Mechanical Characterization in Red Blood Cells Using Optical Tweezers: A Review

1. Introduction

Red blood cells (RBCs), the most abundant cells in human blood, circulate throughout the vascular system and interact with diverse tissues. Therefore, their physical and biochemical properties are essential indicators for biomedical analysis and clinical diagnosis, with mechanical properties serving as key parameters reflecting cellular physiological function and pathological state. For example, high deformability enables RBCs to pass smoothly through capillaries narrower than 3 μm [1].

A number of studies have shown that abnormalities in RBC mechanical properties are closely associated with various diseases. For example, malaria infection leads to a significant increase in RBC membrane’s stiffness [2,3,4]; sickle cell anemia causes a substantial reduction in RBC deformability [5]; and RBC stiffness increases in diabetic patients [6]. This indicates that pathological conditions can significantly alter RBC mechanical properties, including deformability, shape recovery ability, elasticity, stiffness, and aggregability. Conversely, changes in these mechanical properties may serve as potential pathological markers. Because these markers provide mechanical information that cannot be replaced by conventional indicators, offering diagnostic and monitoring value, achieving accurate and quantitative measurements of RBC mechanical parameters has become an important direction in biomedical research.

Current techniques for characterizing the mechanical properties of RBCs can be broadly divided into population-based and single-cell measurements. Population-based techniques include microporous filtration, laser diffraction, microfluidics, and dielectrophoresis, while single-cell techniques include micropipette aspiration, atomic force microscopy (AFM), microfluidics, Brillouin microscopy, magnetic twisting cytometry, and optical tweezers (OT). Each technique has its own advantages and limitations, as summarized in Table 1. And detailed principles can be found in the reviews performed by Matthews et al. [7], Liang et al. [8], and Xing et al. [9]. Among these, OT enables non-invasive, contact-free, piconewton-force manipulation and quantitative measurement of stretching, rotation, and intercellular interaction forces. Due to their flexibility, high precision, minimal photothermal damage and extremely high force sensitivity, OT has become a powerful tool to reveal the microscopic mechanisms governing RBC mechanical behavior.

Recently, the number of studies using OT to investigate RBCs has grown rapidly, and several reviews have discussed the research of OT on RBCs. For instance, the review written by Zhu et al. [10]. provides a detailed introduction to the background and working principles of OT, as well as their various applications in RBC studies. However, it lacks a description of the manipulation modes of RBCs using OT and an introduction to the calculation formula of mechanical parameters. Although the work by Xie et al. [11]. introduces different manipulation modes for RBCs by OT, including controlled deformation, dynamic stretching, aggregation-disaggregation behavior, and blood cell separation, it does not cover the measurement and calculation of mechanical parameters, nor does it provide a systematic analysis of clinical translation. On the other hand, the review performed by Liang et al. [8] focuses on the analysis of RBC deformability in various disease states through different techniques. However, it does not provide a detailed introduction to OT specifically. Furthermore, it does not discuss how the integration of multiple techniques can be applied to study RBC mechanical properties.

However, as attention to OT grows in RBC research, a systematic introduction to the field is still lacking. This review provides a technical and application-focused reference centered on RBCs and OT. It systematically describes the parameters used to characterize RBC mechanical properties and the working principles of OT. Then it highlights two core manipulation modes (direct and indirect manipulation) along with sample preparation methods and the formulas for calculating mechanical parameters for each approach. The review also discusses how OT, combined with other techniques, can assess RBC mechanical properties under physiological and pathological conditions. In addition, it covers the role of OT in basic research on RBC aggregation and environmental stimuli, as well as the clinical significance of OT in blood storage, malaria, anemia, diabetes and so on. This work is expected to support the transition of this field from experimental research to clinical practice.

2. Manipulation of Red Blood Cells by Optical Tweezers

2.1. Mechanical Properties of Red Blood Cells

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The unique mechanical properties of RBCs primarily stem from their distinctive structure and composition. Mature RBCs exhibit a biconcave disc shape, with a diameter of approximately 6~8 μm, a peripheral thickness of 2~2.5 μm, and a central thickness of 0.8 μm. Their primary structure comprises three components: an outer phospholipid bilayer membrane, a submembrane hexagonal scaffold network of ankyrin-actin, and an internal high-concentration hemoglobin solution [35]. These three components work together to maintain the morphological stability of RBCs and determine their mechanical behavior.

The mechanical properties of RBCs can be quantitatively characterized through three types of parameters: macroscopic, membrane microscopic, and rheological.

• Among the macro parameters, the deformation degree of RBCs can be characterized by elongation index (EI), relative elongation ratio (ε), deformation index (DI) and deformation ratio (DR), and the overall stiffness of RBCs was characterized by Young's modulus (E). The following equations are commonly used:

The ratio of stretched RBC length to initial length is defined as elongation index EI (or elongation ratio) [36,37]:

(1)

Then the ratio of elongation to initial length is the relative elongation ratio ε:

(2)

where L₀ is the initial major axis or minor axis, and L is the major axis or minor axis after stretching. EI represents the ratio of the total length of a RBC after being stretched to its original length in a given direction, emphasizing the “overall proportion after elongation”. ε represents the ratio of the increase in length of the RBC to its original length, emphasizing the “incremental part of the deformation”. EI and ε are related by the equation ε=EI−1. In some articles, ε is also called the deformation index [38]. However, to facilitate comparative discussions of results obtained using OT, it is hoped that the nomenclature for the parameter corresponding to this formula will be standardized in the future.

