Biosensing of Red Blood Cell-derived Extracellular Vesicles with the advanced
Bright-Field light Optical Polarization Microscopy
Alexander E. Berezin*1,2, Richard E Mokhnach3, Harry A Byalik3
1Private Clinic “Vita Center”, Zaporozhye, Ukraine
2Internal Medicine Department, Medical University, Zaporozhye, Ukraine
3Technical Department, National Technical University, Zaporozhye, Ukraine
Background: Red blood cell (RBC)-derived extracellular vesicles (EVs) are recognized a sensitive predictive biomarker of cardiovascular risk, which allow distinguishing vulnerable population from healthy and also physiological aging from premature one. Current known methods of determination of RBC-EVs are based on several principles including flow cytometry, culture methods and various visualization techniques (surface plasmon resonance and computer tomography / magnetic resonance imaging), and various types of microscopy, i.g. high sensitive optical coherent microscopy (hs-OCM), atomic microscopy, fluorescent microscopy. However, there are some limitations and broad variabilities in cost of mentioned above methods of EV-determination.
The aim of the study was to compare a capture ability of conventional hs-OCM and advanced bright-field light optical polarization microscopy in detection and measurement of the RBC-EVs.
Methods: The study was retrospectively evolved 26 patients with established stable coronary artery disease who were examined between May 2015 and November 2016. The samples of whole blood were collected before ingestion of the meal at room temperature at
the morning with powder-free gloves. We used a conventional hs-OCM and advanced bright-field light optical polarization microscopy with improved capture features. Conventional hs-OCM and advanced bright-field light optical polarization microscopy was performed with an Olympus® BH-2 microscope (Olympus, Japan). The whole sample was scanned with the Zeiss 10x objective (100x). The monochromatic laser was used to emanation of light with an appropriate wavelength. It has been identified number and relative size distribution of RBC-EVs with further analysis of morphology using original soft.
Results: hs-OCM images supported by yellow-green light allow visualizing cell-free EVs without possibilities for assay their structure and measurement of their number. In contrast, ultraviolet light-enhanced hs-OCM is able to improve capture features of RBC-EVs
including their number, diameter and roughly structure. Using advanced bright-field light optical polarization microscopy associated with original soft allows distinguishing low-contrasted objects in details when we used monochromatic light with λ=370+30 nm with further math modelling.
Conclusions: the advanced bright-field light optical polarization microscopy allows detecting clinically relevant properties of EV in wide ranges and could be determined a new much promising technique, which allows assaying EV in low cost.
Extracellular Vesicles, Red Blood Cells, Measurement, Biosensing, Optical Polarization Microscopy
Red blood cell (RBC)-derived extracellular microvesicles (EVs)
are recently recognized key regulators of cell-to-cell cooperation,
blood cell function, coagulation, and probably inflammation,
proliferation and tissue repair[1, 2]. Recent clinical studies have
shown that the elevated circulating number of RBC-EVs has found in
several cardiovascular diseases including acute coronary syndrome
/ acute myocardial infarction, pulmonary thromboembolism, acute
and chronic heart failure, fibrillation[3-5]. Additionally, a wide
range of other diseases associated with coagulopathy, thrombosis,
anemia (i.e., infections, shock, respiratory distress syndrome,
bleeding, vasculitis, preeclampsia / eclampsia, antiphospholipid
syndrome, HELP-syndrome, malignancy, rheumatic diseases, etc.)
is expressed higher circulating level of RBC-EVs due to activated
secretion or increasing RBC debris. Nowadays there is a large
body of evidence regarding that the RBC-EVs’ number could
be useful circulating predictive biomarker of clinical outcomes
in critical ill patients, individuals with cancer, established CV,
rheumatic, autoimmune and kidney diseases[7-9].
Nowadays conventional transmitted light microscopy technique is
useful and simple method to determine particle size, shape and
structure[10, 11]. A highly sensitive optical coherent microscopy
(hs-OCM) based on objective-type internal reflection regarding
wavelength-modulation may sufficiently improve RBC-EV
determination. Although hs-OCM technique has a serial limitations
for data interpretation predominantly relate to use of light dose
, this method may visualize RBC-EVs with higher accuracy
and measurement limit of 40 nm. To improve a capture ability
of hs-OCM to detect RBC-EVs advanced bright-field light optical
polarization technique might be used.
