Laser tweezers as a biophotonic tool to investigate the efficacy of living sickle red blood cells in response to optical deformation

Abstract

A laser tweezer technique based on single and/or dual-laser beams is proposed as a biophotonic tool to trap single cells and investigate their biophysical and biomechanical characteristics. Optical deformability and changes in size and cellular morphology of living and nonliving cells can be measured using the proposed technique. Representative results of red blood cell (RBC) optical deformability of 20 homozygous patients with sickle cell disease, including follow-up patients after treating with hydroxyurea (HU) for at least 3 months and 20 healthy control groups, are presented and compared. Shape recovery of deformed RBCs and relaxation time are recorded for each RBC. Results showed that healthy blood and patients treated with HU demonstrate significantly higher optical deformability and degree of optical elongation with morphological change of RBCs than untreated patients. Moreover, the healthy control group and patients treated with HU exhibited faster relaxation time for RBCs than untreated patients. A trapping power that reaches 180 mW caused no observable photo-damage at a wavelength 1064 nm.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12551-021-00790-0.

Introduction

Devices that use photons instead of electrons to transmit and process information have many advantages over their solely electronic counterparts (Menzel 2007; Rahma et al. 2017; Ying et al. 2020). As such, photonics is a key technology in the new field of “biophotonics.” Biophotonic products are biomaterials used as photonic media that demonstrate an interaction of photons and biology (Prasad, 2004; Rahma et al., 2017). Biophotonic devices are increasingly used in nanotechnology, biotechnology, disease progression, life science, and other scientific research fields for optical bioimaging and sensing diagnostics and therapies involving fluids, cells, and tissues (Prasad, 2004; Michaelis et al. 2011; Di Bartolo and Collins, 2009; Meglinski 2015; Lichtman and Conchello 2005).These advances in biophotonics can be used as new diagnostic tools to investigate biophysical and biomechanical properties (dimension, size, shape, deformability, and charge properties) of biological materials, specifically biological cells and tissues (Prasad, 2004; Meglinski 2015; Carvalho et al. 2015; Sim et al. 2017) as far as their properties are closely related with significant changes in the cell membrane and cytoskeleton (Bao and Suresh 2003; Hianik 2006; Ofek et al. 2009; Sens and Plastino 2015). Biophysical and biomechanical cell characteristics can be used to distinguish physiological cells from pathological cells and metastasis, respectively (Chu et al. 2005; Johnson et al. 1971; Mudali et al. 2020; Radmacher et al. 1996). At present, optical tweezers (also known as laser tweezers) are used in biophotonics along with related techniques to obtain important properties of cells and molecules (Ashkin 1992; Ashkin and Dziedzic 1987; Blasi et al. 2016; Dholakia et al. 2020; Dinu et al. 2009; Guck et al. 2001; Komoto et al. 2020; Masaeli et al. 2016; Perkins 2009; Wang et al. 2006; Wu et al. 2016). The laser beam applied to the cell causes an optical elongation or stretching that affects its rheological and mechanical properties.

The optical deformability of red blood cells (RBCs) and their membrane mechanical properties are important factors in regulating their circulation and capacity for transporting oxygen and carbon dioxide (Barjas-Castro et al. 2002; Lima et al. 2020; Nascimento et al. 2008; Pozzo et al. 2009). Biconcave disk-shaped RBCs can be subjected to large deformation when passing through microcapillaries during microcirculation due to their highly flexible membrane with high surface-to-volume ratio (Anmar et al. 2018; Madigan and Malik 2006). However, patients with sickle cell disease (SCD) undergo changes to the rheological and mechanical properties of their RBC membranes which reduce cell deformability of the RBCs, and which impairs blood flow and induces other pathophysiological mechanisms of the disease in circulating erythrocytes (Diez-Silva et al. 2010; Humphrey and O’Rourke 2015; Khanna 2015). Thus, the impaired blood flow in SCD is an important factor that affects acute and chronic complications (Alapan et al. 2016; Barabino et al. 2010; Connes et al. 2016; Sundd et al. 2019). Hydroxyurea (HU), a medical treatment for sickle cell patients, is the only effective drug for reduced pain episodes and blood transfusions (Alberts et al. 2020; Charache et al. 1995). Therapeutic benefits of HU are related to increased production of fetal hemoglobin (HbF), which reduces RBC sickling, optimizes lifespan, and therefore reduces hemolysis. Therefore, HU has a remarkable effect on cell optical deformability (Green and Barral 2014; Nader et al. 2018). Here, we propose a laser tweezer (LT) technique to investigate the morphological change of cell shape and membrane mechanical properties using optical forces for single-cell stretching based on dual-selective laser beams to trap the cell. The technique can measure the optical elongation of the cell and size to calculate the cell optical deformability (OD) characterization. The proposed technique has the following advantages: (1) achieves high trapping efficiency, (2) sensitive for patients taking drugs, and (3) can provide information about different properties, such as morphological change of cell shape, size, recovery time, and deformability for single cell. Dual laser beams were directly applied to human erythrocytes (RBCs) in this experiment.