In addition, if RBC is stretched into an ellipse, the DI can be obtained by classical ellipse-fitting method [16]:

(3)

where L_x is the length of RBCs along the stretching direction, and L_y is the length of RBCs in the vertical stretching direction. DI quantifies the extent to which an RBC is deformed into an elliptical shape, ranging from 0 to 1. DI=0 indicates that the RBC remains circular, whereas DI=1 represents an extremely elongated RBC. This equation is commonly used in micropipette aspiration technology, microfluidic technology where the channel size is larger than the diameter of RBCs, and laser diffraction technology.

If the RBC is deformed into a parachute shape, or the channel size is smaller than the RBC diameter, it can be characterized by deformation ratio (DR) [39]:

(4)

DR represents the ratio of the lengths of a RBC in two perpendicular directions, emphasizing the “anisotropy of the shape”.

Four parameters which are used to characterize deformability all describe changes in RBC dimensions and shape. However, RBCs exhibit different deformation behaviors depending on the deformation scenario, which in turn determines the choice of formulas and parameter ranges for quantifying the degree of deformation in the reported articles. In a sample chamber with no spatial restrictions, RBCs can be stretched into elliptical or dumbbell shapes, allowing the use of Equations (1)~(3) to characterize their deformation. In contrast, when stretched through a narrow channel, the cell membrane frequently contacts the channel walls, leading to a parachute-like shape, in which case Equation (4) is appropriate for calculating RBC deformability.

When an RBC is subjected to indentation, its overall stiffness is characterized by the Young's modulus (E) derived from the Hertz model [40]:

(5)

where F is the applied force, ν is the Poisson's ratio of the RBC, R is the radius of the spherical indenter, and h is the indentation depth. In addition, the shear modulus (μ) can be calculated from E using the formula [40]:

(6)

The Young's modulus reflects the combined contributions of the membrane skeleton, cytoplasmic viscosity, and intracellular fluid pressure [41]. It can be derived from force-distance curves. While atomic force microscopy is the main technique for measuring the Young's modulus of RBCs [27], the indentation method based on OT can also be used [3,42]. RBCs under pathological conditions often exhibit a higher Young's modulus than healthy cells, such as diabetes [43], sickle cell disease [44], and malaria [3]. In addition, RBCs with distinct morphologies also differ in their Young's modulus [42,45].

2.

Among the microscopic parameters of the membrane, shear stress and bending stress are the primary external forces applied. The membrane shear modulus (μ) reflects the membrane's resistance to shear deformation; e.g., healthy RBCs exhibit a μ of 2.4~11.3 μN/m, which increases tenfold during malaria infection [4]. Membrane shear viscosity (ηm) and relaxation constant (τ) are typically quantified through post-stretching relaxation processes [46,47,48]; Membrane bending modulus (B≈1.6×10^-19 N•m) is obtained through localized membrane bending operations, with higher values indicating greater resistance to bending of the lipid bilayer [49,50,51].

3.

Rheological parameters reflect the flow behavior of RBCs interacting with the blood environment (such as plasma and other blood cells), including aggregation index, aggregation time, aggregation velocity, aggregation force, disaggregation force, electrophoretic mobility, and suspension viscosity contribution. Aggregation index serves as an indicator of RBC aggregation, typically observed using a rheometer. Cell manipulation techniques can acquire aggregation and disaggregation forces, while high-speed microscopy provides aggregation time and velocity. These parameters can assess blood viscosity and assist in determining thrombotic risk; for example, RBC aggregation force in systemic lupus erythematosus patients is nearly twice as high as that of healthy individuals [52,53].

RBC mechanical properties are sensitive to physiological and pathological changes in internal and external environments. Therefore, they have emerged as valuable biomarkers for biomedical detection and diagnosis.

2.2. Optical Tweezers Technology

Ashkin's team pioneered OT technique, achieving stable capture of microspheres using counter-propagating laser beams [54] and a single highly focused laser beam [55]. They formally named this technique “OT” for capturing, driving, and separating particles.

When a Gaussian beam is focused, the light intensity near the focal point exhibits a three-dimensional gradient distribution. Due to momentum conservation, the incident light upon penetrating or refracting by the particle surface generates a scattering force (along propagation) and a gradient force (along the intensity gradient) [55] (Figure 1a). Stable trapping occurs at the focal point where these forces balance. This condition is determined by optical parameters (wavelength, power, objective NA), particle properties (size, refractive index), and the surrounding medium. In the imaging path, integrated four-quadrant detectors enable sub-nanometer position tracking, transforming the trap into a piconewton-resolution mechanical sensor. For force calculation, Hooke's law is typically used:

F = k × Δx

(7)

where k is the optical trap stiffness, ∆x is the distance between the center of the manipulated object and the center of the optical trap, and F is the optical trap force.

To minimize photothermal damage in biological samples, OT was initially operated at visible wavelengths (e.g., 514.5 nm, 488 nm) [57], which often damaged cell membranes despite successful virus trapping. The approach was subsequently optimized by shifting to a 1064 nm wavelength, which enabled the safe capture and manipulation of RBCs without impairing cellular flexibility or morphology [58]. Comparative studies confirmed that 1064 nm irradiation has a significantly lesser impact on RBC deformability and stiffness than 785 nm [59]. Thus, 1064 nm is the prevalent choice for RBC on OT research, although other near-infrared wavelengths (e.g., 970–1550 nm [60,61]) are also utilized.

Based on differences in beam manipulation methods and system integration configurations, OT can be categorized into traditional optical tweezers, holographic optical tweezers(HOT), fiber-optic tweezers, surface plasmon optical tweezers, standing-wave optical tweezers, and photonic crystal optical tweezers [62,63]. This paper primarily focuses on traditional optical tweezers and holographic optical tweezers.

