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Frequency domain analysis of time-

resolved fluorescence measurements
using the squared distance approach

Yahav, Gilad, Diamandi, Hilel Hagai , Preter, Eyal, Fixler,

Dror

Gilad Yahav, Hilel Hagai Diamandi, Eyal Preter, Dror Fixler, "Frequency
domain analysis of time-resolved fluorescence measurements using the
squared distance approach," Proc. SPIE 10891, Nanoscale Imaging, Sensing,
and Actuation for Biomedical Applications XVI, 108910Z (5 March 2019); doi:
10.1117/12.2507358

Event: SPIE BiOS, 2019, San Francisco, California, United States

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 Frequency domain analysis of time-resolved fluorescence
measurements using the squared distance approach
Gilad Yahav*, Hilel Hagai Diamandi, Eyal Preter, and Dror Fixler

Faculty of Engineering and Institute of Nanotechnology and Advanced Materials, Bar Ilan
University, Ramat Gan, Israel;

ABSTRACT
Frequency domain analysis of time-resolved fluorescence measurements (TRFM) is an extremely rapid technique for
medical diagnostics thanks to its unique sensitivity to a wide variety of physical and chemical features. Nevertheless, the
determination of the underlying fluorescence lifetime (FLT) data of samples by their frequency response data (FRD),
demands fitting algorithms. Therefore, the interpretation of the precise changes in the FLT of complex environments in
term of biochemical processes is a challenge as it involves uncertainties associated with the chosen fitting algorithm.
This research suggests a novel characterization procedure based on the squared distance (D2) between the FRD of the
samples that avoid the inherent blurring caused by the transformation of the FRD into FLT data.
The D2 approach was validated through simulated data of 6 classes with similar FLT characteristics, where the accuracy
of D2 classification was about 96%. In addition, this approach was tested on experimental FRD from 43 individual
samples that their preliminary physician diagnosis divided them into 4 groups: 5 healthy samples served as controls, 9
samples diagnosed with diverse types of bacteria, 16 samples diagnosed with diverse types of viruses and 13 samples
were negatives to any bacterial or viral infection, although presenting related symptoms. Using the D2 analysis, the
classification of 28/30 matched the physician diagnosis and the classification of 41/43 samples matched earlier report.
In conclusion, this work demonstrated that the D2 model can aid in disease identification and increase the specificity and
sensitivity of conventional medical procedures or TRFM-based diagnosis.

Keywords: Fluorescence lifetime (FLT), Frequency response data (FRD), Squared Distance function (D2), Pathogen
detection, time-resolved fluorescence measurements (TRFM).

1. INTRODUCTION
Frequency domain (FD) analysis of time-resolved fluorescence measurements (TRFM) is a valuable technique in a wide
variety of scientific fields such as engineer, physics, and chemistry as well as medical diagnostics and material sciences.
There are two dominant methods of implementing TRFM: the time-domain (TD) and the frequency-domain (FD) both
mathematically equivalent and related by Fourier transform. These two methods provide information regarding the
intensity decay law of the sample (typically single or multi-exponential decay profile), whether the fluorescence
emission is polarized or unpolarized. In addition, both methods used to interpret the decay in terms of the molecular
features of the sample whenever it connects with features that associate with changes in the local fluorophore
microenvironment. In this work, we limit our discussion to the FD method as its FLT measurements and data acquisition
are faster and hence FD- TRFM is more potentially suitable for medical diagnostics 1,2.
Previously, many researchers examined the significance of FLT measurements (whether performed by the TD or the FD)
in medical diagnostics. These include identifications of chromosomal abnormalities in patients with diverse types of
Leukemias 3, or carriers of BRCA1 and BRCA2 mutations 4 and detection of metastatic cell in the cerebrospinal fluid of
children with Medulloblastoma 5. Other works include quantification of the metabolic state in cell-model of Parkinson's
disease 6, tracking apoptosis and stimulation in individual cells 7,8, pH imaging of living cells 9-11 and simultaneous
detection of multiple green fluorescent proteins in live cells 12,13.
In the FD-TRFM measurements, the analysis is performed using the frequency response data (FRD) of the sample,
namely the measures of modulation depth (m) and the phase shift (φ). The FRD is determined by the intensity decay law
of the sample and provide two distinct methods to extract the fluorescence lifetime (FLT) of the sample by the
modulation FLT (τm) and the phase FLT (τφ) 1,2,14.

