ArticlePDF Available

Fiber ATR infrared spectroscopy of kidney tissue during live surgery



More than 90% of solid kidney tumors are cancerous and have to be treated by surgical resection where surgical outcomes and patient prognosis are dependent on the tumor discrimination. The development of alternative approaches based on a new generation of fiber attenuated total reflection (ATR) probes could aid tumor identification even under intra‐surgical conditions. Herein, fiber ATR IR spectroscopy is employed to distinguish normal and cancerous kidney tissues. Freshly resected tissue samples from thirty four patients are investigated under nearly native conditions. Spectral marker bands that allow a reliable discrimination between tumor and normal tissue is identified by a supervised classification algorithm. The absorbance values of the bands at 1025, 1155 and 1240 cm⁻¹ assigned to glycogen and fructose 1,6‐bisphosphatase are used as the clearest markers for the tissue discrimination. Absorbance threshold values for tumor and normal tissue are determined by discriminant analysis. This new approach allows the surgeon to make a clinical diagnosis. This article is protected by copyright. All rights reserved.
Fiber attenuated total reflection infrared spectroscopy of
kidney tissue during live surgery
Valdas Sablinskas
*| Rimante Bandzeviciute
| Martynas Velicka
Justinas Ceponkus
| Vidita Urboniene
| Feliksas Jankevicius
Arvydas Laurinavicˇius
| Darius Dasevicˇius
| Gerald Steiner
Institute of Chemical Physics, Vilnius
University, Vilnius, Lithuania
Faculty of Medicine, Vilnius University,
Vilnius, Lithuania
National Cancer Institute, Vilnius,
National Center of Pathology, Affiliate of
Vilnius University Hospital Santaros
Klinikos, Vilnius, Lithuania
Faculty of Medicine Carl Gustav Carus,
Clinical Sensoring and Monitoring,
Dresden University of Technology,
Dresden, Germany
Valdas Sablinskas, Faculty of Medicine,
Vilnius University, Santariskiu str.
2, Vilnius, LT-08661, Lithuania.
Funding information
Research Council of Lithuania, Grant/
Award Number: SEN-16/2015
More than 90% of solid kidney
tumors are cancerous and have to be
treated by surgical resection where
surgical outcomes and patient prog-
nosis are dependent on the tumor
discrimination. The development of alternative approaches based on a new
generation of fiber attenuated total reflection (ATR) probes could aid tumor
identification even under intrasurgical conditions. Herein, fiber ATR IR spec-
troscopy is employed to distinguish normal and cancerous kidney tissues.
Freshly resected tissue samples from 34 patients are investigated under nearly
native conditions. Spectral marker bands that allow a reliable discrimination
between tumor and normal tissue are identified by a supervised classification
algorithm. The absorbance values of the bands at 1025, 1155 and 1240 cm
assigned to glycogen and fructose 1,6-bisphosphatase are used as the clearest
markers for the tissue discrimination. Absorbance threshold values for tumor
and normal tissue are determined by discriminant analysis. This new approach
allows the surgeon to make a clinical diagnosis.
ATR, fiber probe, FTIR, kidney cancer, resected tissue
During the past decades, many efforts were made to char-
acterize and to distinguish tissue by vibrational spectros-
copy. Although a huge number of successful reports were
published dealing with this topic [13], the transfer into
clinic appears to be limited due to the lack of instruments
and systems that allow in situ measurements. While usu-
ally Raman spectroscopy is considered as method of
choice for an in situ characterization of tissue [4], infra-
red (IR) spectroscopy is often seen as laboratory testing
method. However, Raman spectroscopy requires often
few minutes measurement time to record qualitative
spectra and, more important, the question of photo toxic-
ity or photo damaging of the excitation laser is not
answered yet. Reasons why IR spectroscopy did not
found broad application for in situ diagnosis might be
among other things. The strong absorption bands of
Received: 17 January 2020 Revised: 6 March 2020 Accepted: 24 March 2020
DOI: 10.1002/jbio.202000018
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Biophotonics. 2020;e202000018. 1of7
water and the fact that IR light is difficult to transport
to the point of measurements. However, recent develop-
ments in IR fiber spectroscopy allow now quick and reli-
able spectroscopic measurements even of native tissue
and opens the door for in situ applications in the clinic.
This is mainly driven by the fact that there is big need for
surgeons to determine malignancy of tissue during the
surgical operation in order to make final decision about
exact place of the surgical cut. On the other hand, the
evaluation of spectra in regard to classify tissue as normal
or pathological is still a challenge.
IR absorption spectra of biological tissue are rather
complex and difficult to analyze; nevertheless, during the
last decades, there are many successful attempts to apply
IR spectroscopy for detection of tumorous tissue areas
[58], for elucidating structure of kidney, bladder or gall
stones [912], for analysis of sediments in various bodily
fluids [1315]. Main drawback of this method is that sam-
the instrument. Using IR fibers in combination with an
attenuated total reflection (ATR) crystal may overcome the
time consuming ex vivo measurements. However, main
drawback of the ATR-FTIR method as a tool for detection
of tumorous tissue areas was a lack of suitable fiber probes
which provide quick and reliable measurements of native
crystal probe and loop probe. Generally, both types of probe
could be used for tissue examination, but an application of
fiber probes for tissue measurements is restricted by the
requirement to sterilize them after each use to avoid the
contamination of the tissue. This requirement restricts the
design of the probes to be easily changeable and sterilizable.