• Traditional optical tweezers 

Traditional optical tweezers primarily comprise a near-infrared laser, beam expander, high numerical aperture (NA≥1) oil- or water-immersion objective, and a three-dimensional nanoscale-translation stage. Among these, a high numerical aperture microscope objective and a single optical trap are the main characteristics of traditional setups. The expanded laser beam is focused onto the entrance pupil of the objective (Figure 2a), where the oil immersion medium reduces spherical aberration and forms a three-dimensional optical trap with a focal-spot radius of ~0.6 λ/NA. Piezoelectric mirrors or PID-controlled displacement stages enable millisecond-scale nanoscale repositioning, supporting dragging, positioning, and stretching of RBCs. A beam-splitter prism at the objective’s back focal plane monitors transmitted light intensity. However, conventional systems generate only a single trap from one light source. Multiple optical trapping requires additional components such as an acousto-optic deflector (AOD) for time multiplexing or a polarizing beam splitter (PBS) and scanning galvanometer for spatial multiplexing [64,65,66] (Figure 2b). Owing to switching-speed and diffraction-efficiency limitations, the number of traps remains restricted, and crosstalk between two closely positioned traps remains difficult to avoid [67].

2.

Holographic optical tweezers 

Inserting a spatial light modulator (SLM) before the laser enters the objective can effectively produce multiple dynamic optical traps that are parallel in both space and time [68,69] (Figure 2c). The SLM modulates the wavefront phase through a liquid-crystal pixel array, encoding the designed target dynamic optical traps into a phase hologram. The position of each focal spot is determined by the phase grating period, while the intensity is adjusted by the local phase depth. The spots can be spaced below the diffraction limit, which not only improves diffraction efficiency but also generates a dense trap array. Additionally, HOT is primarily used to grasp and drag non-spherical objects. With computer-generated holograms, tens of independent optical traps can be created to achieve the capture and parallel manipulation of large non-spherical cells as well as multiple spherical particles [70]. Millisecond-level refresh rates enable real-time adjustment of these arrays, reducing experimental time and trapping errors. The ability to perform parallel, three-dimensional, and independent manipulation makes HOT an ideal tool for single-cell mechanical characterization.

By applying external forces and monitoring real-time deformation, OT enables characterization of RBC mechanical responses under varying stress conditions and elucidation of associated structural mechanisms. Two primary manipulation approaches are employed: (1) direct manipulation, in which optical forces induce small cellular deformations, and (2) indirect manipulation, where microbeads attached to RBCs membrane are trapped and moved to deform cells.

2.3. Direct Manipulation

In direct manipulation studies, to maintain RBCs in suspension, RBCs are typically suspended in solutions including 0.9% sodium chloride solution, 0.01 M phosphate-buffered saline free of Mg²⁺ and Ca²⁺, and plasma [25,59,73]. Following suspension, sample chambers are frequently pre-treated with bovine serum albumin (BSA) (applied and air-dried) [73,74] or other agents such as polylysine and casein [75,76] to prevent adhesion and ensure reliable measurements.

Under direct trapping by one or two optical traps, an RBC rotates from its flat-lying state to make its cell plane parallel to the optical axis. However, with three or more optical traps, the RBC remains stably flat-lying. This phenomenon was first numerically validated by Tognato et al. using a ray-optics model of a native biconcave RBC [77]. Their findings indicate that with one or two traps, optical torque drives RBC rotation, and the beam focus tends to stabilize at the thickest part of the cell; increasing the number of optical traps allows control over the RBC's orientation.

To closely replicate RBC physiological conditions and acquire both the imaging and mechanical data during deformation, two direct optical manipulation methods are broadly applied: (1) single-tweezer trapping and dragging; (2) multi-tweezer stretching and manipulation.

2.3.1. Trap-and-Drag Operation Based on a Single Optical Trap

When one end of an RBC is held by an optical trap, its elasticity enables reversible deformation from a discocyte to a bullet-like shape under fluid shear stress (Figure 3). The membrane shear elasticity, μ, is calculated based on changes in the major axis [78] by:

(8)

with

(9)

where L is the length of the RBC after deformation, L₀ is the length before deformation. η is the viscosity of the serum, plasma, or RBC suspension, ν is the velocity at which the optical trap drags the cell, Z₁ is the distance from the RBC to the bottom of the sample chamber, and Z₂ is the distance from the cell to the top of the chamber.

The shape recovery relaxation time, τ, is calculated from the exponential decay curve of the cell length [79] by:

(10)

and the membrane viscosity, η_m, is then determined [79] by:

(11)

Membrane shear elasticity, relaxation time and membrane viscosity together characterize the ability and rate of RBCs to resist deformation.

Since it was proposed by Ashkin in 1987 [58], the single-beam OT “drag-and-deform” method has matured into a reproducible technique for quantifying RBC mechanical properties. To improve accuracy and reliability, a viscous drag-based calibration method based on the single trap was also developed to precisely measure optical trapping forces on RBCs [74]. Its applications span from measuring membrane elasticity and viscosity to assessing the effects of laser damage [59], storage lesions [78,81], and diseases like sickle cell anemia [5,82] and thalassemia [83]. Despite its established role in biophysical research, the method’s low experimental throughput remains a limitation for broader application.

2.3.2. Stretch-and-Squeeze Operation Based on Dual Optical Tweezers and Multiple Optical Tweezers

Another common method for probing RBC mechanics uses two optical traps: one stationary and one movable, to apply stretching force (Figure 4a). By measuring the cell’s initial length, maximum extension, and trapping force, key mechanical parameters can be derived, such as deformation-index [38,74,76], stiffness [45,73], shear modulus [74,84], and relaxation time [46,73]).