* Gilad.Yahav@gmail.com; phone +972-54-5375253; fax +972-3-7384050;

Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XVI, edited by
Dan V. Nicolau, Dror Fixler, and Ewa M. Goldys · Proc. of SPIE Vol. 10891, 108910Z
© 2019 SPIE · CCC code: 1605-7422/19/$18 · doi: 10.1117/12.2507358

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 In addition, working in the FD-FLT involves two main challenges. The first challenge is the demand of a reference that
should hold the same properties as the requested sample to ensure proper FRD calibration and accurate calculation of the
extracted FLTs. These properties include similar FLT, same modulation frequencies, same gain amplification factors,
and same fluorescence emission. However, frequently, FLT measurements are performed in complex environments,
where multiple FLTs exist in the samples with multiple properties. Therefore, finding a reference that suits all these
properties is a challenge. For this purpose, we previously suggested the crossing point (CRPO) method that provides a
method to extract the FLT values and species fraction (FLT data) of a sample without the request of using appropriate
reference. The main principles of this technique were described elsewhere 15. The second challenge is that the
determination of the underlying FLT data in complex environments usually involves iterative nonlinear fitting
algorithms, which connects the FRD that span the frequency response of the sample to its FLT data. Therefore, there are
inherent uncertainties in FRD to FLT data transformation that are connected to the chosen fitting algorithm 16.
The goodness of fit is determined by a nonlinear least square method, which is a reduced chi-square (χ2), calculated by:
n  c
i  mi   mc i  mmi  
2 2
1 
 
2
  
2n  k  1 i 1       m

 
(1)
  
The terms n and k refer to the number of modulation frequencies and a number of free variables need to be determined
by the suggesting model, respectively. The parameters φωi and mωi are the phases and modulation data of each angular
modulation frequency ωi. The superscript c and m indicate the calculated values for an assumed fitting model with define
values of FLT data and measured values, respectively. The terms σφ and σm are the standard deviations (STD) from these
measures and are typically 0.20 and 0.004, respectively. Nevertheless, these values may change depending on the chosen
FD instrumentation 16. The acceptability of a suggested model is determined by the model producing a reduced χ2 close
to 1. Nonetheless, there are situations where the χ2 cannot distinguish between different models. The presence of even
more components will significantly increase the uncertainty in the transformation of the FRD to FLT data 16.
Several works were developed to characterize samples by the FLT perspective without the need of the nonlinear fitting
algorithms such as the polar plot, CRPO, and the noise corrected principal component analysis (NC-PCA) but in the
practical use up to date do not utilize most of the frequency response information 15,17-21.
To address the challenge of characterizing samples by their FRD without the uncertainties that result from the fitting
algorithms, this research suggests the squared distance (D2) approach. The D2 performs the samples classification based
on the squared distance between their raw FRD. Therefore, it does not require the determination of the FLT data and
hence it avoids the possible blurring of the data in the analysis process and the effect of analysis and model assumptions
on the obtained results. These include the number of fluorescing species and the chosen initial values of the iterative
process. As a result, the D2 approach increases the classification resolution. This may be used to distinguish between
samples in situations where the FLT analysis or the conventional existing methods fail and produce false negative or
false positive results.
The following section will present the equation for measuring the squared distance between the samples FRD using FD
measurements.

2. THEORY
2
The D approach uses the direct raw FRD to determine sample classification without the need of its transformation to
FLT data and hence without the fitting algorithms. Therefore, it increases the accuracy of the analysis as well as the
classification resolution. The standard Euclidean distance (D) is squared to highlight whether the tested samples have
similar FRD (typically D<1) or different FRD (typically D>1). The difference between the FRD is normalized
(according to the variance) based on the number of the modulation frequencies (N), which the FRD was extracted from.
Generally, the D2 between 2 samples, A and B, is calculated by:
 A 2 2
N    A
mi  mi  
B
 
B
1  i i 
D 
2
   (2)
2 N i 1   T    Tm  
    


N is the number of modulation frequencies used to achieve the FRD. The terms  i and mi represent the arithmetical
mean of the measured phase shifts and the modulations for each sample, A and B. For example, if the number of
subsamples in sample A is denoted by nA, then:

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 1 nA A

A
  (3)
i
nA l 1 i ,l
nA
A 1
mi   mA (4)
nA l 1
i ,l

B B 
Using the number of subsamples (e.g. cells) in sample B, nB,  i and mi are similarly described. The terms  T and
m
 T refer to the total STDs of the FRD measures. The STDs are generated from the FRD measures and from taking their

averages.  T is described by:
   T
 T   sample _ A   sample _ B   system
2 2 2
(5)

m
In a similar manner,  T is described by:
m
 T   sample
m
_ A   sample _ B   system
2 m 2 mT 2
(6)

T
The term  system is derived from the STD in all FRD measures.

