Laser trap ionization for identification of human erythrocytes with variable hemoglobin quantitation

Michele Kelley; James Cooper; Daniel Devito; Robert Mushi; Maria del Pilar Aguinaga; Daniel B. Erenso

An approach to an established technique that is potentially applicable for a more comprehensive understanding of the electrical properties of red blood cells (RBCs) is presented. Using a high-intensity gradient laser trap, RBCs can be singly trapped and consequentially ionized. The subsequent dynamics of the ionized cell allows one to calculate the charge developed and the ionization energy (IE) through a Newtonian-based analysis. RBCs with two different hemoglobin (Hb) types were ionized. The first sample was identified as carrying Hb HbAA (normal Hb) and the second one was identified as carrying HbAC (HbC trait). By analyzing the charge developed on each cell and several other related factors, we were able to discern a difference between the main Hb types contained within the individual RBC, independent of cell size. A relationship between the charge developed and the IE of the cell was also established based on the electrical properties of RBCs. Thus, we present this laser trapping technique as a study of the electrical properties of RBCs and as possible biomedical tool to be used for the differentiation of Hb types.

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Changes in hemoglobin–oxygen affinity with shape variations of red blood cells

Aniket Chowdhury; Raktim Dasgupta; Shovan K. Majumder

Shape variations of red blood cells (RBCs) are known to occur upon exposure to various drugs or under diseased conditions. The commonly observed discocytic RBCs can be transformed to echinocytic or stomatocytic shape under such conditions. Raman spectra of the three major shape variations, namely discocyte, echinocyte, and stomatocyte, of RBCs were studied while subjecting the cells to oxygenated and deoxygenated conditions. Analysis of the recorded spectra suggests an increased level of hemoglobin (Hb)–oxygen affinity for the echinocytes. Also, some level of Hb degradation could be noticed for the deoxygenated echinocytes. The effects may arise from a reduced level of intracellular adenosine triphosphate in echinocytic cells and an increased fraction of submembrane Hb.

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Assessment of the “cross-bridge”-induced interaction of red blood cells by optical trapping combined with microfluidics

Kisung Lee; Christian Wagner; Alexander V. Priezzhev

Red blood cell (RBC) aggregation is an intrinsic property of the blood that has a direct effect on the blood viscosity and circulation. Nevertheless, the mechanism behind the RBC aggregation has not been confirmed and is still under investigation with two major hypotheses, known as “depletion layer” and “cross-bridging.” We aim to ultimately understand the mechanism of the RBC aggregation and clarify both models. To measure the cell interaction in vitro in different suspensions (including plasma, isotonic solution of fibrinogen, isotonic solution of fibrinogen with albumin, and phosphate buffer saline) while moving the aggregate from one solution to another, an approach combining optical trapping and microfluidics has been applied. The study reveals evidence that RBC aggregation in plasma is at least partly due to the cross-bridging mechanism. The cell interaction strength measured in the final solution was found to be significantly changed depending on the initial solution where the aggregate was formed.

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Poly(3-hydroxybutyrate) anabolism in Cupriavidus necator cultivated at various carbon-to-nitrogen ratios: insights from single-cell Raman spectroscopy

Zhanhua Tao ; Pengfei Zhang ; Zhaojun Qin ; Yong-Qing Li ; Guiwen Wang

Cupriavidus necator accumulates large amounts of poly(3-hydroxybutyrate) (PHB), a biodegradable substitute for petroleum-based plastics, under certain nutrient conditions. Conventional solvent-extraction-based methods for PHB quantification only obtain average information from cell populations and, thus, mask the heterogeneity among individual cells. Laser tweezers Raman spectroscopy (LTRS) was used to monitor dynamic changes in the contents of PHB, nucleic acids, and proteins in C. necator at the population and single-cell levels when the microorganism cells were cultivated at various carbon-to-nitrogen ratios. The biosynthetic activities of nucleic acids and proteins were maintained at high levels, and only a small amount of PHB was produced when the bacterial cells were cultured under balanced growth conditions. By contrast, the syntheses of nucleic acids and proteins were blocked, and PHB was accumulated in massive amount inside the microbial cells under nitrogen-limiting growth circumstances. Single-cell analysis revealed a relatively high heterogeneity in PHB level at the early stage of the bacterial growth. Additionally, bacterial cells in populations at certain cultivation stages were composed of two or three subpopulations on the basis of their PHB abundance. Overall, LTRS is a reliable single-cell analysis tool that can provide insights into PHB fermentation.