To minimize photodamage, an innovative “tug-of-war” optical tweezer configuration generating divergent and elongated beams via SLM was developed (Figure 4b). This system achieves a trapping force of 15 pN at just 20 mW, nearly double that of conventional dual Gaussian-beam traps [74].

By varying the movement speed of the optical traps, it is possible to establish the correlation between DI and different cell morphologies, achieving cell classification [38,76,86]. In contrast to the macroscopic characterization provided by deformation-index, the shear modulus reflects microscopic membrane properties and is calculated from the measured deformation-index and trapping forces. Recent studies have shown that μ decreases under hypotonic conditions due to increased surface area and reduced tension [74]. Furthermore, finite element modeling and analysis, performed with COMSOL simulation software, enabled precise calculation of the stress distribution on the surface of deformed RBCs [84]. This work provided theoretical foundations for understanding the mechanical response of spherical RBC membranes in chemical environments.

Research on RBC mechanical dynamics has been advanced through new stretching modes and parameter definitions. Dual HOT enables stiffness measurement under small-strain conditions (<20%). Fatigue studies revealed that biconcave RBCs exhibit significantly greater stiffness increment (Δk=+2.3 pN/μm per cycle) than echinocytes during repeated stretching in early storage [45]. Here, the slope of the force-elongation curve under small-strain conditions is defined as the linear stiffness k, given by:

(12)

where F is the optical trapping force and l is the RBC’s major-axis length. Furthermore, successive stretch-relaxation cycles show a progressive linear decrease in DI, a power-law increase in stiffness, and a 2.5-fold prolongation of relaxation time [73].

Although the two studies differ in how stiffness is defined, both approaches reveal consistent trends that capture the evolving elastic behavior of RBCs. Advancing beyond dual-trap systems, multi-trap configurations have enabled more complex RBC deformation studies. Three traps were used to form a “parachute-like” shape in RBCs, deriving an angular relaxation time that provides a unique metric for shape recovery dynamics [46] (Figure 4c). Recently, four holographic optical traps were also employed to symmetrically stretch RBCs while simultaneously monitoring deformation and forces in multiple directions [72] (Figure 4d).

Researches on RBC mechanical properties using multi-trap OT collectively establish a multidimensional framework for analyzing RBC mechanics, which offers key biomechanical indicators for applications in blood disease diagnostics, radiotherapy monitoring, and blood transfusion quality assessment.

2.4. Indirect Manipulation

Unlike direct optical stretching, the indirect method applies force non-invasively to minimize photodamage and avoid cell flipping. This maintains the RBC’s disc plane parallel to the focal plane, allowing CCD image acquisition and simultaneous analysis of both lateral and axial deformation.

Experimental sample preparation focuses on creating a “mechanical manipulation medium” by incubating RBCs with microbeads at a set ratio, forming stable attachments that act as “handles” for optical trapping. To prevent nonspecific adhesion of cells or beads to chamber walls, which would interfere with measurements, BSA can be applied to coat the chamber interior or be added directly to the suspension [87,88,89].

In early stages, unmodified silica or polystyrene microbeads with diameters ranging from 1~4 µm were commonly used. Later, microbeads functionalized with IgG [93], carboxyl [94], streptavidin [95], or amino groups [89] were developed to enhance binding specificity and stability. Meanwhile, force calibration methodologies evolved from hydrodynamic flow to stage displacement, Boltzmann statistics, and thermal fluctuation analysis, improving measurement precision from the nanonewton to the piconewton scale [96].

The dual-bead attachment strategy was first introduced in 1999 with two operation modes: the “single-tweezer pulling and single-bead anchoring” mode and the “dual-tweezer trapping, anchoring and translation” mode [90]. By gradually increasing the distance between the two optical traps, a gradient tensile force was applied (Figure 5a). The shear modulus (μ) was derived from the relationship between the applied force and the change in cell diameter at the cell poles under small deformations, given by:

(13)

where L_y0 is the initial lateral diameter of the RBC (vertical to the force direction), L_y is the lateral diameter after deformation, and F is the applied tensile force. In the same year, Sleep et al. adopted a “fixed-translation” dual-tweezer configuration to investigate the contribution of the cytoskeletal network to the elasticity of nearly spherical RBCs [91]. Periodic triangular-wave or stepwise tensile forces were applied (Figure 5b), the parameters BH² and shear modulus H were calculated based on Parker’s [97] axisymmetric shell elasticity theory:

(14)

with

(15)

where a is the bead radius, B is the bending modulus, F is the applied force, and ε is the axial relative elongation ratio of the cell, L₀ and L are the initial and deformed axial diameters, respectively. Collectively, these two pioneering studies established a fundamental framework for calculating the RBC shear modulus, which has since become a reference standard for subsequent investigations. However, the spherical membrane model under small strain is different from the actual biconcave morphology of native cells.

Since 2003, OT stretching techniques have advanced toward large-deformation studies with improved precision. A finite element-based biconcave RBC model with and without cytoplasm enabled accurate mechanical quantification under forces up to 600 pN, overcoming earlier spherical simplifications [47]. Subsequent refinements included bead height correction for reliable cell-length measurements and deformation data under high force ~193 pN [92] (Figure 5c). More recently, a microbead indentation method was developed to probe lateral membrane stiffness, yielding results consistent with micropipette aspiration while avoiding strong cell-substrate interactions [42] (Figure 5d).

With continuous advances in OT manipulation strategies, measurable parameters have expanded from the membrane shear modulus under small deformations [90] to a diverse set of multidimensional mechanical indicators, including the shear modulus under large deformations [47], relaxation time [47], intercellular adhesion forces [98], apparent membrane viscosity [98], stiffness [42], membrane fluctuation power spectral density [94], and dynamic changes in cell height during stretching [95].