A laser tweezer technique with near-infrared and visible lasers at wavelengths of 1064 and 532 nm, respectively, is presented and designed in the present work to investigate and analyze the efficacy of blood transfusions in single RBCs from healthy, SCD, and follow-up after HU treatment patients by measuring the response of RBCs to optical deformability, morphological change of cell shape, and membrane mechanical property modifications. We found that RBCs from both SCD and follow-up after HU treatment patients demonstrate significantly different morphological and mechanical properties compared with healthy RBCs.

Generation of letter-specific data

Building the experimental system and its principles

Optical tweezers, also as known as optical traps, can be formed with a tightly focused Gaussian laser beam using objective lens with high numerical aperture (NA) for optically trapping and manipulating micron-sized objects, including biological materials. Optical tweezers are based on piconewton (PN) force via the interaction of laser beam with particles (Whitley et al. 2017; Yaakov et al. 2016).

A laser tweezer technique based on single and/or dual-laser beams for investigating the optical deformability and cell interaction dynamics was presented to trap RBCs in our system. As shown in Fig. 1, the technique was designed, constructed, and controlled as follows:

1. Trapping laser system. Two diode-pumped solid-state (DPSS) lasers in the Gaussian TEM₀₀ mode with a maximum output power of 600 and 300 mW are used as the trapping laser. Neutral density filter (NDF) is used to attenuate the laser beam intensity.

2. Beam steering. Adjustable steering mirrors are used to match and adjust the focal plane of laser beams with the sample, extend the path of the laser beam for sufficient laser beam diameter or diffraction-limited spot at the sample to overfill back aperture of the objective lens, and achieve tight focus. CW DPSS operating at a wavelength of 532 nm is reflected by a mirror (M1), attenuated by an NDF, and reflected by a dichroic mirror (DM1). CW DPSS operating in the near-infrared region at 1064 nm is reflected by another mirror (M2). Upon reflection by M2, the beam is attenuated by an NDF and reflected by another dichroic mirror (DM2). DM1 and DM2 have high reflectivity at these wavelengths and fixed at 45°.

3. Microscopic system. An optical trapping setup typically consists of a microscopic system, which includes many lenses with infinity correction, and an illumination system with a translation stage (TS) that controls XYZ directions and holds and moves the test sample. The illumination system in this study is a halogen lamp (HL) with a variable power of 150 W. Two laser beams individually reflected by DM1 and DM2 will be focused using an oil-immersion objective lens with high NA (NA = 1.25) and ×100 magnification. After the sample is illuminated by HL, a condenser lens focuses the light for uniform distribution and strong illumination at the sample. However, the optical image of the sample will be recorded by a charge-coupled device (CCD) camera after passing through an optical component (OC) and optical filter (OF), and the transmitted image is monitored by a computer in our design.

Fig. 1.