Therefore, the tip with ATR crystal is more appropriate for
the tissue examination. For this reason, this type of tips was
used in designing our fiber ATR FTIR system.
In order to use IR spectroscopy in operating room
(OR) there are some requirements concerning size of the
instrument and ability to do measurements in situ. There
is some choice of portable IR spectrometers in the mar-
ket, but they are not suitable for the in situ ATR mea-
surement, since they are equipped with compact detector
(usually DTGS) which has limited sensitivity. Such in situ
measurements require a fiber probe with ATR tip
attached to the end of the fiber. Usually, in such fiber
arrangement loses of optical signal are 90% or even
larger, what makes the measurements meaningless.
In an early study, we have demonstrated that very
small areas of infiltrated kidney tumor can be detected by
IR spectroscopic imaging and supervised classification
[16]. In the next study, we found a particular spectral
region that contains spectroscopic signals from extracellu-
lar and intracellular molecules such as fatty acids, glycerol
and glycogen. The signals can be used as spectral markers
for classification of healthy and tumor cells of kidney tissue
[17]. These initial studies were focused mainly on deter-
mining whether and where reliable spectral differences
between normal and tumor kidney tissue are located.
Also, we have demonstrated that kidney tumor tissue
can be identified by measuring spectra of dried tissue
smears by using a portable IR spectrometer equipped
with a new type of a fiber ATR probe [18]. In the present
study, we demonstrate that kidney tumor tissue can be
identified during live surgery using this fiber setup for
the measurements of wet tissue nearly in its native condi-
tions. In contrast to other studies, the spectra are not
classified and results are not translated into a computer
based diagnose like tumoror healthy.Aimed on the
clinical application and in accordance to the regulations
in medicine, the concrete absorbance values of the
marker spectral bands are defined and represented with-
out any algorithm-based classification.
Spectra of tissue were measured using ATR silver halide
fiber probe attached to the standard FTIR spectrometer
Alpha (Bruker Optik GmbH, Ettlingen, Germany).
Changeable fiber probe tips with single reflection germa-
nium ATR crystal were used. The schematics of the ATR
fiber probe (Art Photonics GmbH, Berlin, Germany)
accessory is presented in Figure 1.
The development of the complete system was
implemented by our researchers' group in cooperation
with Art Photonics.Optical fibers used in the setup are
made from silver halide. The focusing and directing of
the light in this accessory is done only by two elliptical
and four flat mirrors. The germanium ATR crystal is
fixed to the fiber by means of detachable plastic holder
(tip). Such approach gives an opportunity for a new and
sterile tip to be used for every successive measurement
and ensures that no contaminants are introduced to the
tissue thus reducing the risk of the complications during
FIGURE 1 Schematic diagram of fiber attenuated total
reflection (ATR) probe with changeable ATR crystal
the surgery. The ATR fiber accessory is made in a com-
pact way and allows the quick interchanging from the
conventional ATR setup of the portable FTIR spectrome-
ter and the ATR fiber setup without the need to use two
different spectrometers.
A liquid nitrogen cooled MCT detector (Infrared
Associates, Inc. Model IRA-20-00131) was coupled with
the whole system in order to compensate the loss of opti-
cal signal in the fiber which leads to the reduction of
signal-to-noise ratio. This spectroscopy system is rather
light and compact thus it can be easily fitted on a mobile
table for maneuverability.
The main advantage of the application of fiber probes
is that the sample could be analyzed in situ conditions
and it does not have to be transferred to the device. Also,
there are some drawbacks of the fiber ATR setup. The
spectral region is restricted due to scattering of light in
the fiber but it does not have impact while analyzing the
fingerprint spectral region. While performing measure-
ments in fiber ATR configuration the loss of optical sig-
nal is current; however, it could be compensated by using
more sensitive MCT detector. Fibers made of silver halide
are fragile and degradation of the fiber could be observed.
The IR absorption spectra of freshly resected human kid-
ney tissues, taken from 34 patients, were measured immedi-
ately after surgical resection inside the operation theater of
the Vilnius university hospital Santaros Clinics urology
department. The protocol for spectroscopic studies was
approved by Vilnius regional bioethics committee (approval
No. 158200-15-803-312). Before each measurement, a back-
ground spectrum was recorded from the clean ATR fiber
probe. Small amounts of suspected tumorous and normal
the freshly cut area of resected kidney tissue sample and
spectra of the tissue were collected. IR absorption spectra of
tumorous and normal human kidney tissue were measured
in 400 to 4000 cm
spectral region with 4 cm
spectral res-
olution. Sixty-four interferograms were averaged and Fourier
transformed into a spectrum applying three-term Blackman-
Harris apodization function and zero filling factor of 2.
Evaluation of spectral data was performed using the
MATLAB Package (Version 7, Math Works Inc. Natick,
Massachusetts). In order to minimize the data volume and
to exclude the strong absorption bands of water only the
region between 950 and 1350 cm
was considered. Data
preprocessing involved a linear baseline correction by using
the msbackadj function of the Statistics Toolbox of
MATLAB. The baseline correction was performed to reduce
influences of light scattering. Spectra with a maximum
absorbance larger than 1.8 or smaller than 0.02 were identi-
fied as outliers and removed from the data set. Finally, the
selected spectra were area-normalized to eradicate spectral
differences due to different sampling conditions during the
measurements of different samples. Different overall absorp-
tion values of different samples are influenced by several
factors. One of the main factors is the contact between the
sample and the ATR crystal; in different measurements, it is
impossible to ensure the same pressing force on the sample.