T
 system is extracted by:

2 2
 nA i   nB i 
 system 
T
   system _ A     system _B  (7)
 i 1   i 1 

In a similar manner,  system

mT
is described by:

2 2
 nA   nB 
 system
mT
   system
mi
_ A      system _ B 
mi
(8)
 i 1   i 1 

The D2, as presented in Eq.(2), reminds the χ2, as presented in Eq.(1). However, contrary to the χ2, in the D2 approach,
there is no demand for any suggested model for comparison. The analysis is only based on the D2 between the raw FRD
of two different samples, A and B.

3. MATERIALS AND METHODS

The preceding section presented the equation for computing the D2 between the FRD of two samples. The following
section will describe the analyzed samples as well as the optical setup used to extract the FRD and the methodology by
which D2 is calculated and implemented.

3.1 Samples collection, preparation, and diagnostics

The controls of this research were 5 samples of peripheral blood (PB) cells taken from 5 normal subjects who were found
negative to oncological or bacterial and viral infectious diseases. These samples were harvested, prepared, and analyzed
as part of a routine clinical evaluation of the patients according to the standard protocol developed in the Laboratory of
Hematology, Sheba Medical Center, Israel. The controls collection and preparation were previously described 3,22.
The 37 samples study was conducted at a tertiary medical center. This study design adhered to the tenets of the
Declaration of Helsinki and it was approved by the local review board of the institute before its initiation. The
anonymous CSF samples were obtained under separate institutional review board approval and exempted from consent.
Pathogen samples of CSF with inflammatory cells derived from patients with infectious diseases were collected from the
microbiology laboratory, Rabin Medical Center, Israel. The samples collection and preparation were previously

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 described 5. Both the 5 controls and the 37 samples slides were stained with 20µl of 4′,6-diamidino-2-phenylindole
(DAPI) in antifade solution (Q-Biogene, Cambridge, UK) and covered with a coverslip.

3.2 Group Classification

The study group of this research included 4061 cells taken from 43 samples divided into 4 groups distinguished by their
preliminary diagnoses.
In the first group, there were 5 healthy samples that served as the controls (denote as Patient # 1-5).
In the second group, there were 9 samples diagnosed with diverse types of bacteria: 3 samples with S. aureus (+), and 6
samples with the following: E. coli (-), Agromyces (+), Citrobacter (-), Haemophilus influenzae (-), Bacillus (-), and
Klebsiella pneumonia (-) (denote as Patient # 6-14).
In the third group, there were 16 samples diagnosed with diverse types of viruses: 13 samples with enterovirus, 2
samples with VZV, and 1 sample with Herpes1 (denote as Patient # 15-30).
In the fourth group, there were 13 samples that were suspected to have an infection with a virus or microbe (denote as
Patient # 31-43) but found negative to either of them (for more information regarding the samples please see 23).