Experimental laser tweezer technique for investigating the morphological change of cell shape and membrane mechanical properties. M1and M2 are reflected mirrors; DM1 and DM2 are dichroic mirrors. NDF natural density filter, BE beam expander, OL objective lens (×100, 1.25NA), TS translation stage, HL halogen lamp, OC optical component, CCD charge-coupled device

Patient selection

Twenty-seven sample adult patients with SCD were candidates for HU treatment in the present study. All the patients were informed about and agreed to their participation in the study. Some patients were excluded because of their irregular use of HU. Also all patients were clinically stable and did not have any sickle cell crises before sampling. The healthy control group with matched age and gender was also selected at the same time. The patients with SCD were classified into two groups: group 1 (G1) consists of blood samples taken from patients before starting the HU treatment, and group 2 (G2) samples were taken again from patients on regular and restricted use of HU after three months for comparison.

Sample preparation

Fresh blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes. There are five steps in the preparation of washed RBCs.

1. Centrifuge the whole blood at 3500 rpm for 3 min.

2. Remove and separate the plasma and buffy coat layer.

3. Diluting 30 μl of RBCs by 1000 ml in phosphate-buffered saline (PBS).

4. Centrifuge for 3 min at 3500 rpm and removed the supernatant.

5. Repeat Steps 3 and 4 twice for a total of 3 washes until the supernatant is clear.

Then, washed RBC samples were diluted in PBS, and 1% bovine serum albumin was added dropwise to avoid aggregation formation and prevent cells from adhering to the slides. Finally, a cover glass was placed over microscope slides containing the prepared solutions at room temperature. All measurements were performed at a room temperature within 3 h.

Laboratory parameters

As part of the recommended work up by BCHBD for patients, all patients should have hemoglobin variant (Hb variant) and complete blood count (CBC) tests before starting HU treatment and during the follow-up period. CBC includes the following parameters: Hb concentration, RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), white blood cell (WBC) count, platelet count, and variant hemoglobin.

Data analysis

Optical images of RBCs before and after laser beam trapping indicated free (original) and elongating (stretching) cells, respectively, and OD in terms of elongation was analyzed by measuring the diameter (size) of each RBC as follows (Agrawal et al. 2016);

$$\text{Optical deformability}\left(\mathrm{OD}\right)=\frac{X\text{trans}-X0}{X0},$$ 1

where X_trans is the elongation size in the transverse configuration and X₀ is the original cell before laser trapping. As example, Fig. 2 shows the deformation profile of a single cell before and after laser beam trapping. Relative cell deformation is measured along transverse (X_trans) and longitudinal (X_long) diameter axes along the laser beam and perpendicular directions, respectively, to characterize the deformation of RBCs (Fazal and Block 2011). The relative change in diameter can be expressed as follows:

$$\text{Relative change in diameter}=\frac{X0-X\text{trans},\text{long}}{X0}\%$$ 2

Fig. 2.

Optical RBC image before trapping at RBC size diameter (X₀) and after trapping along the laser beam direction at RBC size diameter (X_trans) and perpendicular the laser beam direction at RBC size diameter (X_long)

Statistical Package for Social Science (SPSS version 20) was used to determine the difference value of healthy control and patient groups. Correlation test was applied between original RBC and RBC elongation sizes after laser beam trapping as well as given hematological parameters in this study. One-way analysis of variance (ANOVA) with several post hoc comparisons of the mean and least significant difference (LSD) was performed. Intradifferences in parameters between two of the different groups were evaluated using independent t test at a probability level of 0.05.

Letter-specific results and discussion

Characteristics of patients and control groups are listed in Table 1. Control nonsickle patients were matched to patients in gender and age. G2 demonstrated significantly higher Hb levels, MCV, MCH, and Hb F levels than G1 along with reduced Hb S levels, WBC, and platelet counts in G2 due to the effect of HU. These findings are consistent with the results of other studies (Barma et al. 2020; Lee et al., 2012; Okoye et al. 2015; Strouse and Heeney 2012)

Table 1.