3. Application of Optical Tweezers in Red Blood Cell Research

3.1. Measurement and Study of Intercellular Interaction Forces

In physiological environments, RBCs usually do not exist in isolation; their functions rely heavily on intercellular interactions. The mechanical characteristics of RBC populations are directly reflected by their aggregation, disaggregation, and adhesion behaviors, which not only affect blood rheology and microcirculatory function but are also closely associated with various diseases. Therefore, the research scope of OT has extended beyond single-cell manipulation toward the investigation of collective mechanical properties of cell ensembles.

Using dual-trap and four-trap OT configurations to operate RBCs is a classical method for studying the aggregation characteristics of RBCs. Two RBCs were captured by dual traps and lifted 40 μm above the chamber bottom. The aggregation and disaggregation force of RBC-doublets were then measured by adjusting the light power and pulling dual optical traps (Figure 6a) [66]. This direct manipulation method can also be realized with only a set of double optical traps [80], which not only simplifies the operation steps, but also reduces the light damage of RBCs.

However, the traditional methods only focus on the aggregation and disaggregation behavior of RBC dimers under shear tension, ignoring the influence of the adhesive force between RBC discs on the aggregation behavior. A more physiological adhesion model was proposed by researchers: four traps generated by HOT were positioned pairwise on two RBCs to apply forces perpendicular to the disc plane, simulating “collision and separation” under full contact (Figure 6b) [99]. Through the evaluation of the aggregation and disaggregation of RBCs by OT, the regulatory effects of laser radiation, nanoparticles, the life span of RBCs, adhesion factors and other factors on the interaction of RBCs have been explored [66,102,103,104].

These studies have advanced quantitative measurements of RBC interaction forces from various perspectives. However, most are in vitro experiments that involve extensive dilution, washing, and resuspension. Although beneficial for single-cell manipulation and mechanical measurements, such treatments do not replicate the shear forces, complex plasma composition, and multicomponent interactions found in real blood flow. Thus, current results are more appropriate as methodological validation.

Due to antigen variant recognition technology can promote early disease diagnosis, quantitative analysis of cell surface antigen variation has attracted more and more attention. Traditional flow cytometry requires a high number of samples, and cannot achieve quantitative analysis of a small number of samples and single cells. The cell-tearing operation based on OT can solve this problem (Figure 6c) [100]. This approach enables highly sensitive quantitative analysis only with minimal fingertip blood volume, offering a novel approach for detecting surface antigen variations. In addition, the interaction behavior and related mechanisms between RBC and endothelial cells were explored by the same means [105].

These two studies have extended OT applications in RBC interaction research, showing strong methodological innovation. However, limitations remain: (1) Using laser power as a substitute for absolute force simplifies experiments but reduces comparability with other mechanical studies. (2) The change in RBC–endothelial adhesion force was weak and not statistically supported in all experimental groups.

In addition, OT can also quantitatively study RBCs-merozoites adhesion forces through a triple-cell system (“RBC-Plasmodium falciparum merozoite-RBC”) (Figure 6d). The traditional viewpoint holds that PfMSP1 protein dominates the initial attachment of Plasmodium, but high-precision OT experiments showed no significant change in adhesion forces after PfMSP1 knockdown [101]. In Dantu blood group, it was also found that RBCs resist merozoite invasion not by reducing adhesion but by increasing membrane tension, providing key mechanical evidence for the screening of vaccine targets for malaria [75].

The samples in these two studies are representative, and cross-validation using multiple biological techniques makes their conclusions mechanistically convincing. However, such representativeness also introduces single-cell heterogeneity, requiring a large sample size to ensure data stability.

3.2. Study on Bioinformation of Red Blood Cells

The mechanical parameters of RBCs directly reflect membrane structural characteristics, physiological status, and species adaptability. With integration of biochemical analysis methods, OT has advanced RBC bioinformation research from qualitative mechanism exploration to quantitative characterization.

This technology has enabled quantitative analysis of key determinants in RBC aggregation. It was demonstrated that Ca²⁺ influx and phosphatidylserine exposure, rather than lysophosphatidic acid, are responsible for irreversible adhesion, excluding group-effect interference [99]. Using OT combined with laser aggregation assays and flow cytometry, researchers showed that inhibition of glycoprotein receptor IIbIIIa (GPIIbIIIa) significantly reduced aggregation forces (Figure 7a), revealing a protein-dependent regulatory mechanism [104]. Subsequent studies further clarified the distinct roles of plasma proteins such as fibrinogen and albumin in modulating aggregation [16] (Figure 7b). Additionally, experiments demonstrated that nitric oxide regulates RBC-endothelial adhesion via the soluble guanylate cyclase (sGC) pathway, exhibiting a bell-shaped dependence on L-arginine concentration [105] (Figure 7c).

Although these studies included good negative and pharmacological controls (e.g., Ca²⁺ removal, GPIIbIIIa inhibitors, NO donors, or sGC inhibitors), some mechanisms remain largely inferred from pharmacological inhibitors, lacking direct molecular evidence to confirm specific receptors or binding sites.

RBCs exhibit varying lifespans within blood vessels, and experiments have shown that aggregation force increases with cell age [103] (Figure 7d), and that RBCs of varying densities exhibit significant differences in shear modulus [89]. This suggests that density-gradient centrifugation can effectively minimizes sample heterogeneity and reduces measurement errors. However, Percoll gradient separation reflects RBC density rather than precise cell age. Thus, the classification of "young" versus "aged" RBCs requires further validation.