The consistency and hardness of the samples are also differ-
ent, especially between normal and tumorous tissues due to
different amount of water and biochemical composition. A
training set of 24 spectra of each class was built for super-
vised classification. Aim of the supervised classification was
used to explore optimal spectral regions for discrimination
of normal and tumor tissue. The approach uses a genetic
algorithm to maximize the classification rate with the itera-
tive optimization of selected features and is similar to a
method described elsewhere [19]. Each spectrum was
reexpressed as a set of three intensity values, which were
used for the subsequent classification by quadratic discrimi-
nant analysis, done using the classify function available in
the Statistics Toolbox of MATLAB. The performance of the
classification was assessed with the leave-one-out-validation
method. An independent test set of 10 patients was used to
test the discrimination parameters.
Examples of resected kidney tissue are represented in
Figure 2. While general differences between normal
FIGURE 2 Photographs of normal, A, and tumorous, B, tissue
samples, microscopy image of H&E stained tissue, C. After
microscopic histopathological examination, the borderline between
normal and tumorous kidney tissues is clearly visible
(Figure 2A) and tumor tissue (Figure 2B) were in the
most cases visible, a sharp borderline is not observable.
The borderline becomes clearly visible after microscopic
histopathological examination (Figure 2C).
For each case, one part of the resected tissue was
examined by standard histopathological analysis; another
part of sample was used for the measurements of IR
absorption spectra. Diagnosed types of kidney tumors
and number of cases are summarized in Table 1.
Figure 3A shows the recorded raw ATR spectra of all
tumor (red) and normal (green) tissue samples. Due to
the strong water absorption bands, spectra were reduced
to the spectral region from 950 to 1350 cm
. At the first
glance, the most important spectral bands that may allow
to discriminate tumor from normal tissue are located
around 1025 and 1155 cm
Between 1000 and 1250 cm
absorption bands mainly
due to carbohydrates, glycoproteins and phosphate groups
occur. Clearly, the IR spectrum captures a wealth of chemi-
cal information and slight variations in the band positions
and intensities reveal heterogeneity across the samples.
The key question addressed here is whether the biochemi-
cal information latent in these spectra is able to discrimi-
nate normal from tumor tissue. The absorption profile
between 1000 and 1050 cm
, in particular the absorption
band around 1025 cm
is stronger for tumor tissue than
for normal tissue. Furthermore, tumor tissue exhibits also
stronger absorption around 1150 cm
and weaker signals
between 1200 and 1275 cm
. The question that then arises
is which constituents could be responsible for these bands.
To illustrate the spectral changes more clearly, Figure 3B
shows the average spectra and standard deviation of both
types of the tissue. Table 2 summarizes the vibrational
modes in this spectral range.
The spectral band at 1025 cm
is assigned to
ν(C O), ν(C C) stretching and δ(C O) bending
vibrations of C OH groups of glycogen while the band at
1155 cm
is attributed to the ν(C O) stretching vibra-
tions [24]. Spectra of tumor tissue clearly show a higher
intensity at 1025 and 1155 cm
. It is known that kidney
tumor cells tend to store glycogen in their cytosol
[25]. Generally, the stronger absorption signals arise
because tumor cells have a higher demand of energy than
normal cells because of their fast proliferation.
In case of clear cell renal cell carcinoma tumors, the
amount of fructose 1,6-bisphosphatase is decreased [25, 26].
Tumor cells express less of fructose 1,6-bisphosphatase thus
reinforces Warburg-like metabolic shift. Decreased amount
of this enzyme is associated with changed cellular meta-
bolic processes and increased amount of glycolytic flux in
tumorous cells as fructose 1,6-bisphosphatase antagonizes
TABLE 1 Diagnosed types of kidney tumors
Number of
Clear cell renal cell carcinoma 22
Clear cell renal cell carcinoma,
retention cysts
Papillary renal cell carcinoma 1
Papillary urothelial carcinoma 1
Chromophobe renal cell carcinoma 3
Chromophobe renal cell carcinoma,
retention cysts
Oncocytoma, retention cysts 1
SDHB renal cell carcinoma 1
Abbreviation: SDHB, succinate dehydrogenase deficient.
FIGURE 3 A, Spectra of normal (green) and tumor tissue
(red). Spectra were preprocessed as described and area
normalized. B, Plot of the mean (μ) spectra (bold) and standard
deviation (δ) bands. Differences between the spectral profiles of
tumor and normal tissue become clearly visible. C, Box whisker
plot of selected spectral regions. The three spectral regions were
identified as best positions by a supervised classification algorithm
as described elsewhere [27]
the glycolytic flux and inhibits the nuclear function of
HIF-αmetabolic regulator [25, 26]. The band located at
1240 cm
is assigned to νPO
asymmetric stretching
vibrations and to the amide III mode of proteins
[24]. Hereby, the decreased absorbance value of this band
possibly could be related with lower concentration of
fructose 1,6-bisphosphatase.