General characteristic and hematological parameters for healthy control and patient study groups

RBC samples
Groups	Control	G1	P-value
		G2
No. of samples	20	20/20
Gender M/F	10/10	11/9	0.92
Age	25.45	27.4091	0.75
HG (g/dl)	10.98	9.725	0.035
		10.5995	0.515
RBC × 106/μl	4.2415	3.7677	0.091
		3.6927	0.052
WBC × 103/μl	7.153	10.8286	0.008
		9.2759	0.119
PLT × 103/μl	253.05	317.0773	0.125
		244.0909	0.829
MCV (fl)	90.7	77.0273	0.028
		84.6455	0.001
MCH (pg)	25.75	27.1455	0.441
		29.2955	0.054
Hb variants
	G1	G2	P value
S	79.23182	72.08636	0.043
F	14.97727	16.92273	0.319

OD was determined using Eq. (1) and the presented technique at room temperature. The OD distribution of all the sample groups is analyzed and illustrated in Fig. 3. Figure 3 a shows that at laser wavelength of 1064 nm, the OD of patients G1 (0.02531) was significantly lower than that of control group (0.0582) (P = 0.0001). OD in G1 was 56% less than control group. In contrast, there is no significant difference in OD of G2 (0.0521) compared with control group (0.0582) (P = 0.074). OD in G2 was 10% less than healthy control, which is referring to improve OD in G2 compared with G1. At the wavelength of 532 nm (Fig. 3b), patients in G1 demonstrated slightly lower OD (0.0099) compared with the control group (0.017). OD of G1 was 41% less than the healthy control while the patients of G2 exhibited a slightly higher OD (0.012) compared with G1 (but still lower than the healthy control group). OD of G2 was 29% less than control group, but that both patients groups (G1and G2) were no statistically significant difference from healthy control. Figure 3 c shows that the OD distribution in G1 is narrow with high population density while healthy control and G2 demonstrate a heterogeneous population density. Figure 3 d presents that the deformation distribution and population density have the same behavior in patient groups (G1and G2) but different tendency in the control group. Hence, changes were evident in the wavelength of 1064 nm. The improvement of RBC deformability in HU-treated patients can be explained by the increased MCV, production of HbF, and reduced HbS.

Fig. 3.

Distribution of red blood cell optical deformability in all sample groups: laser wavelength for sets (I) λ = 1064 nm and (II) λ = 532 nm

Figure 4 shows OD as a function of the trapping laser power at wavelengths of 1064 and 532 nm for the all sample groups. OD increases with increasing power for the two lasers. Figure 4 a illustrates that healthy control and G2 exhibit higher deformability values than G1 at a laser wavelength of 1064 nm. In addition, patients in G1 exhibited photo-damage at power values beyond 200 mW. However, RBCs of all sample groups exhibited photo-damage at power levels less than 15 mW at laser wavelength of 532 nm with OD less than the value at a wavelength of 1064 nm (Fig. 4b). Furthermore, increased laser trapping power demonstrated no relation with time of returned RBCs to initial size. The results are listed in Table 2.

Fig. 4.

Optical deformability as a function to laser trapping power for all sample groups: laser wavelength for a λ = 1064 nm and b λ = 532 nm

Table 2.

Optical deformability as a function to laser trapping power in healthy control and patients groups G1and G2 at laser wavelength of 1064 nm and 532 nm

Laser trapping wavelength (nm)	Trapping power (mW)	Optical deformability of RBC sample ± SE
		Control	G1	P value
			G2
1064	20	0.0241 ± 0.00382	0.0058 ± 0.00061	0.0001
			0.0208 ± 0.00263	0.016
	60	0.0389 ± 0.00462	0.0124 ± 0.00106	0.0001
			0.0339 ± 0.00407	0.806
	100	0.0515 ± 0.00582	0.0239 ± 0.00185	0.0001
			0.044 ± 0.00429	0.046
	140	0.0575 ± 0.00778	0.033 ± 0.0024	0.0001
			0.0564 ± 0.00569	0.130
	180	0.0701 ± 0.00817	0.047 ± 0.0037	0.0001
			0.0699 ± 0.00618	0.04
	220	0.09 ± 0.00804	Photo-damaged	—
			0.0886 ± 0.00809	0.217
532	5	0.002 ± 0.00065	0.00243 ± 0.00075	0.957
			0.0027 ± 0.00456	0.151
	7	0.0062 ± 0.0019	0.0038 ± 0.00369	0.455
			0.0038 ± 0.00369	0.849
	9	0.0098 ± 0.00318	0.0054 ± 0.00224	0.351
			0.0066 ± 0.00231	0.192
	11	0.0142 ± 0.00269	0.0099 ± 0.00394	0.227
			0.0124 ± 0.00129	0.099
	13	0.0185 ± 0.00355	0.013 ± 0.00479	0.832
			0.0153 ± 0.00091	0.675
	15	0.0681 ± 0.02091	0.0124 ± 0.00484	0.003
			0.0313 ± 0.00091	0.001