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Damage induced in red blood cells by infrared optical trapping: an evaluation based on elasticity measurements

Marcos A. S. de Oliveira ; Diógenes S. Moura ; Adriana Fontes ; Renato E. de Araujo

We evaluated the damage caused to optically trapped red blood cells (RBCs) after 1 or 2 min of exposure to near-infrared (NIR) laser beams at 785 or 1064 nm. Damage was quantified by measuring cell elasticity using an automatic, real-time, homemade, optical tweezer system. The measurements, performed on a significant number (hundreds) of cells, revealed an overall deformability decrease up to ∼104%∼104% after 2 min of light exposure, under 10 mW optical trapping for the 785-nm wavelength. Wavelength dependence of the optical damage is attributed to the light absorption by hemoglobin. The results provided evidence that RBCs have their biomechanical properties affected by NIR radiation. Our findings establish limits for laser applications with RBCs.

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Effect of neighboring cells on cell stiffness measured by optical tweezers indentation

Muhammad S. Yousafzai ; Giovanna Coceano ; Alberto Mariutti ; Fatou Ndoye ; Ladan Amin ; Joseph Niemela ; Serena Bonin ; Giacinto Scoles ; Dan Cojoc

We report on the modification of mechanical properties of breast cancer cells when they get in contact with other neighboring cells of the same type. Optical tweezers vertical indentation was employed to investigate cell mechanics in isolated and contact conditions, by setting up stiffness as a marker. Two human breast cancer cell lines with different aggressiveness [MCF-7 (luminal breast cancer) and MDA-MB-231 (basal-like breast cancer)] and one normal immortalized breast cell line HBL-100 (normal and myoepithelial) were selected. We found that neighboring cells significantly alter cell stiffness: MDA-MB-231 becomes stiffer when in contact, while HBL-100 and MCF-7 exhibit softer character. Cell stiffness was probed at three cellular subregions: central (above nucleus), intermediate (cytoplasm), and near the leading edge. In an isolated condition, all cells showed a significant regional variation in stiffness: higher at the center and fading toward the leading edge. However, the regional variation becomes statistically insignificant when the cells were in contact with other neighboring cells. The proposed approach will contribute to understand the intriguing temporal sequential alterations in cancer cells during interaction with their surrounding microenvironment.

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Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions

Kisung Lee ; Matti Kinnunen ; Maria D. Khokhlova ; Evgeny V. Lyubin ; Alexander V. Priezzhev ; Igor Meglinski ; Andrey A. Fedyanin

Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

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Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy

Surekha Barkur; Aseefhali Bankapur; Madhura Pradhan; Santhosh Chidangil; Deepak Mathur; Uma Ladiwala

Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O−P−O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.

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Different Raman spectral patterns of primary rat pancreatic β cells and insulinoma cells

Qiu-Li Zhou ; Xi Rong ; Fang Wei ; Rui-Qiong Luo ; Hong Liu

As a noninvasive and label-free analytical technique, Raman spectroscopy has been widely used to study the difference between malignant cells and normal cells. Insulinomas are functional β-cell tumors of pancreatic islet cells. They exhibit many structural and immunohistochemical features in common with normal pancreatic β cells; thus, they are typically difficult to distinguish under the microscope, especially in vivo. We investigated insulinoma and primary rat pancreatic β-cell populations using Raman spectroscopy. The details of the optical heterogeneity between these two populations were determined based on different Raman regions primarily involving nucleic acid and protein contents, which are the most distinct cellular contents in these two types of cells. Using principal component analysis–linear discriminant analysis, these two cell types can be readily separated. The results of this work indicate that Raman spectroscopy is a promising tool for the noninvasive and label-free differentiation of insulinoma cells and normal pancreatic β cells.

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Overview of single-cell elastic light scattering techniques

Matti Kinnunen; Artashes Karmenyan

We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed.

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Identification of individual biofilm-forming bacterial cells using Raman tweezers

Ota Samek ; Silvie Bernatová ; Jan Ježek ; Martin Šiler ; Mojmir Šerý ; Vladislav Krzyžánek ; Kamila Hrubanová ; Pavel Zemánek ; Veronika Holá ; Filip Růžička

A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

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Birefringence of a normal human red blood cell and related optomechanics in an optical trap

Belavadi Venkatakrishnaiah Nagesh; Yogesha; Ramarao Pratibha; Praveen Parthasarathi; Shruthi Subhash Iyengar; Sarbari Bhattacharya; Sharath Ananthamurthy