The optimization classification procedure of the algo-
rithm selects the best number of spectral bands and their
spectral positions. In respect to a practical use, in particu-
lar that the surgeon has to define the diagnosis based on
the intensity of spectral marker bands, the maximum
number of selected bands used for the genetic algorithm
was set to five. An optimized classification result could
be obtained by three selected bands. When the algorithm
involves more than three bands the accuracy of the train-
ings set becomes not better and the risk of an over deter-
mination increases. Therefore, a leave-one-out validation
was used to avoid an over-determination by too many
classifiers. It has to be noted that all three bands are nec-
essary for a successful classification and all bands have
the same importancefor the classification result or
diagnosis, respectively. The optimal separation between
normal and tumor tissue was determined by linear dis-
criminant analysis. Thresholds of absorbance values
(A) are listed in Table 3. It has to be noted that the
defined thresholds are referred to area normalized spec-
tra as described above.
The absorbance values were determined and plotted
in Figure 4. After tissue discrimination according to the
defined thresholds, all normal tissue samples were identi-
fied correctly. Then, 27 of 34 kidney tumor samples were
correctly classified as tumor tissues. In two cases (#8 and
#33) tumor tissue and partly normal tissue exhibit absor-
bance values of the other, wrong tissue class. These two
cases are diagnosed as papillary renal cell carcinoma and
succinate dehydrogenase-deficient renal cell carcinoma
with retention cysts. Only single cases of papillary renal
cell carcinoma and succinate dehydrogenase-deficient
renal cell carcinoma with retention cysts were observed
during the study, while the most frequent type of kidney
tumors is clear cell renal cell carcinoma. Due to specific
biochemical processes in different tumor types, different
spectral markers are required for discrimination of vari-
ous tumor types.
In three cases (patients #7, #27 and #29), tumorous
tissue samples were classified as questionable. In these
cases, patients were diagnosed with chromophobe renal
cell carcinoma (patient #29), chromophobe renal cell car-
cinoma with retention cysts (patient #27) and clear cell
renal cell carcinoma with retention cysts (patient #7).
Specific morphological features of chromophobe renal
cell carcinoma may produce specific IR spectra that dis-
criminate this tumor type from other kidney tumors.
During the study 50% (patient #17 and #22) of chromo-
phobe renal cell carcinoma samples were identified as
tumorous tissue and 50% (patients #29 and #27) as ques-
tionable tissue. It could be linked to the fact that chromo-
phobe renal cell carcinoma morphologically has classic
and eosinophilic types. The latter has significant overlap
with oncocytoma and often poses a diagnostic problem.
The basic chromophobe cell type is characterized by large
polygonal cells with a transparent, slightly reticulated
cytoplasm with prominent cell membranes leading to a
plant cell-like appearance. Electron microscopically, the
cytoplasm is crowded by loose glycogen deposits and
numerous, sometimes invaginated and studded vesicles.
The second cell type in chromophobe renal cell carci-
noma is also characterized by an increased cytoplasmic
eosinophilia or granularity, due to an augmentation of
mitochondria. Both cell types can occur singly or in com-
bination within a given tumor. On the assumption that
in one part of chromophobe renal cell carcinoma tumors,
the amount of glycogen is altered, those tissues could be
classified as tumorous tissue as in case of clear cell renal
cell carcinoma which has specific biochemical feature of
increased amount of lipids and glycogen. In case when
loose glycogen is not apparent in the cells, it leads to the
misclassification of tissue. Chromophobe renal cell carci-
noma is infrequently occurring type of kidney tumors
TABLE 2 IR absorption spectral bands of kidney tissue in the
spectral region from 950 to 1350 cm
and their
assignments [2023]
Spectral position (cm
) Assignment
1025 ν(C O), ν(C C), δ(C O)
1045 ν(C O), δ(C OH)
1080 ν
1155 ν(C O)
1164 ν(C C), ν(C O), δ(C OH)
1205 ν(C O C), ν(C O), amide III
1240 ν
1270 CH
1332 CH
Abbreviation: IR, infrared.
TABLE 3 Defined thresholds of absorbance values (A)to
discriminate tumor from normal tissue
Spectral position (cm
) Tumor Normal
1025 A0.0064 A< 0.0064
1155 A0.005 A< 0.005
1240 A0.01 A< 0.01
and different spectral markers are required for tissue dis-
crimination. For the more detailed conclusions, more
cases of chromophobe renal cell carcinoma should be
In two cases (patients #2 and #26), tissue samples
were identified as suspected to be tumorous. In these
cases, patients were diagnosed with clear cell renal cell
carcinoma with retention cysts. In most cases, when the
retention cysts are present, tissues are not classified as
tumorous. The presence of retention cysts disables to rec-
ognize the tissue type according to changed concentra-
tions of biochemical components that could be assigned
as markers of cancer.
The results indicate that fiber ATR IR spectroscopy
could be used to aid clinical differentiation of normal
and tumor kidney tissue in a fast and sensitive way
during live surgery. It should be noted that the presen-
tation of spectral markers values in the form of labora-
tory data, without computer-based classification,
enables a real clinical application of the spectroscopic
approach. The final goal of such spectroscopic approach
is to detect the hard-to-see borderlines under
intraoperative conditions, because an incomplete
removal of the tumor is linked to recurrences which
dramatically reduce the prognosis of the patient. This is
the preclinical trial, so validation by a larger sample set
is the next step followed by an adaption of the fiber
optic probe for in situ applications.
The initial results of this study are promising and
demonstrate that the core idea is rather round.