3.3 Experimental Setup

The FRD were acquired by Lambert instruments fluorescence lifetime attachment system (LIFA, The Netherlands) 24.
In this system, a homodyne phase sensitive detection scheme is used. The excitation light source, a light emitting diode
(LED) with three different wavelengths 403nm, 468nm, and 537nm, is modulated in a sinusoidal fashion by a signal
generator/power supply unit (LIFA, The Netherlands). This sine wave (AC modulation superimposed upon DC) is
characterized by a high frequency that is reciprocal to the FLT of the sample 24.
The fluorescence emission responds with the same modulation frequency but presents a phase shift and decrease in
modulation in respect to the excitation. These FRD can be acquired by using a gain-modulated detection device
operating at the same frequency as the fluorescence emission but different numerous phase angles between them 24.
An Olympus IX-81 inverted microscope with a 10X, NA=0.4 objective (OLYMPUS, Japan) was used to focus the
sample. Then, a dichroic mirror located within the microscope filtered the excitation so that only the fluorescence
emission arrived at the image intensifier. The output of the image intensifier is coupled to a charge-coupled device
(CCD) camera with a resolution of 1392x1040 pixels (LI2CAM).
FRD measurements were calibrated using a reference of a fluorescein dye (prepared by dissolving it in water, Sigma-
Aldrich, St. Louis, MO). The fluorescent marker DAPI that was used to stain the nucleus of the 43 leukocytes samples,
has two fluorescing species and hence the FRD that was extracted for DNA-DAPI complex in each leucocyte cell's
nuclei, yielded apparent FRD values 24.
As in all fluorescence-based measurements, the FRD measurements may be influenced by the fluorescence anisotropy.
This can be avoided by using an excitation polarizer oriented in the vertical direction and an emission polarizer oriented
54.7o from the vertical direction (known as the magic angle) 25-27. However, using control experiments, measurements of
the parallel and perpendicular intensities from the reference fluorescein dye and from the 43 samples, were found
identical. Therefore, we concluded that the fluorescence anisotropy in our samples is negligible and hence the
inaccuracies that caused by not controlling it.

4. RESULTS AND DISCUSSION

4.1 Simulation
A series of 6 classes with similar FLT data was generated (Table 1). The last 3 classes were differentiated by closely
spaced FLT values relate to the first 3 classes. Each class was tested 50 times differing by additive white Gaussian noise
(AWGN). Class 1 is presented through tests # 1-50; class 2 is presented through 51-100# etc. The D2 function measured
the FRD taken from the 300 tests and performed the classification based on the class presenting the minimal D2 value.
The 6 classes, as presented in Table 1, can represent the sample with 3 homogeneous subsamples or one sample with 3
FLT components.
The FLT data were used to extract the FRD and further the apparent modulation and phase FLTs using 10 modulation
frequencies linearly spaced between 10MHz-100MHz.
The methodology by which the FLT data is used to extract the FRD and the apparent τm and τφ is discussed elsewhere
16
.

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 As known, the optimal modulation frequencies should be about reciprocal to the FLTs. Otherwise, the extracted FLTs
might be incorrect 24. However, this demand is not always possible as sometimes there is a great variety of FLT values.

Table 1 FLT data for the 6 classes (each class was tested 50 times).
Class (test#) FLT components species fractions
1 2 3 1 2 3
1 (#1-50) 2.00 4.00 6.00 0.20 0.20 0.60
2 (#51-100) 2.00 4.00 6.00 0.10 0.30 0.60
3 (#101-150) 2.00 4.00 6.00 0.20 0.23 0.57
4 (#151-200) 2.80 3.10 3.40 0.20 0.20 0.60
5 (#201-250) 2.80 3.10 3.40 0.00 0.00 0.10
6 (#251-300) 2.80 3.10 3.40 0.22 0.25 0.53

The classification of the 300 tests was performed using the apparent FLT values, τm (blue) and τφ (orange) (Fig. 1a) and
through the D2 approach (Fig. 1b). However, contrary to the apparent FLT analysis, which provided difficulties in the
classification due to the similar FLT data (such as between classes 1,3 and 4,6), the D2 approach matched the 300 tests
into their right 6 classes inaccuracy of 96%. The accuracy of the D2 approach is presented through a confusion matrix.
The architecture of a confusion matrix is presented hereinafter: the first upward diagonal cells (green) show the number
and percentage of the correct classifications using the D2 approach. For instance, the top left diagonal cell indicated that
49/50 tests were correctly classified as class 1. This corresponds to 16.3% of all the 300 tests. However, all tests were
correctly classified as class 2. This corresponds to 16.7% of the 300 experiments. The last 4 classes are similarly
described.
a) b)

Fig.1. Classification of the six classes comprised of three widely and three closely spaced FLTs (Table 1) through apparent τm (blue)
and τφ (orange) as well as through minimal D2 classification. Each class is tested 50 times (with AWGN) in a sequential manner (tests
# 1-50 belong to class 1, etc.). In general, τma ≥ τφa and their equality should exist only for class with single FLT component (class 5).
However, this phenomenon is not observable for the last 3 classes (tests # 150-300) due to the inappropriate modulation frequencies.
The apparent FLTs cannot distinguish between classes with similar FLT data (classes 1, 3 and 4, 6) (a). However, the D2 classification
clearly distinguishes all classes in good accuracy. Out of the 300 predictions, 96.0% are correct (b).