Additionally, the marked differences in DI and Raman spectral also revealed the differences between various morphologies of RBCs [76] and different species [86], such as camel (0.024±0.019) and human (0.215±0.061), offering new insights into the adaptive mechanisms of RBCs in extreme environments.

3.3. Study on the Effect of Different External Environmental Stimuli on Red Blood Cells

Investigating RBC intrinsic mechanical properties under complex microenvironments enhances understanding of relationships between their structure and function.

Laser irradiation studies demonstrated that wavelength and exposure dose modulate RBC stiffness and deformability: 785 nm near-infrared light induces greater stiffening than 1064 nm [59], and visible-light irradiation triggers oxidative damage through photodissociation of hemoglobin-bound oxygen [106]. Continuous low-dose 450 nm exposure enhances deformability, whereas short-pulse He-Ne irradiation reduces aggregation force [66,107]. In radiobiology, high-dose irradiation decreases DI, making it a potential biomarker for radiation responses [85].

Osmotic stress also plays a critical role in influencing RBC mechanical behavior. OT–based stretching and indentation experiments have shown that shear modulus and stiffness of RBC increase proportionally with osmotic pressure [42,93]. The deformability ranking follows: hypotonic > isotonic > hypertonic conditions [74] (Figure 8a). The study of laser trapping with Raman spectroscopy (LTRS) at the molecular level revealed that osmotic changes alter the concentration of intracellular heme, reorganize membrane structures, and induce protein conformational variations, thereby regulating overall mechanical responses of RBC [108].

Regarding chemical stimulation, LTRS demonstrated that bisphenol A damages membrane and hemoglobin structures [109] (Figure 8b); ethanol exposure induces deoxygenation and irreversible morphological loss [88]; and hydrogen peroxide increases shear modulus while reducing deformability [87], offering insights into environmental toxicology and blood-disease mechanisms.

With expanding biomedical applications of nanomaterials, nanoparticle (NP) biocompatibility with RBCs has drawn increasing attention. LTRS showed that nanoparticles, including gold and silver, induce mechanical alterations in RBC membranes [111] (Figure 8c). Nanodiamonds exhibit strongest interference on RBC interactions, whereas polymer-based NP and nitride-based plasmonic NP show superior biocompatibility, guiding nanodrug carrier design [102,110].

In mechanical fatigue studies, RBCs of different morphologies were repeatedly stretched using dual HOT, demonstrating that storage duration and stretching cycles increase stiffness and prolong recovery time (Figure 8d). These findings validated universal principles of “stretch-induced stiffness enhancement” and “rheological non-scalability”, revealing that accumulated mechanical strain drives rheological degradation of RBCs [45,73].

These studies have revealed RBC functional changes under external stimuli at multiple levels, including single-cell mechanics, morphology, and hemoglobin molecular state. However, most mechanistic interpretations rely on inferences from mechanical characterization, Raman shifts, or morphology, lacking direct validation of membrane skeleton proteins, ATP levels, oxidative damage markers, hemoglobin oxygen affinity, and ion channel function.

3.4. Study on Related Hematological Diseases

Hematological diseases are caused by abnormalities in blood cells, plasma proteins, or hematopoietic functions. OT and their combined technologies have been widely applied to the study of diseases related to RBCs, such as thalassemia [83,112,113] and sickle cell disease (SCD) [5,48,82].

Thalassemia induces abnormal mechanical and molecular properties of RBCs. LTRS has revealed that RBCs from β-thalassemia patients have significantly lower oxidative capacity and 40% higher stiffness than normal RBCs [112,115]. Additionally, RBC elasticity and membrane viscosity have shown decreased elasticity and increased membrane viscosity in diseased cells (Figure 9a). Combined with quantum dot detection, a reduction in the negative surface charge of pathological RBC membranes has also been observed [83]. These findings suggest that disease-induced damage to membrane structures (such as surface glycoproteins and the cytoskeleton-associated band 3 protein) alters both the mechanical and electrical properties of RBCs.

In sickle cell disease (SCD), the HbS mutation leads to increased RBC rigidity and reduced deformability. Compared to healthy RBCs, the elastic modulus of RBCs from sickle cell trait donors (HbAS) significantly increased during storage, while hydroxyurea (HU) treatment restored deformability of RBCs [5,82] (Figure 9b). In addition to measuring elastic modulus, 1064 nm optical traps allow non-destructive measurement of folding angle, rotational features, and deformation relaxation time of RBCs with SCD, showing more obvious distinctions in HU-treated samples [48,116]. The Raman tweezers systems also revealed that RBCs with SCD are more prone to deoxygenation under mechanical stress [117]. Besides, mannose-binding lectin (MBL), by binding to the membrane, alters both the membrane and the cytoskeleton, thereby increasing membrane stability and reducing the elasticity of RBCs with HbSS [118]. This reveals the regulatory role of the immune molecule MBL in RBC mechanical properties.

However, the number of mechanical investigations on other disorders like acute myeloid leukemia (AML) [119], paroxysmal nocturnal hemoglobinuria (PNH) [120], hereditary elliptocytosis (HE) [121], iron deficiency anemia (IDA) [122] remain relatively limited.

3.5. Study on Red Blood Cells in Non-Hematological Diseases

Beyond hematological disorders, diseases such as malaria, cardiovascular disease, and diabetes profoundly also affect the RBC microenvironment, leading to abnormalities in aggregation, deformability, and elasticity.

