Application of digital holographic microscopy to evaluate the dynamics of a single red blood cell influenced by low-power laser light

Highlights

• Light from a Low-Level-Laser-Therapy scanner is invasive for erythrocyte dynamics.

• A coherent red light strongly impacts the cells drift speed.

• A standard deviation is a sensitive statistical marker of the erythrocyte dynamics.

• An open-source image processing algorithm is essential for data reproduction.

• The light-cell interaction was studied because of the everyday use of LLLT scanners.

Abstract

In the paper, a shift of individual human erythrocytes drifts speed, stimulated by various low-power lasers irradiation, was studied by digital holographic microscopy (DHM). Well known in physiotherapy, a low-level-laser-therapy (LLLT) scanner was used as the stimulation light source, with diode and He-Ne lasers to compare.

In the article, an open-source image processing algorithm for simultaneous dynamics and geometrical properties evaluations of the red blood cells is proposed. A strong influence of the low-level red light on the red blood cells dynamics was observed. The effect is important for a better understanding of the laser treatment mechanism.

The proposed phase image analysis algorithm significantly improves the individual cells tracing precision for a life science application. The programme might be useful for optical tweezer, imaging flow cytometry, or high precision cells-photon interaction research.

Introduction

Light is modified in amplitude and phase when travelling through a sample, in general. Cells, mostly as transparent objects, do not absorb light, do not change the wave amplitude, but affect its phase. Therefore, cells are not detectable by classical bright-field microscopy. The conventional bright-field imaging measures amplitude information only. As the QPI (Quantitative Phase Imaging), the holography or interferometry retrieves both amplitude and phase information [25].

A first conception of the holographic measurement technique was proposed already in 1920 by Wolfke [53], but with no experimental proof. Later, the holographic microscopy principle was independently realised by the Nobel prize winner Denis Gabor in 1948 [15]. He described a research story of the holography, in details, in his Nobel lecture [16]. Kim [21] presented an updated, comprehensive review of a subset of research and development in digital holography, focusing on microscopy techniques and applications. Park et al. [34] showed a review on the more general QPI application in biomedicine.

Researchers are using digital holographic microscopy (DHM) extensively nowadays. Kemper and von Bally [20] introduced a powerful application of DHM for long-term observation of living cells (red blood and pancreas tumour cells) and engineered surface imaging. They used a setup based on a laser’s coherent light (frequency-doubled Nd:YAG laser, $\lambda =532$ nm).

Rappaz et al. [42] used DHM for a comparative study of human erythrocytes with confocal microscopy, and impedance volume analyser. Later, Rappaz [43] with another team, used DHM for spatial analysis of red blood cell membrane fluctuations. Comprehensive applications of digital holographic microscopy for measuring biophysical parameters of living cells were described in [44].

Holographic microscopy is a useful technique for surface metrology. For semi-transparent object measurements, transmission DHM is highly useful. Charrière et al. [9] described a precision characterisation of micro-optics with DHM transmission imaging. They showed comparison measurements with white-light interferometry (WLI), with differences less than $1\%$. Holmes and Pedder [18] showed a remarkable application of DHM for laser machined microlenses metrology. Montfort et al. [31] presented high precision roughness measurements on micro-balls by reflection DHM, compared with a stylus profilometer. The similar reflection technique was introduced by Sandra et al. [47] for the determination of local defects on the outer surface of microshell. Colomb et al. [11] presented in details digital holographic reflectometry.

The holography technique allows retrieving optical topography information from a single image grab, without any scanning. Without a typical beam focusing on a sample, offers the intervention of digital processing at a level that can not be handle by the classical microscopy. In the DHM, a fast CCD camera records a hologram produced by the interference between a reference wave and a wave emanating from a specimen. That solution allows real-time measurements, which is crucial for biomedical applications. Cuche et al. [12] gave the complete description of the method in the reviewed paper.

Transmission DHM is a technique with significant advantages for cells and tissues imaging. The microscope can perform high accuracy, non-invasive measurements of volume, refractive index, and haemoglobin content of single living red blood cells (RBC’s), and more. Pavillon et al. [36] developed an imaging system combining digital holographic microscopy (DHM) with epifluorescence microscopy. They used it for cell morphology and intracellular calcium homeostasis investigation.