A normal human red blood cell (RBC) when trapped with a linearly polarized laser, reorients about the electric polarization direction and then remains rotationally bound to this direction. This behavior is expected for a birefringent object. We have measured the birefringence of distortion-free RBCs in an isotonic medium using a polarizing microscope. The birefringence is confined to the cell’s dimple region and the slow axis is along a diameter. We report an average retardation of 3.5±1.5  nm for linearly polarized green light (λ=546  nm). We also estimate a retardation of 1.87±0.09  nm from the optomechanical response of the RBC in an optical trap. We reason that the birefringence is a property of the cell membrane and propose a simple model attributing the origin of birefringence to the phospholipid molecules in the lipid bilayer and the variation to the membrane curvature. We observe that RBCs reconstituted in shape subsequent to crenation show diminished birefringence along with a sluggish optomechanical response in a trap. As the arrangement of phospholipid molecules in the cell membrane is disrupted on crenation, this lends credence to our conjecture on the origin of birefringence. Dependence of the birefringence on membrane contours is further illustrated through studies on chicken RBCs.

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Comparative study of methods to calibrate the stiffness of a single-beam gradient-force optical tweezers over various laser trapping powers

Mohammad Sarshar; Winson T. Wong; Bahman Anvari

Optical tweezers have become an important instrument in force measurements associated with various physical, biological, and biophysical phenomena. Quantitative use of optical tweezers relies on accurate calibration of the stiffness of the optical trap. Using the same optical tweezers platform operating at 1064 nm and beads with two different diameters, we present a comparative study of viscous drag force, equipartition theorem, Boltzmann statistics, and power spectral density (PSD) as methods in calibrating the stiffness of a single beam gradient force optical trap at trapping laser powers in the range of 0.05 to 1.38 W at the focal plane. The equipartition theorem and Boltzmann statistic methods demonstrate a linear stiffness with trapping laser powers up to 355 mW, when used in conjunction with video position sensing means. The PSD of a trapped particle’s Brownian motion or measurements of the particle displacement against known viscous drag forces can be reliably used for stiffness calibration of an optical trap over a greater range of trapping laser powers. Viscous drag stiffness calibration method produces results relevant to applications where trapped particle undergoes large displacements, and at a given position sensing resolution, can be used for stiffness calibration at higher trapping laser powers than the PSD method.

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Polarized Raman spectroscopic investigations on hemoglobin ordering in red blood cells

Sunita Ahlawat; Aniket Chowdhury; Nitin Kumar; Abha Uppal; Ravi Shanker Verma; Pradeep Kumar Gupta

We have investigated the dependence of the Raman spectrum of an optically trapped red blood cell (RBC) on the orientation of the cell, relative to the polarization direction of the Raman excitation beam. The Raman scattered light polarized parallel to the polarization direction of the excitation beam was observed to depend upon the orientation of the cell. In particular, the heme bands at ∼754  cm−1 and in the 1500 to 1700  cm−1 region were observed to become maximum when the cells’ equatorial plane was parallel to the excitation beam polarization direction and minimum when the cells’ plane was normal to the polarization direction. In contrast, no significant orientational dependence was seen in the Raman scattered light polarized orthogonal to the polarization direction of the excitation beam. Theoretical simulations carried out to investigate these observations suggest that inside the RBCs, the hemoglobin molecules must be present in an ordered arrangement, such that heme-porphyrin planes become preferentially orientated parallel to the RBCs’ equatorial plane.

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Optical clearing at cellular level

Matti Kinnunen ; Alexander V. Bykov ; Juho Tuorila ; Tomi Haapalainen ; Artashes V. Karmenyan ; Valery V. Tuchin

Strong light scattering in tissues and blood reduces the usability of many optical techniques. By reducing scattering, optical clearing enables deeper light penetration and improves resolution in several optical imaging applications. We demonstrate the usage of optical tweezers and elastic light scattering to study optical clearing [one of the major mechanisms—matching of refractive indices (RIs)] at the single particle and cell level. We used polystyrene spheres and human red blood cells (RBCs) as samples and glycerol or glucose water solutions as clearing agents. Optical tweezers kept single microspheres and RBCs in place during the measurement of light scattering patterns. The results show that optical clearing reduces the scattering cross section and increases g. Glucose also decreased light scattering from a RBC. Optical clearing affected the anisotropy factor g of 23.25-μm polystyrene spheres, increasing it by 0.5% for an RI change of 2.2% (20% glycerol) and 0.3% for an RI change of 1.1% (13% glucose).