In conclusion, this work has shown that fiber ATR IR
spectroscopy is suitable to obtain meaningful spectra of
normal and tumorous kidney tissues. The spectra of both
types of tissue contain enough information for the tissue
discrimination. Spectral bands at 1025 and 1155 cm
assigned to glycogen and the band at 1240 cm
to fructose 1,6-bisphosphatase were considered as spec-
tral markers for tumorous tissue identification. In case of
FIGURE 4 Representing spectroscopic diagnostic information similar to the standard form of laboratory-analyzed findings. The figure
shows a plot of the absorbance values at the selected spectral regions of normal (green) and tumor (red) tissue. Spectra of patients #1 to #24
were used as training set for determination of optimal spectral regions. Ten Spectra (#25 to #34) were classified as independent test set. In
this figure, the spectral data of patients #1 to #24 are reclassified
tumorous tissue of the intensity of the spectral bands,
corresponding to glycogen gets higher, while the absor-
bance of the band corresponding to fructose
1,6-bisphosphatase gets lower. Concrete intensity values
of marker spectral bands used for the discrimination of
normal and tumorous tissue were defined. Identification
of tumorous tissue in case of presence of retention cysts
in it can leads to misclassification due to changed con-
centrations of biochemical components that are present
in cancerous tissue. The newly designed fiber probe could
be developed in the future; method has potential to be
moved towards intrasurgical applications for the more
efficient surgical treatment.
This research was funded by a grant SEN-16/2015 from
the Research Council of Lithuania.
The authors declare no financial or commercial conflict
of interest.
F. J., A. L. and D. D. were involved in sample preparation
and histopathological analysis, V. S. was involved in con-
ceptualization and project management, G. S. was
involved in conceptualization, statistical analysis, writing
and editing, R. B. was involved in investigation and writ-
ing and J. C., M. V. and V. U. were involved in the experi-
ment setup.
Data can be requested from the authors.
Gerald Steiner
[1] R. K. Sahu, S. Mordechai, Future Oncol. 2005,1, 635.
[2] D. P. Lau, Z. Huang, H. Lui, C. S. Man, K. Berean,
M. D. Morrison, H. Zeng, Lasers Surg. Med. 2003,32, 210.
[3] F. M. Lyng, D. Traynor, T. N. Q. Nguyen, A. D. Meade,
F. Rakib, R. Al-Saady, E. Goormaghtigh, K. Al-Saad,
M. H. Ali, PLoS One 2019,14, e0212376.
[4] C. Krafft, J. Popp, J. Anal. Bioanal. Chem. 2015,407, 699.
[5] G. Bellisola, C. Sorio, Am. J. Cancer Res. 2012,2,1.
[6] S. E. Taylor, K. T. Cheung, I. I. Patel, J. Trevisan,
H. F. Stringfellow, K. M. Ashton, N. J. Wood, P. J. Keating,
P. L. Martin-Hirsch, F. L. Martin, Br. J. Cancer 2011,104, 790.
[7] X. Sun, Y. Xu, J. Wu, Y. Zhang, K. Sun, J. Surg. Res. 2013,
179, 33.
[8] S. Kumar, A. Srinivasan, F. Nikolajeff, Curr. Med. Chem. 2018,
25, 1055.
[9] S. Tamosaityte, V. Hendrixson, A. Zelvys, R. Tyla,
Z. A. Kucinskiene, F. Jankevicius, M. Pucetaite,
V. Jablonskiene, V. Sablinskas, J. Biomed. Opt. 2013,18, 027011.
[10] M. Pucetaite, S. Tamosaityte, A. Engdahl, J. Ceponkus,
V. Sablinskas, P. Uvdal, Cent. Eur. J. Chem. 2014,12, 44.
[11] O. Kleiner, J. Ramesh, M. Huleihel, B. Cohen, K. Kantarovich,
C. Levi, B. Polyak, R. S. Marks, J. Mordehai, Z. Cohen,
S. Mordechai, BMC Gastroenterol. 2002,2, 2.
[12] C. B. J. Ha, S. Park, Biomater. Res. 2018,22, 22.
[13] J. R. Hands, G. Clemens, R. Stables, K. Ashton, A. Brodbelt,
C. Davis, T. P. Dawson, M. D. Jenkinson, R. W. Lea,
C. Walker, M. J. Baker, J. Neurooncol 2016,127, 463.
[14] A. Takamura, K. Watanabe, T. Akutsu, T. Ozawa, Sci. Rep.
2018,8, 8459.
[15] K. V. Oliver, A. Vilasi, A. Maréchal, S. H. Moochhala,
R. J. Unwin, P. R. Rich, Sci. Rep. 2016,6, 34737.
[16] V. Sablinskas, V. Urboniene, J. Ceponkus, A. Laurinavivius,
D. Dasevicius, F. Jankevicius, V. Hendrixson, E. Koch,
G. Steiner, J. Biomed. Opt. 2011,16, 096006.
[17] V. Urboniene, M. Pucetaite, F. Jankevicius, A. Zelvys,
V. Sablinskas, G. Steiner, J. Biomed. Opt. 2014,19, 087005.
[18] V. Sablinskas, M. Velicka, M. Pucetaite, V. Urboniene,
J. Ceponkus, R. Bandzeviciute, F. Jankevicius, T. Sakharova,
O. Bibikova, G. Steiner, Proc. SPIE 2018,10497, 1049713.
[19] A. E. Nikulin, B. Dolenko, T. Bezabeh, R. L. Somorjai, NMR
Biomed. 1998,11, 209.
[20] G. I. Dovbeshko, N. Y. Gridina, E. B. Kruglova,
O. P. Pashchuk, Talanta 2000,53, 1, 233.
[21] G. Socrates, Infrared and Raman Characteristic Group Fre-
quencies, Wiley, West Sussex 2001.