4.2 Experiment
The D2 approach used to classify the 43 samples, which were described in section 3.2, as follows:
1. The FRD and their STDs were obtained in 8 frequencies between 20-90MHz linearly spaced for all cells taken
from the 43 samples.
2. Next, we created average FRD and hence additional STDs per frequency for each of the 43 samples.
3. We repeated the first 2 steps for the first 3 groups. Thus, receiving the FRD database for each group.
4. We used Eq. (2) to calculate the squared distance between each of the 43 samples and the 3 groups. Each
sample was classified by the group having the minimal D2.
2
The D classification, as appeared in Fig 2a, matched between the first 30 samples to their physician diagnosis. A
matched is denoted by V. All first 5 control samples were correctly classified. Eight samples were correctly classified to
have bacteria (patient # 6-11, 13, 14) and 15 samples were correctly classified to have a virus (patient # 15-25, 27-30).

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 One bacterial sample (patient # 12) and one virus sample (patient # 26) were miss-classified to have another type of
infection. The 2 miss classifications might result from a relatively small number of measured cells (data not shown).
In conclusion, The D2 approach classified between the infected and the non-infected samples in 100% accuracy and the
overall accuracy between the 3 groups was 93.3%.
a)

b)

Fig.2. Classification using the D2 of the 30 samples taken from the first 3 groups, as presented in section 3.2. Patients #1-5 were the
controls of this research, patients #6-14 were diagnosed with diverse types of bacteria and patients #15-30 were diagnosed with
diverse types of viruses. The D2 classification matched between 28 samples out of 30 to its pre-diagnosis (a). In addition, the 3 groups
were used by the D2 approach to classifying 13 additional samples that were negatives to any virus or bacterium, although presenting
related symptoms (b). The D2 classification divided these 13 samples into 3 groups: one group that matched the controls (patient # 31-
34, 36 and 39), the second group matched the bacteria group (patient# 40, 42 and 43), and the last group matched the viruses’ group
(patient # 35, 37, 38 and 41). These findings matched the earlier report23,28,29.

In the second stage, the 13 negatives (patient # 31-43) were classified by the D2, using the FRD database of the first 3
groups (Fig 2b). A matched is denoted by V. The D2 classification divided the negatives into 3 groups; Patient # 31-34,
36, 39 were classified as controls, patient # 35, 37, 38 and 41 were classified to have viruses and patient# 40,42 and 43
were classified to have bacteria. It is instructive to note that the difference in the minimal D2 might suggest another type
of pathogens or diseases. Patient # 37 returned to the second diagnosis that had found him to have a virus as the D2
analysis suggested.

5. CONCLUSION
This research demonstrates how diagnostic classification can be achieved in a more accurate and efficient manner using
the D2 approach. The originality of this approach is that it performs the classification using the FRD, which today is only
used to extract the apparent FLTs. This transformation is based on nonlinear fitting algorithms and hence can distort
existing valuable information that may be essential to perform correct classifications. As a result, the D2 approach
provides further information in complex biological systems, as applied in this paper to differentiate between the presence
of viral and bacterial infection.
This added information may also be used to distinguish two samples, separated by slight changes in their FLTs, which
may result from small changes in the sample’s conditions (such as FRET, quenching or interacting ligands) 30-39.
In addition, the D2 approach may shed new light regarding the ideal conditions and parameters that need to be
determined before the samples’ classifications.

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 In this research, we probed changes of the FLTs. However, the D2 approach may also be applied for FD anisotropy decay
measurements, in the same manner, using the FRD of the parallel and the perpendicular fluorescence emission.

Acknowledgments

We would like to thank Dr. Luba Trakhtenbrot who provided the control samples, as well as to Dr. Haim Ben-Zvi and
Gabriel Mircus who provided the pathogen samples and the negatives. We would also like to thank Prof. Nitza
Goldenberg-Cohen, Dr. Mali Salmon-Divon, and Sivan Gershanov.
In addition, we would like to thank the Levi-Eshkol Fund, Ministry of Science, Technology & Space, Israel (grant
number 3-12624) for providing S.G scholarship. The funding organization had no role in the design or conduct of this
research. H. Hagai. Diamandi is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship.

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