As shown in Fig. 4, OD for both healthy control and patient groups increased linearly with increasing trapping power and response for all sample groups. G1 demonstrated lower OD responses with increasing laser beam power than healthy control and G2 due to the therapeutic effect of HU and improving RBC deformability. Optical images for trapped RBC were taken at different laser powers and compared with the optical images before trapping. The results are shown in Fig. 5. The results at wavelength of 1064 nm (Fig. 5a) for all the trapped cells exhibited elongation along the optical axis of laser beam compared with the cells before trapping. But, patient with G1 had a photo-damaged at power higher than 200 mW. In contrast, Fig. 5b at laser wavelength 532 nm appeared a little increasing in value of RBC size related to the increasing in the laser trapping power for all sample groups as well as the RBCs at power less than of 15 mW exhibit a photo-damage.

Fig. 5.

Optical images of RBCs before and after trapping of laser beam at different powers for all sample groups. Laser wavelength for a λ = 1064 nm and b λ = 532 nm

Investigating the ability of RBCs to change shape or the shape recovery process (relaxation) of cells after removal from a deformed parachute shape is important. The relaxation time of sample groups on OD was explored at two different wavelengths of 1064 and 532 nm. The results are displayed in Fig. 6. As shown in Fig. 6a, OD of relaxed cells increased significantly immediately after direct laser trapping at 1 s and returned rapidly to their initial size at a wavelength of 1064 nm. It was observed that at an approximate time of t = 12 s healthy RBCs returned to 98% of their initial size compared with RBCs of G2 and G1, which returned to 97% and only 55%, respectively, of their original size. RBCs of healthy control and patients in G2 returned to their original size and shape after t = 16 s compared with RBCs of G1 patients that slowly returned to 88% of their initial size but achieved their original size after t = 24 s. In addition, relaxation time demonstrated no statistically significant difference for all groups, and RBCs failed to return to their original shape at a laser wavelength of 532 nm (Fig. 6b). Figure 7 shows the optical images for each trapped RBC that were taken during the relaxation time and returning to original shape and size at two laser wavelengths of 1064 nm and 532 nm. It is noticed clearly that in Fig. 7a at laser wavelength 1064 nm, the RBC returned to its original size and shape for all sample groups, and RBC for patients in G1 had a long time to return to its original size and shape compared with the healthy control and G2. Furthermore, at laser wavelength 532 nm (Fig. 7b), the RBC did not return to their original shape at frame time of 24 s. The results are summarized in Table 3.

Fig. 6.

Optical deformability as a function to RBC relaxation time for health control and patient groups G1and G2. Laser wavelength for a λ = 1064 nm and b λ = 532 nm

Fig. 7.

Optical images of RBCs before (zero time) and after trapping of laser beam according to the relaxation time for all sample groups. Laser wavelength for a λ = 1064 nm and b λ = 532 nm

Table 3.