The aim of the study was to compare a capture ability of
conventional hs-OM and advanced bright-field light optical
polarization microscopy in detection and measurement of the
The study was retrospectively evolved 26 patients with established
stable coronary artery disease (positive contrast-enhanced
multispiral tomography angiography and determination of stable
angina pectoris according contemporary clinical guideline )
who were examined between May 2015 and November 2016. All
patients have given their informed written consent for participation
in the study. The study was approved by the local ethics committee
of State Medical University, Zaporozhye, Ukraine. The study was
performed in conformity with the Declaration of Helsinki
Blood collection and storing:
Blood samples were collected before ingestion of the meal at room
temperature at the morning with powder-free gloves (Latex, soft-
hand). We optionally used “Vacutainer sets” (BD, Europe) with 22
gauge needles and 3ml plastic syringes to collect 2ml of the whole
blood in the plastic tubes with EDTA. The first 2ml of blood where
discarded to prevent RBC-MC shaping due to microvascular
damage. Finally we chose a 0.1ml of whole blood placed on
one microscope slide with a cover slip for light microscopy
examination. We used a conventional hs-OCM and advanced
bright-field light optical polarization microscopy with improved
capture features and original soft for further analysis of images.
Conventional hs-OCM and advanced bright-field light optical
polarization microscopy was performed with an Olympus® BH-2
microscope (Olympus, Japan). The whole sample was scanned
with the Zeiss 10x objective (100x). Only glass particles or micro-
bubbles made the distinction difficult. In these cases we used the
40x Zeiss objective (400x) to be beyond doubt. RBC-EVs were
determined as intra RBC-shaping vesicles with diameter less 400
nm. On the actual step of optical detection of the RBC-EVs we
identified their number and relative size distribution, although the
methods allowed determining the morphology as mean shape and
ultrastructure and measure the concentration of using original soft
called advanced highly dynamic resolution capture system(R).
Advanced bright-field light optical polarization
RBC-EVs could be identified by their formation in RBCs in various
polarized lights. The limit of detection was 10 nm. We found an
optimal reflected tight attachment that sufficiently expands scope
of research through flexible combinations of polarizing light
with various wavelengths and considerably simple switchover
of multiple observation method. The monochromatic laser was
used to emanation of light with an appropriate wavelength. All
measurements were done as blinded duplicative performed by
Statistical analysis of the results obtained was performed in SPSS
system for Windows, Version 22 (SPSS Inc, Chicago, IL, USA).
The data were presented as median (Ме) and 25%-75% interquartile
range (IQR). To compare the main parameters of patient cohorts
Mann - Whitney U-test were used. The intra assay and inter assay
coefficients were calculated. A two-tailed probability value of
<0.05 was considered as significant.
Determination of cell-free EVs and EVs (arrows) at
the stage of active secretion by RBCs in yellow-green (λ=560-580
nm) light using hs-OCM.
Figure 1a and 1b are reported hs-OCM images of mono-layered
Determination of cell-free EVs and EVs (arrows) at
the stage of active secretion by RBCs in violet (λ=450-470 nm)
light using hs-OCM.
Determination of cell-free EVs and EVs (arrows)
using advanced bright-field light optical polarization microscopy
with monochromatic ultraviolet (λ=370+30 nm)
whole blood sample received from the patients. One can see
cell-free EVs and EVs at the stage of active secretion by RBCs
in yellow-green (λ=560-580 nm) and ultraviolet (λ=450-470 nm)
lights (Fig 1a and Fig 1b respectively). The difference between
both images affects capture ability regarding determination
of RBC-EVs. Indeed, hs-OCM images supported by yellow-
green light allow visualizing cell-free EVs without possibilities
for assay their structure and measurement of their number. In
contrast, ultraviolet light-enhanced hs-OCM is able to improve
capture features of RBC-EVs including their number, diameter
and roughly structure. Consequently, contemporary used hs-ECM
is not able to distinguished cell-free RBC-derived EVs in blood
samples and does not maintain much more pretty accurate capture
ability to determine RBC-EVs from debris. As a result, the prompt
to calculate the number of cell-free RBC-derived EVs using a math
model based on data received from hs-ECM-enhanced method
may lead to increased falsely-positive results.