During the malaria merozoite stage, the shear modulus of infected RBCs increases up to tenfold (≈53.3 N/m), indicating cytoskeletal remodeling by parasite-derived proteins [4]. Microsphere indentation experiments similarly revealed elevated shear stiffness [3]. Assuming stiffening of the local membrane skeleton, these experimental findings agree well with numerical calculations based on the Skalak constitutive model for RBCs, providing further insight into how Plasmodium infection affects the RBC membrane [123]. Crick’s group directly captured parasite invasion events and measured a detachment force of ~40 pN, further demonstrating that heparin significantly reduces adhesion strength and invasion rate [124]. Using OT combined with flicker spectroscopy, Dantu blood group RBCs were shown to display higher membrane tension (1.22×10^-6 N/m) and an invasion threshold of ~3.8×10^-7 N/m, supporting a “high-tension invasion-resistance” mechanism [75]. Additionally, the combination of gene knockout and OT identified the PfEBA/PfRH protein family, not PfMSP1, dominates adhesion, clarifying the molecular biomechanics of malaria invasion [101] (Figure 9c).

Cardiovascular diseases significantly modify RBCs rheology. In hypertensive patients, aggregation time decreased by 24%, disaggregation force increased by 28%, providing one of the explanation for the elevated blood viscosity in hypertension, with the F_D/F_A ratio showing diagnostic value [125]. Through combined OT-aggregometry-capillaroscopy analysis, aggregation time in CHD patients decreased by 27%, and those with CHD comorbid T2DM exhibited even stronger aggregation [114] (Figure 9d).

In diabetes mellitus, mechanical impairment of RBCs is strongly associated with microangiopathy. OT measurements indicated significantly reduced DI in type 2 diabetes and diabetic retinopathy, negatively correlated with initial RBCs size [38]. Fetal RBCs from gestational diabetes displayed increased membrane tension and bending modulus using a dual time-resolved membrane fluctuation spectroscopy based on OT [126]. Raman tweezers (785 nm) further detected distinct spectral differences at 1003 cm^-1 and 753 cm^-1, achieving 100% classification accuracy from diabetes erythrocyte using PCA-LDA [127]. Furthermore, OT technology provides a method for the early diagnosis of diabetes. Acute high-glucose exposure (early common symptoms of diabetes) enhanced oxidative stress, reduced membrane elasticity, and decreased elongation ratio with rising glucose concentration [128].

Although these studies provide multiparametric support for early diagnosis and disease monitoring, they lack in-depth exploration of the underlying molecular mechanisms.

3.6. Study on Drug Evaluation and Development

Drugs generally reach all parts of body through the blood circulatory system, which also affect mechanical properties of RBCs.

For pharmacological research, integrated with fluorescence imaging and flow cytometry, OT experiments revealed several drugs can reduce RBC membrane elasticity and stiffness, such as lithium salts, the first-line treatment for bipolar disorder [129]; vitamin E, an antioxidant used for stored RBC preservation [81]; and doxorubicin, a chemotherapy agent for acute myeloid leukemia [119]. These findings provide new biophysical evidence for understanding drug-induced cytotoxicity mechanisms.

Though Raman tweezers, common intravenous infusion solutions (such as 0.9% saline, Ringer's lactate, and Plasmalyte-A) induced hemoglobin deoxygenation, impairing RBCs oxygen-carrying capacity, whereas RBCs in plasma demonstrated superior anti-deoxygenation properties [130,131]. Notably, hydroxyethyl starch, a solutions with safety concerns, was observed to impair both oxygenation state and membrane structure of RBCs [132].

These studies indicate that saline and other crystalloids should not be treated as "uninfluential backgrounds" when assessing drug effects on RBCs. Plasma or more physiological control conditions are recommended. Otherwise, drug and diluent effects may be confounded.

3.7. Application in Quality Evaluation of Artificial Red Blood Cells

Cultured RBCs (cRBCs) offer a promising solution to growing the demand for safe blood, yet their morphology and mechanical properties remain distinct from native RBCs (nRBCs), demanding high-precision evaluation.

The oscillatory OT system can employ an hourglass-shaped optical trap to capture cRBCs and apply axial oscillations. By analyzing high-frequency scattering signals and membrane fluctuations, results showed that cRBCs cultured with human platelet lysate exhibited biomechanical properties most similar to nRBCs, consistent with measurements from digital holographic microscopy [133]. Despite comparable membrane elasticity and protein expression, a lipid deficiency in cRBCs caused their morphological and mechanical impairments, analysed by integrated OT-AFM-SEM, also highlighting the utility of OT as a critical tool for evaluating cRBC quality [41].

However, these conclusions are mainly based on in vitro single-cell experiments, and factors like maturation stage, cell heterogeneity, and measurement methods may influence the mechanical parameters. Thus, further validation (e.g., causal lipid interventions, high-resolution membrane skeleton analysis, and in vivo models) is needed to confirm whether these mechanical similarities can assess cRBC quality.

4. Study of Red Blood Cells by Optical Tweezers Combined with Other Techniques

In conventional LTRS experiments, one laser beam is dedicated to Raman signal detection, while the other beams function as optical traps to capture RBCs, apply mechanical forces, and induce deformation, thereby enabling the acquisition of deformation and mechanical data. Thus, LTRS has been used to simultaneously study the mechanical and chemical properties of RBCs (Figure 10a).

Using dual-trap stretching, an LTRS study revealed that β-thalassemia RBCs have a 40% higher membrane shear modulus and a weaker oxygenating capacity compared to normal RBCs [115]. These results indicate that the genetic defects underlying thalassemia, which primarily affect hemoglobin structure, also significantly impact RBC mechanical properties. Moreover, LTRS has been applied to investigate RBCs in malaria [136], sickle cell disease [137], and diabetes [127], highlighting its potential as a diagnostic tool.