[22] G. I. Dovbeshko, V. I. Chegel, N. Y. Gridina, O. P. Repnytska,
Y. M. Shirshov, V. P. Tryndiak, I. M. Todor, G. I. Solyanik, Bio-
polymers 2002,67, 470.
[23] M. Jackson, H. H. Mantsch, Pathology by Infrared and Raman
Spectroscopy, Handbook of Vibrational Spectroscopy, John
Wiley & Sons, Hoboken, New Jersey, 2006.
[24] Z. Movasaghi, S. Rehman, I. U. Rehman, Appl. Spectrosc. Rev.
2008,43, 134.
[25] I. J. Frew, H. Moch, Annu. Rev. Pathol. 2015,10, 263.
[26] Y. Xiao, D. Meierhofer, Int. J. Mol. Sci. 2019,20, 3672.
[27] G. Steiner, S. Küchler, A. Hermann, E. Koch, R. Salzer,
G. Schackert, M. Kirsch, Cytometry A 2008,73A, 1158.
Additional supporting information may be found online
in the Supporting Information section at the end of this
How to cite this article: Sablinskas V,
Bandzeviciute R, Velicka M, et al. Fiber attenuated
total reflection infrared spectroscopy of kidney
tissue during live surgery. J. Biophotonics. 2020;
... Details of the system are described elsewhere. 17,18 The cone-shaped Ge ATR crystal tip was pressed onto the sample tissue during the measurement covering a surface of ∼5 mm 2 . Before each measurement, tip cleaning was performed by wiping the tip surface by a soft cotton swab dampened with distilled water followed by ethanol. ...
Full-text available
Significance: Pancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. Frozen section analysis for intraoperative evaluation of tissue is still time consuming and laborious. Aim: We describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous, and pancreatitis tissue. A pilot study for the intraoperative application was performed. Approach: The method was applied for unprocessed freshly resected tissue samples of 58 patients, and a classification model for differentiating between the distinct tissue classes was established. Results: The developed three-class classification model for tissue spectra allows for the delineation of tumors from normal and pancreatitis tissues using a probability score for class assignment. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room. Conclusion: Our study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.
... Some authors report results of application of IR spectroscopy for human BC investigation, however, most of them cover applications for specially prepared tissue samples such as formalin-fixed and paraffin-embedded tissues [20,21] or cultured cell lines fixed in glutaraldehyde in Phosphate-Buffered Saline (PBS) [22]. In our previous studies we showed that fiber-based IR spectroscopy can be successfully applied for examination of various freshly resected human tissues [23,24]. Here we represent the study of 54 patients by using fiber-based IR spectroscopy to delineate normal and tumorous human bladder tissues under ex vivo conditions. ...
Full-text available
Surgical treatment is widely applied curative approach for bladder cancer. White light cystoscopy (WLC) is currently used for intraoperative diagnostics of malignant lesions but has relatively high false-negative rate. Here we represent an application of label free fiber-based attenuated total reflection infrared spectroscopy (ATR IR) for freshly resected human bladder tissue examination for 54 patients. Defined molecular spectral markers allow to identify normal and urothelial carcinoma tissues. While methods of statistical analysis (Hierarchical cluster analysis (HCA) and Principal component analysis (PCA)) used for spectral data treatment allow to discriminate tissue types with 91% sensitivity and 96–98% specificity. In the present study the described method was applied for tissue examination under ex vivo conditions. However, after method validation the equipment could be translated from laboratory studies to in situ or even in vivo studies in operating room.
... Mert et al. [95] employed SERS (830 nm) to examine normal and abnormal homogenized tissue samples collected from 40 patients at different cancer stages. In a range of different diagnostic comparisons, the study demonstrated sensitivity, specificity, and total accuracy as high as 100% Sablinskas et al. [96] employed the less explored technique of Fiber ATR-IR to examine fresh kidney tissue, resected from patients undergoing surgery. Spectra of tissue were measured inside the operation theater, immediately after resection, using an ATR silver halide fiber probe. ...
Full-text available
Analytical technologies that can improve disease diagnosis are highly sought after. Current screening/diagnostic tests for several diseases are limited by their moderate diagnostic performance, invasiveness, costly and laborious methodologies or the need for multiple tests before a definitive diagnosis. Spectroscopic techniques, including infrared (IR) and Raman, have attracted great interest in the medical field, with applications expanding from early disease detection to monitoring and real-time diagnosis. This review highlights applications of IR and Raman spectroscopy, with a focus on cancer and infectious diseases since 2015, and underscores the diverse sample types that can be analyzed, such as biofluids, cells and tissues. Studies involving more than 25 participants per group (disease and control group; if no control group >25 in disease group) were considered eligible, to retain the clinical focus of the paper. Following literature searches, we identified 94 spectroscopic studies on different cancers and 30 studies on infectious diseases. The review suggests that such technologies have the potential to develop into an objective, inexpensive, point-of-care test or facilitate disease diagnosis and monitoring. Up-to-date considerations for the implementation of spectroscopic techniques into a clinical setting, health economics and successful applications of vibrational spectroscopic tests in the clinical arena are also discussed.