Charrière et al. [10] presented practical use of transmission DHM for different pollen particles identification. Boss et al. [7] applied a dual-wavelength digital holographic microscopy to measure the absolute volume of living cells, such as human embryonic kidney cells, Chinese hamster ovary cells, human red blood cells, mouse cortical astrocytes, and neurons.

For life science, DHM has a unique feature, perform numerically vary the focus from a single hologram, with no necessarily moving the sample. In this way, also rapid 3D cells imaging can be performed [14]. As one of the QPI (Quantitative Phase Imaging) technique, holographic microscopy can retrieve cellular metabolism and activities that can play a role in human diseases pathophysiology. Lee et al. [25] showed a comprehensive overview of the QPI application and RBC’s dynamics investigations’ importance.

Red blood cells dynamics is intensively investigated [50] as well as their interaction with a coherent light source by using various techniques. An in-depth study of the effects is necessary because widely lasers applications in a biomedical field are observed. Some of the RBC’s-laser-light interactions are already fairly described. Makropoulou et al. [27] suggested that He-Ne laser irradiation did not cause any critical change to human red blood cell membranes. In 2009, Al-Yasiri [2] concluded that exposure of red blood cells to low-level diode laser ($\lambda =650$ nm, $W=50$ mW) for an extended time equal or more than 40 min led to denaturation of the membrane protein of red blood cells.

New insights into the understanding of the RBC interaction mechanism and pulsed He-Ne laser irradiation effects on blood cell disaggregation process was described by Zhu et al. [55]. Al Musawi et al. [1] showed an influence of low power violet laser irradiation on red blood cell volume and sedimentation rate in human blood. Cui et al. [13] studied the effect of low-intensity He-Ne laser on the human erythrocyte membrane’s structure by atomic force microscopy.

A promising research direction of the DHM application in the field was given by Marquet et al. [28], where RBC’s membrane eigenmode energy distribution was investigated for biomarkers application.

Cacace et al. [8] introduced the QPI techniques and the holographic method as still developing biomedical application tools. Automatisation of the reconstruction procedures is recommended to make these techniques more accessible to non-specialised personnel. Our proposed R algorithm is a step to achieve this goal.

A promising technique for imaging the cells was reviewed by Balasubramani et al. [5]. Holographic tomography (HT) gives the possibility for biophysical erythrocytes imaging together with their metabolic activities.

However, no comprehensive study of the individual cell’s dynamics in the laser field was presented yet in the literature.

Therefore, considering the above research gaps, the dynamics of a human single red blood cell influenced by low-power laser light has been presented in this work. The commercial transmission digital holographic microscopy (Lyncèe Tec DHM T-1000) was used, with external phase image processing for the first time for the investigation. Fast, automatic, open-source phase image processing R algorithm [40], for single red blood cells analysis, is proposed.

The various type of lasers stimulated the cells; well known in physiotherapy, a low-level laser therapy (LLLT) scanner [24], a He-Ne laser, and a diode laser module. Data analysis showed a strong influence of the laser light with different wavelength. A significant drift speed shifting of the single red blood cells was observed. At the same time, the geometry of the cells was monitored.

The proposed image processing algorithm might be useful for other cells imaging techniques, such as optical tweezer, flow cytometry or more advanced, where precise cells tracing is a priority. For all of the microscopy investigations, and especially for DHM applications, accurate phase image processing is an essential part of the data analysis. Fast, open-source algorithms for biomedical images processing are highly recommended.

The investigations with DHM showed for the first time the importance of research with multiple wavelengths on the interactions between red blood cells and the low-level-laser-light. The paper shows that commercial laser scanners for a biomedical application can be invasive, not “non-invasive” as they usually called. Results offer a revision possibility of the daily use of commercial biomedical scanners for better patients protection. We showed the influence of coherent light on erythrocytes dynamics is complex, wavelength connected and might be physiological disturbing. Moreover, research on the cell-light interaction by DHM application may provide a new approach for a possible biomarkers investigation.

The proposed experiment is the first whistleblower, and research is continuing. We will investigate the biophysics of thermal, excitation and scattering effects in a long-term study in progress, based on the presented experience.