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Localized cell stiffness measurement using axial movement of an optically trapped microparticle

Mary-Clare Dy; Shigehiko Kanaya; Tadao Sugiura

A simple optical tweezers design is proposed to manipulate particles in the axial direction and estimate particle position with nanometer sensitivity. Balb3T3 cell is probed using two different-sized particles, and the localized cell stiffness is evaluated using Hertz model. A series of experiments are performed to obtain the necessary parameters for the cell stiffness computation: particle displacement, trapping stiffness, force exertion, and cell deformation. The computed cell stiffness measurements are 17 and 40 Pa using 4 μm- and 2 μm-sized particles, respectively. Results suggest that the proposed optical tweezers scheme can measure the stiffness of a particular cell locale using Hertz model, offering insights about how cells respond to outside mechanical stimulus.

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Laser guidance–based cell detection in a microfluidic biochip

Wan Qin; Lucas Schmidt; Xiaoqi Yang; Lina Wei; Ting Huang; Julie X. Yuan; Xiang Peng; Xiaocong Yuan; Bruce Z. Gao

We developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain neurons and spinal cord neurons. Observation of neurons under a high-magnification microscope, which we obtained from these two cell types, showed no difference in morphology. However, when flowing in the microfluidic channel and simultaneously being laser guided, the two cell types gained quite different guidance speeds under the same experimental conditions. The results demonstrate that different cell types with the same morphology (e.g., size, shape, etc.) can be effectively distinguished from each other by measuring the difference in guidance speeds (the maximum flow speed minus the initial flow speed). This technique is expected to provide a new approach to high-throughput, label-free cell sorting with high sensitivity.
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Optical micromanipulation of active cells with minimal perturbations: direct and indirect pushing

Chenlu Wang ; Sagar Chowdhury ; Satyandra K. Gupta ; Wolfgang Losert
The challenge to wide application of optical tweezers in biological micromanipulation is the photodamage caused by high-intensity laser exposure to the manipulated living systems. While direct exposure to infrared lasers is less likely to kill cells, it can affect cell behavior and signaling. Pushing cells with optically trapped objects has been introduced as a less invasive alternative, but the technique includes some exposure of the biological object to parts of the optical tweezer beam. To keep the cells farther away from the laser, we introduce an indirect pushing-based technique for noninvasive manipulation of sensitive cells. We compare how cells respond to three manipulation approaches: direct manipulation, pushing, and indirect pushing. We find that indirect manipulation techniques lessen the impact of manipulation on cell behavior. Cell survival increases, as does the ability of cells to maintain shape and wiggle. Our experiments also demonstrate that indirect pushing allows cell–cell contacts to be formed in a controllable way, while retaining the ability of cells to change shape and move.

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Purple sea urchin Strongylocentrotus purpuratus gamete manipulation using optical trapping and microfluidics

Charlie Chandsawangbhuwana ; Linda Z. Shi ; Qingyuan Zhu ; Michael W. Berns
A system has been developed that allows for optical and fluidic manipulation of gametes. The optical manipulation is performed by using a single-point gradient trap with a 40× oil immersion PH3 1.3 NA objective on a Zeiss inverted microscope. The fluidic manipulation is performed by using a custom microfluidic chamber designed to fit into the short working distance between the condenser and objective. The system is validated using purple sea urchin Strongylocentrotus purpuratus gametes and has the potential to be used for mammalian in vitro fertilization and animal husbandry.
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Orientational dynamics of human red blood cells in an optical trap

Praveen Parthasarathi; Belavadi V. Nagesh; Yogesha Lakkegowda; Shruthi S. Iyengar; Sharath Ananthamurthy; Sarbari Bhattacharya
We report here on studies of reorientation of human red blood cells (RBCs) in an optical trap. We have measured the time required, tre, for the plane of the RBC entering the optical trap to undergo a 90-deg rotation to acquire an edge on orientation with respect to the beam direction. This has been studied as a function of laser power, P, at the trap center. The variation of tre with increasing P shows an initial sharp decrease followed by a much smaller rate of further decrease. We find that this experimentally measured variation is not in complete agreement with the variation predicted by a theoretical model where the RBC is treated as a perfectly rigid circular disk-like body. We argue that this deviation arises due to deformation of the RBC. We further reason that this feature is dominated by the elastic behavior of the RBC membrane. We compare the studies carried out on normal RBCs with RBCs where varying conditions of membrane stiffness are expected. We propose that the value of energy used for maximum deformation possible during a reorientation process is an indicator of the membrane elasticity of the system under study.
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