Fourier Transform Infrared (FTIR) Spectroscopy, in particular ATR-FTIR, is a widely used technique that allows, in a very short time, to screen biological samples and to identify its specific spectral signature, being an important tool for clinical diagnosis and biomarker discovery. FTIR spectroscopy is used to screen cells, tissues and biofluids and is already implemented in biomedicine, mainly in pre-clinical setting. Although the experimental procedure is easy to implement, sample preparation, definition of spectra acquisition parameters and spectral analysis are crucial steps to obtain reliable and reproducible results. However, the selection of experimental conditions for spectral analysis can be a difficult task for researchers because the information is dispersed and often the choice is made in an empirical and inaccurate way. This review gathers and summarizes studies using FTIR in pre-clinical and clinical setting and aims to systematize information and propose some guidelines for FTIR spectroscopy studies of biological samples. This will help new users to prepare samples for FTIR analysis and understand the critical steps to correctly perform spectra pretreatment, preprocessing and statistical analysis and to implement appropriate and evidence-based experimental designs.
Full-text available
A significantly increased level of the reactive oxygen species (ROS) scavenger glutathione (GSH) has been identified as a hallmark of renal cell carcinoma (RCC). The proposed mechanism for increased GSH levels is to counteract damaging ROS to sustain the viability and growth of the malignancy. Here, we review the current knowledge about the three main RCC subtypes, namely clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC), at the genetic, transcript, protein, and metabolite level and highlight their mutual influence on GSH metabolism. A further discussion addresses the question of how the manipulation of GSH levels can be exploited as a potential treatment strategy for RCC.
Full-text available
Breast cancer is the most common cancer among women worldwide, with an estimated 1.7 million cases and 522,000 deaths in 2012. Breast cancer is diagnosed by histopathological examination of breast biopsy material but this is subjective and relies on morphological changes in the tissue. Raman spectroscopy uses incident radiation to induce vibrations in the molecules of a sample and the scattered radiation can be used to characterise the sample. This technique is rapid and non-destructive and is sensitive to subtle biochemical changes occurring at the molecular level. This allows spectral variations corresponding to disease onset to be detected. The aim of this work was to use Raman spectroscopy to discriminate between benign lesions (fibrocystic, fibroadenoma, intraductal papilloma) and cancer (invasive ductal carcinoma and lobular carcinoma) using formalin fixed paraffin preserved (FFPP) tissue. Haematoxylin and Eosin stained sections from the patient biopsies were marked by a pathologist. Raman maps were recorded from parallel unstained tissue sections. Immunohistochemical staining for estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2/neu) was performed on a further set of parallel sections. Both benign and cancer cases were positive for ER while only the cancer cases were positive for HER2. Significant spectral differences were observed between the benign and cancer cases and the benign cases could be differentiated from the cancer cases with good sensitivity and specificity. This study has shown the potential of Raman spectroscopy as an aid to histopathological diagnosis of breast cancer, in particular in the discrimination between benign and malignant tumours. © 2019 Lyng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Full-text available
Background Gallstones have conventionally been classified by gross inspection into 4 categories: cholesterol gallstones, black pigment (calcium bilirubinate) gallstones, brown gallstones, and mixed gallstones that contain both cholesterol and calcium bilirubinate. Classification using Fourier-transform infrared (FT-IR) spectroscopy supplements gross inspection; however, the issue of ambiguity in gallstone classification has not been fully addressed to date. Methods Twenty-six gallstones obtained after surgical gallbladder removal were examined using FT-IR spectroscopy and digital photography, and classified into 6 gallstone groups according to characteristic FT-IR absorption bands. Results FT-IR spectra of nine gallstones matched well with that of pure cholesterol, and the gallstones were thus classified as cholesterol stones. Twelve gallstones were classified as calcium bilirubinate stones as they showed characteristic absorption bands of calcium bilirubinate. However, the FT-IR spectra of these gallstones always showed a broad absorption band of bound water at 3600–2400 cm− 1. The other five gallstones were classified as mixed stones with combinations of cholesterol, calcium bilirubinate, and calcium carbonate. Conclusion FT-IR spectroscopy is a powerful and convenient method for gallstone classification. Nevertheless, one should take serious note of the superposition of FT-IR absorption bands of different chemical components of gallstones including that of bound water.
Full-text available
Body fluid (BF) identification is a critical part of a criminal investigation because of its ability to suggest how the crime was committed and to provide reliable origins of DNA. In contrast to current methods using serological and biochemical techniques, vibrational spectroscopic approaches provide alternative advantages for forensic BF identification, such as non-destructivity and versatility for various BF types and analytical interests. However, unexplored issues remain for its practical application to forensics; for example, a specific BF needs to be discriminated from all other suspicious materials as well as other BFs, and the method should be applicable even to aged BF samples. Herein, we describe an innovative modeling method for discriminating the ATR FT-IR spectra of various BFs, including peripheral blood, saliva, semen, urine and sweat, to meet the practical demands described above. Spectra from unexpected non-BF samples were efficiently excluded as outliers by adopting the Q-statistics technique. The robustness of the models against aged BFs was significantly improved by using the discrimination scheme of a dichotomous classification tree with hierarchical clustering. The present study advances the use of vibrational spectroscopy and a chemometric strategy for forensic BF identification.