Optical deformability as a function of the relaxation time in healthy control and patients groups G1and G2 at laser wavelength of 1064 nm and 532 nm

Laser trapping wavelength (nm)	Relaxation time (s)	Optical deformability of RBC sample ± SE
		Control	G1	P value
			G2
1064	Zero time	0.00	0.00	—
	1	0.0947 ± 0.00608	0.0428 ± 0.00301	0.0001
			0.0943 ± 0.00441	0.002
	4	0.0896 ± 0.00618	0.0367 ± 0.00277	0.0001
			0.0706 ± 0.00442	0.002
	8	0.0357 ± 0.00345	0.0278 ± 0.00236	0.0001
			0.0379 ± 0.0034	0.711
	12	0.0013 ± 0.00134	0.0193 ± 0.00206	0.0001
			0.0022 ± 0.00111	0.392
	16	0.00	0.0049 ± 0.00145	—
			0.00	—
	20	—	0.0027 ± 0.00189	—
			—	—
	24	—	0.00	—
			—	—
532	Zero time	0.00	0.00	—
	1	0.0311 ± 0.00619	0.0243 ± 0.00456	0.191
			0.0269 ± 0.00347	0.354
	4	0.0227 ± 0.00514	0.0201 ± 0.00369	0. 118
			0.0211 ± 0.00352	0.977
	8	0.0118 ± 0.00358	0.0094 ± 0.00231	0.006
			0.0098 ± 0.00322	0.003
	12	0.0008 ± 0.00045	0.0024 ± 0.00129	0.019
			0.0035 ± 0.00175	0.005
	16	0.0152 ± 0.01043	0.0013 ± 0.00091	0.015
			0.0014 ± 0.0014	0.003
	20	0.0014 ± 0.02091	0.0019 ± 0.00091	0.006
			0.0013 ± 0.0014	0.321

The results showed the reduced elongation and long shape recovery time of OD in G1 patients. The optical trapping at the region of infrared (IR) wavelength (1064 nm) demonstrated better than visible wavelength (532 nm) to understand biological implications and therapeutic effectiveness, due to, no heat damage to living cells, Since the absorption of molecules like hemoglobin falls rapidly in the IR region. This result agrees with the findings of other biological tissues (Ashkin 2000; Avsievich et al. 2020; Konig et al. 1996; Chen et al. 2020; Leitz et al. 2002; Ying et al. 2020).

The ability of RBCs to change shape or go through the shape recovery process (relaxation) of cells after removal from a deformed parachute shape was examined. The RBC shape recovery time as a function of the laser trapping power using a laser wavelength of 1064 nm is plotted in Fig. 8 for all the sample groups. We found that the shape recovery time of RBCs between control and G2 groups was 0.35 s (narrow bath) while the shape recovery time difference of RBCs between control and G1 groups was extended to approximately 6 sec (wide bath). The shape recovery time of G1 patient exhibits reduced deformable morphological changes in the presence of optical trapping. Morphological change properties of RBCs improve the different degrees of shape recovery time.

Fig. 8.

Shape of RBC recovery time to initial size as a function to laser trapping powers in all sample groups

Notably, the change in RBC diameters is influenced by measured deformations along transverse (maximum) and longitudinal (minimum) axes in the laser beam and perpendicular directions. The percentage change in the maximum and minimum diameters of all the sample groups was explored under different laser trapping powers for infrared laser with a wavelength of 1064 nm. The results are illustrated in Fig. 9. The healthy group exhibited a maximum RBC elongation of 10% compared with patients in G1 and G2 at 3% and 9%, respectively. A similar behavior of the RBC change was calculated in (Pellizzaro et al. 2012).

Fig. 9.

The percentage RBC change diameter as a function to laser trapping power for health control and patient groups G1 and G2. a Maximum change diameter and b minimum change diameter

Conclusion

We presented a laser tweezer technique based on single and/or dual-laser beams as a biophotonic tool to trap a single biological cell in this work. The results confirmed that the technique can detect the subtle change of RBC deformability between normal and SCD blood with high sensitivity and has the advantage of measuring the real-time life of individual cells. Thus, the constructed system successfully measured physical parameters and shape recovery of RBCs. Characteristics of biophysical and biomechanical properties of sample groups were investigated using the proposed technique. The near-infrared laser at a wavelength of 1064 nm demonstrated better optical trapping and clearer RBC characteristics than the laser at 532 nm. Furthermore, healthy blood and HU-treated patients exhibited higher OD than untreated patients. Both healthy control and HU-treated patients showed faster RBC relaxation time than untreated patients.