55

Definition of texture Definition of texture featuresfeatures

– EntropyEntropy• A statistical measure of randomness that can A statistical measure of randomness that can

be used to characterize the texture of input be used to characterize the texture of input imageimage

– Inverse difference moment (IDM)Inverse difference moment (IDM)• A measure of image texture with value A measure of image texture with value

ranging from 0 for highly textured image to 1 ranging from 0 for highly textured image to 1 for untextured imagefor untextured image

P(i,j) is defined as the matrix of relative frequencies with which two neighboring resolution cells separated by distance d occur on the image, one with gray tone i and the other with gray tone j

Evolution of yeast Evolution of yeast fluorescence with fluorescence with

culture ageculture age

A9

Y275

24 hrs 48 hrs 72 hrs

Histograms for cells at Histograms for cells at 48hrs48hrs

A9

Y275

Capturing and Capturing and quantifying quantifying

heterogeneity (2)heterogeneity (2)• Statistical descriptionStatistical description

– Empirical cumulative distribution Empirical cumulative distribution function (ecdf)function (ecdf)• ecdf(xecdf(x00)=fraction of population )=fraction of population

characterised by the relevant parameter characterised by the relevant parameter x<xx<x00

• Statistical analysisStatistical analysis– Kolmogorov-Smirnov testKolmogorov-Smirnov test

• One of the most useful nonparametric One of the most useful nonparametric tets for comparing two distributionstets for comparing two distributions

Materials and methods Materials and methods (1)(1)

• Cell cultureCell culture– Yeast strains (Yeast strains (Saccharomyces cerevisiaeSaccharomyces cerevisiae) A9 ) A9

(baking strain) and Y275 (brewing (baking strain) and Y275 (brewing strain) grown in non-fluorescent strain) grown in non-fluorescent mediummedium

– Standardized inoculation, ODStandardized inoculation, OD600nm = 600nm =

0.006 for starter culture0.006 for starter culture– Each strain cultured in two different Each strain cultured in two different

flasks at room temperature (25flasks at room temperature (2500C), 3 C), 3 images taken for each as reproducibility images taken for each as reproducibility checkcheck

Materials and methods Materials and methods (2)(2)

• Microscopy of cellsMicroscopy of cells– 100100 oil objectives were used oil objectives were used– Fluorescence emission and lifetime data Fluorescence emission and lifetime data

collected at 440 – 540 nm for 405 nm collected at 440 – 540 nm for 405 nm excitation for cells grown for 24, 48 and excitation for cells grown for 24, 48 and 72 hrs (6 replicates)72 hrs (6 replicates)

– Fluorescence images were collected in Fluorescence images were collected in z-stack of 10 slices then cell images z-stack of 10 slices then cell images with maximum intensity selected for with maximum intensity selected for analysisanalysis

Analysis of cell size - ecdf Analysis of cell size - ecdf and KS testand KS test

The cumulative distribution function for size of the yeast strain A9 (dots) and Y275 The cumulative distribution function for size of the yeast strain A9 (dots) and Y275 (line) at 24 hrs of age. Thick lines represent combined data for all six replicates. (line) at 24 hrs of age. Thick lines represent combined data for all six replicates. Inset: ecdfs of combined data with the KS test band in grey.Inset: ecdfs of combined data with the KS test band in grey.

P=0.05

24 h culture

Analysis of entropy & Analysis of entropy & intensity (24 hrs)intensity (24 hrs)

The cumulative distribution function for entropy and fluorescence intensity of the yeast strain A9 The cumulative distribution function for entropy and fluorescence intensity of the yeast strain A9 (dots) and Y275 (line) at 24 hrs of age. Thick lines represent combined data for all six replicates. (dots) and Y275 (line) at 24 hrs of age. Thick lines represent combined data for all six replicates. Inset: ecdfs of combined data with the KS test band in grey.Inset: ecdfs of combined data with the KS test band in grey.

P=0.05 P=0.05

Size evolution with ageSize evolution with age

P=0.05

P=0.05

P=0.05

Entropy evolution with Entropy evolution with ageage

P=0.50

P=0.50P=0.05

dots –A9, lines -Y275

Intensity evolution with ageIntensity evolution with age

P=0.05

P=0.50

P=0.05

Dots – A9

Lines - Y275

Significance levels Significance levels

D0 p0 n0 D1 p1 n1

24 h 0.20 0.05 87 0.38 <0.001 522

48h 0.16 0.05 125 0.39 <0.001 750

72 h 0.15 0.05 128 0.42 <0.001 768

24 h 0.20 0.05 87 0.45 <0.001 522

48h 0.10 0.50 125 0.20 <0.001 750

72 h 0.10 0.50 128 0.27 <0.001 768

24 h 0.20 0.05 87 0.17 <0.001 522

48h 0.09 0.50 125 0.20 <0.001 750

72 h 0.15 0.05 128 0.29 <0.001 768

Size

Entropy

Intensity

Age Maximum distance (D) and probability (p) values for the k-s test applied to yeast images.

D1 is the maximum difference between combined ecdf and individual ecdfs of six replicates (1/2 width of grey stripe), p1 is relevant to intra-strain variability given in preceding column, n1 is the sample size for single data set of each strain, D2 is the maximum difference between combined ecdfs for two strains, p2 is relevant to intra-strain variability given in preceding column, n2 is the sample size for cumulative data set of each strain.

FLIM resultsFLIM results

Color coded Color coded fluorescencfluorescence lifetime e lifetime image (a) , image (a) , distribution distribution of lifetimes of lifetimes (b) (b)

and lifetime and lifetime decay curve decay curve (c) of yeast (c) of yeast strain A9 at strain A9 at 48 hrs of 48 hrs of age.age.

(a)

(b)

(c)

FLIM textureFLIM texture

ecdf plot of inverse difference moment (IDM) for fluorescence ecdf plot of inverse difference moment (IDM) for fluorescence lifetime of yeast strain A9 (dots) and Y275 (line) at 48 hrs of age. lifetime of yeast strain A9 (dots) and Y275 (line) at 48 hrs of age.

P<0.001 for n=522

ConclusionsConclusions

• We carried out detailed characterisation We carried out detailed characterisation of yeast cell populations by fluorescence of yeast cell populations by fluorescence microscopy and FLIMmicroscopy and FLIM

• It is possible to distinguish the two It is possible to distinguish the two examined yeast strains by population examined yeast strains by population propertiesproperties

• Strain identities and differentiation Strain identities and differentiation demonstrated at acceptable confidence demonstrated at acceptable confidence levelslevels

• A novel method for comprehensive A novel method for comprehensive characterization of yeast strains through characterization of yeast strains through data mining of microscopy imagesdata mining of microscopy images

• Evolution of cell features with culture Evolution of cell features with culture age examinedage examined..