Using advanced bright-field light optical polarization microscopy
associated with original soft allows distinguishing low-contrasted
objects in details when we used monochromatic light with
λ=370+30 nm (Fig 2a). At the figure 2b we can see EVs with diameter less 1 mcm secreting by RBCs. However, cell free RBC-
EVs and cell debris could not be distinguished with the method,
although the dynamic diapason of the advanced polarization
microscopy was higher than this that was achieved through hs-
ECM. Additionally we consequently applied ultraviolet emanation
with high sensitive polarized capture through original soft to
construct the image with improved contrasted features suitable for
analysis of shaping, number and structure of RBC-EVs (Fig 2b).
Determination of cell-free EVs and EVs (arrows)
using advanced bright-field light optical polarization microscopy
after math modelling
Scattering of EV diameter measured by both methods is reported
in Table 1. We found that advanced bright-field light optical
polarization microscopy with original soft is able to distinguish
as none-free RBC-derived EVs as well as RBC-free EVs in pretty
broad ranges of diameter. Therefore the intra assay and inter assay
coefficients which were determined as the standard deviation of a
set of measurements were found as 7.6% and 9.5% respectively.
Scattering of diameter of RBC-derived EVs measured by hs-OCM and advanced bright-field light optical polarization microscopy
In the study we first found that the advanced bright-field light
optical polarization microscopy could be an alternate free-labeled
optical method for quantified measured of sizes and size-related
characteristics of EVs derived from RBCs. Increased resolution
of new method of optical microscopy is based on interaction
of polarized light with thin film of monolayer of whole blood
with further mathematical analysis of image through mutual
superposition each next layer over previous one. All these relate to
sufficient increased capture ability and dynamic diapason extension
in a way of use the same optical magnification. This is an essential
advantage of advanced bright-field light optical polarization
microscopy in comparison with conventional hs-OCM [15, 16].
Therefore, technically we have confirmed that the advanced
bright-field light optical polarization microscopy exhibited pretty
potential to accurately obtain all clinically relevant properties of
single EV at a high speed, although reproducibility requires more
investigations in future. Created by us mathematic model has now
implemented into original soft that allow managing number and
diameter of even small-sized EVs with higher sensitivity. Finally,
advanced bright-field light optical polarization microscopy is
a low cost method of real time detection of both types of EVs
derived from RBCs (cell-free and none ell free).
These evidence may have a serious advantages regarding low
depending on capture features and quality of blood sample
preparation and refractive index of material that are considerable
critical for conventional hs-OCM[17, 18]. It is well known that
the quantity of light scattered by a single EV is proportional to the
diameter of one that should optimally be at least 10 times smaller
than the wavelength. Therefore, the value of refractive index
of prepared samples is critical to distinguish very variable in their
diameter EVs [20, 21]. Indeed, recent studies have shown that
the scattering intensity from EVs is periodically modulated by
shifting the intensity fringes of the standing evanescent field. It has
been postulated that to improve measuring contrast of scattering
intensity variation during one cycle of modulation, particle sizes
could be estimated easily [21, 22]. We have overcome these
obstacles using original soft especially created for new method
of polarized optical microscopy, which allows improving contrast
of previously unrecognizable objects. Thus, we suggest that our
method proposing first for easily determination of EV in pretty
wide rages could be deserved further investigations.
Limitations and future directions:
There is need to merge sensitivity, selectivity and reproducibility
of final detection of EVs using new method of advanced bright-
field light optical polarization microscopy with further comparison
with other routinely used analytical methods, i.e. flow cytometry
and magnetic resonance technology. However, biosensing of EVs
with advanced bright-field light optical polarization microscopy
requires standardization and more investigations in field of quality
In conclusion, advanced bright-field light optical polarization
microscopy allow detecting clinically relevant properties of EV
in wide ranges and could be determined a new much promising
technique, which allows assaying EV in low cost.
Conflict of interests:
Authors declare no conflict of interest.
We thank all patients for their participation in the investigation,
staff of the Regional Zaporozhye Hospital (Ukraine), and the
doctors, nurses, and administrative staff in Regional Center of
cardiovascular diseases (Zaporozhye, Ukraine) and Private Clinic
“Vita Center” (Zaporozhye, Ukraine), general practices, and site-
managed organizations that assisted with the study. We also thank
the staff of the Technical Department of the Zaporozhye National
Technical University (Zaporozhye, Ukraine) for preforming EV
measurements and technical assistance.
Legends for figures
Abbreviations: hs-OCM, high sensitive optical coherent
microscopy; EVs, extra vesicles; RBCs, red blood cells.
Notes: dimension between both nearly arranged lines on a top of
the figure is 5 micrometers
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