However, during optical trapping, the effect of light-induced protein oxidation on shear modulus is unavoidable. To overcome this problem, Raj’s group [138] used an indirect stretching approach in which RBCs were tethered to micro-beads, which significantly reduced laser-induced damage to the cells. They found that once cell deformation exceeded 10%, several Raman peaks corresponding to hemoglobin and protein vibration modes rose and then saturated, suggesting that external force markedly alters the chemical structure of intracellular molecules.

To minimize laser-induced damage to RBCs in LTRS, a light-sheet Raman tweezers system that uses a single laser beam for both Raman excitation and optical trapping was developed. The system can achieve stable capture at a low power density of 3.8×10⁴ W/cm², cutting power demand seven-fold [139], and enhancing biocompatibility and clinical potential by minimizing photodamage and heme aggregation.

With its “molecular fingerprint” characteristic, Raman spectroscopy enables non-destructive detection of structural changes in intracellular biomolecules. Its introduction endows OT with an additional capability about label-free chemical information detection.

Conventional OT systems are incapable of three-dimensional imaging of RBCs, making it impossible to track the underlying causes of the shape changes observed from a restricted perspective (top-down view) after RBCs are captured. To overcome this limitation, Mohanty’s team [140] integrated confocal fluorescence microscopy into an OT system, achieving fluorescence imaging and dynamic tracking of RBCs, revealing laser-induced orientation aligned with polarization rather than folding into rods.

Given that OT cannot discriminate between physical or chemical adhesion during RBC aggregation, flow cytometry was also combined with OT, demonstrating fibrinogen accumulation at cell-contact regions and confirming that GPIIbIIIa-mediated adhesion governs RBC aggregation, which can be reduced by GPIIbIIIa inhibitors [104].

To investigate the composition of RBC membrane tethers, researchers used a complementary setup of OT and confocal microscopy. Along with viscosity and bending modulus measurements, they showed that the membrane tether is mainly supported by the lipid bilayer, with no F-actin signal detected in tether regions [134] (Figure 10b).

Through specific labeling, fluorescence microscopy enables highly sensitive visualization of cellular structures, molecular distribution, and dynamic processes. The integration of OT with confocal fluorescence imaging and flow cytometry has progressively expanded RBC mechanics researches from the overall elasticity of whole cells to morphological changes and mechanical regulatory mechanisms.

Conventional OT enables non-contact mechanical measurements but suffer from low throughput and difficulties in continuous analysis, whereas microfluidics offers high-throughput advantages but lacks precise single-cell manipulation. Combining high-precision optical manipulation with high-throughput microfluidics can break through the constraints of a single technology.

By integrating periodic chopped laser beams with microfluidic channels, researchers achieved non-contact stretching of RBCs during continuous transport [135] (Figure 10c). To overcome the low efficiency of single-channel capture and frequent clogging in multi-channel systems, a multi-channel microfluidic chip with pressure-release structures and time-shared OT was designed, achieving 100 cells/min throughput and >90% capture efficiency, and revealing membrane stiffening in pathological RBCs [65].

Beyond mechanical measurements, OT-microfluidic technology has also achieved label-free cell sorting. By exploiting differences in escape velocity, researchers obtained 95% sorting efficiency for healthy and thalassemic RBCs at a lower cost [113]. Another design, using an arc-protrusion chip with stationary tweezers, simplified the system while maintaining high biocompatibility, attaining >95% purity and >90% viability for sorting B16F10 cells from RBCs [141].

The combination of OT and microfluidics overcomes their limitations, enabling efficient measurement of RBC mechanical properties and supporting clinical diagnosis. However, the feasibility of this method in complex and heterogeneous biological samples requires further validation.

5. Conclusions and Outlook

OT has developed into a critical tool in RBC mechanics research. Their evolution is marked by three key advances: the transition from basic manipulation to performance-optimized systems, the integration with complementary techniques for multidimensional analysis, and the expansion from studying healthy cells to clinical applications in disease diagnosis. These developments have revealed the relationship and differences between the global and local mechanical properties of RBCs, and their interaction mechanisms, providing new insights for medical diagnostics.

However, OT technology still faces several challenges in RBC mechanics research. (1) Although novel systems have been developed to reduce photothermal damage[74,139], manipulation flexibility of system remains limited. (2) Current studies mainly focus on highly RBC-associated diseases (e.g., sickle cell disease, malaria, and diabetes), whereas research on diseases with lower correlation but potential value (e.g., Alzheimer's disease) remains insufficient. (3) Limited clinical samples hinder the development of its clinical application. (4) OT has been demonstrated to trap and manipulate RBCs in the zebrafish caudal artery and mouse ear capillaries [142,143]. However, due to multiple light scattering in tissue, a Gaussian beam cannot form an effective optical trap at depth, making in vivo manipulation in humans currently unachievable. (5) More critically, most OT systems are low-throughput, unsuitable for rapid clinical testing and large-scale studies.

Looking ahead, the development directions of OT in RBC mechanical studies are becoming clear. (1) Achieving light focusing, trapping, and imaging in deep tissue is a key research direction for applying OT to the non-invasive study of RBC mechanical behavior within the human body [144]. (2) The integration with microfluidics shows greater potential than flow cytometry, with label-free RBC sorting already achieved to overcome single-cell throughput limitations [113]. However, only the deformation index is currently obtained in this direction; future work is expected to enable high-throughput analysis of more RBC mechanical parameters. (3) Culturing RBCs in vitro allows OT, together with transcription, gene editing, and membrane composition control, to explore the connection between RBC mechanical parameters and molecular [41]. (4) Additionally, the combination with machine learning will significantly enhance data analysis of force and spectroscopic measurements, enabling faster and more accurate interpretation [145,146]. These advances are expected to open new avenues for both fundamental research and clinical applications.