Full-text available
Background: Cancer is a major global health issue. It causes extensive individual suffering and gives a huge burden on the health care in society. Despite extensive research and different tools have been developed it still remains a challenge for early detection of this disease. FTIR imaging has been used to diagnose and differentiate the molecular differences between normal and diseased tissues. Methods: Fourier Transform Infrared Spectroscopy (FTIR) is able to measure biochemical changes in tissue, cell and biofluids based on the vibrational signature of their components. This technique enables to the distribution and structure of lipids, proteins, nucleic acids as well as other metabolites. These differences depended on the type and the grade of cancer. Results: We emphasize here, that the FTIR spectroscopy and imaging can be considered as a promising technique and will find its place on the detection of this dreadful disease because of high sensitivity, accuracy and inexpensive technique. Now the medical community started using and accepting this technique for early stage cancer detection. We discussed this technique and the several challenges in its application for the diagnosis of cancer in regards of sample preparations, data interpretation, and data analysis. The sensitivity of chemotherapy drugs on individual specific has also discussed. Conclusion: So far progressed has done with the FTIR imaging in understanding of cancer disease pathology. However, more research is needed in this field and it is necessary to understand the morphology and biology of the sample before using the spectroscopy and imaging because invaluable information to be figured out.
Full-text available
Cystinuria is the commonest inherited cause of nephrolithiasis (~1% in adults; ~6% in children) and is the result of impaired cystine reabsorption in the renal proximal tubule. Cystine is poorly soluble in urine with a solubility of ~1 mM and can readily form microcrystals that lead to cystine stone formation, especially at low urine pH. Diagnosis of cystinuria is made typically by ion-exchange chromatography (IEC) detection and quantitation, which is slow, laboursome and costly. More rapid and frequent monitoring of urinary cystine concentration would significantly improve the diagnosis and clinical management of cystinuria. We used attenuated total reflection - Fourier transform infrared spectroscopy (ATR-FTIR) to detect and quantitate insoluble cystine in 22 cystinuric and 5 healthy control urine samples. Creatinine concentration was also determined by ATR-FTIR to adjust for urinary concentration/dilution. Urine was centrifuged, the insoluble fraction re-suspended in 5 μL water and dried on the ATR prism. Cystine was quantitated using its 1296 cm⁻¹ absorption band and levels matched with parallel measurements made using IEC. ATR-FTIR afforded a rapid and inexpensive method of detecting and quantitating insoluble urinary cystine. This proof-of-concept study provides a basis for developing a high-throughput, cost-effective diagnostic method for cystinuria, and for point-of-care clinical monitoring
Full-text available
The ability to diagnose cancer rapidly with high sensitivity and specificity is essential to exploit advances in new treatments to lead significant reductions in mortality and morbidity. Current cancer diagnostic tests observing tissue architecture and specific protein expression for specific cancers suffer from inter-observer variability, poor detection rates and occur when the patient is symptomatic. A new method for the detection of cancer using 1 μl of human serum, attenuated total reflection—Fourier transform infrared spectroscopy and pattern recognition algorithms is reported using a 433 patient dataset (3897 spectra). To the best of our knowledge, we present the largest study on serum mid-infrared spectroscopy for cancer research. We achieve optimum sensitivities and specificities using a Radial Basis Function Support Vector Machine of between 80.0 and 100 % for all strata and identify the major spectral features, hence biochemical components, responsible for the discrimination within each stratum. We assess feature fed-SVM analysis for our cancer versus non-cancer model and achieve 91.5 and 83.0 % sensitivity and specificity respectively. We demonstrate the use of infrared light to provide a spectral signature from human serum to detect, for the first time, cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and the organ of origin of metastatic disease from the same sample enabling stratified diagnostics depending upon the clinical question asked. Electronic supplementary material The online version of this article (doi:10.1007/s11060-016-2060-x) contains supplementary material, which is available to authorized users.
A critical review is presented on the use of linear and nonlinear Raman microspectroscopy in biomedical diagnostics of bacteria, cells, and tissues. This contribution is combined with an overview of the achievements of our research group. Linear Raman spectroscopy offers a wealth of chemical and molecular information. Its routine clinical application poses a challenge due to relatively weak signal intensities and confounding overlapping effects. Nonlinear variants of Raman spectroscopy such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have been recognized as tools for rapid image acquisition. Imaging applications benefit from the fact that contrast is based on the chemical composition and molecular structures in a label-free and nondestructive manner. Although not label-free, surface enhanced Raman scattering (SERS) has also been recognized as a complementary biomedical tool to increase sensitivity. The current state of the art is evaluated, illustrative examples are given, future developments are pointed out, and important reviews and references from the current literature are selected. The topics are identification of bacteria and single cells, imaging of single cells, Raman activated cell sorting, diagnosis of tissue sections, fiber optic Raman spectroscopy, and progress in coherent Raman scattering in tissue diagnosis. The roles of networks-such as Raman4clinics and CLIRSPEC on a European level-and early adopters in the translation, dissemination, and validation of new methods are discussed.
The von Hippel-Lindau (VHL) tumor suppressor gene is mutated as an early event in almost all cases of clear cell renal cell carcinoma (ccRCC), the most frequent form of kidney cancer. In this review we discuss recent advances in understanding how dysregulation of the many hypoxia-inducible factor α-dependent and -independent functions of the VHL tumor suppressor protein (pVHL) can contribute to tumor initiation and progression. Recent evidence showing extensive inter- and intratumoral genetic diversity has given rise to the idea that ccRCC should actually be considered as a series of molecularly related, yet distinct, diseases defined by the pattern of combinatorial genetic alterations present within the cells of the tumor. We highlight the range of genetic and epigenetic alterations that recur in ccRCC and discuss the mechanisms through which these events appear to function cooperatively with a loss of pVHL function in tumorigenesis. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 10 is January 24, 2015. Please see for revised estimates.