Cell Refractive Index Research Articles

A PhC-SOA based cancerous cell detection biosensor

In this paper, we present a novel method to design an ultra-small photonic integrated biosensor to detect cancerous cells. The proposed biosensor is based on the self-phase modulation in PhC-SOA, inducing a frequency shift on a pulse traveling through the device. The amount of the frequency chirp depends on the group velocity of the active medium waveguide being determined by the refractive index of the microfluidic infiltrating the holes around the waveguide. The refractive index of the microfluidic is also determined by the cell type that can be normal or cancerous. Since the refractive index of a cancerous cell is higher than that of a normal one, the group index of the waveguide and the output chirp will decrease. By measuring the amount of the output chirp, we can detect the cell type. The Simulation results showed that for a 0.02 change in the refractive index of the cell, a 3.71 nm central wavelength shift occurred for a 10-ps 7-mW gaussian pulse input with a central wavelength of 1533.53 nm. In terms of the wavelength shift, the sensitivity and figure of merit are 185.5 and 530, respectively. To detect the cell type, we integrated a PhC channel drop filter to drop the chirped signal due to the cancerous cell infiltration. Designing an appropriate PhC-CDF leads to achieving an ultra-small cancerous detection cell biosensor with more than 97% precision.

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux.

Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell's refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells' division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images.

A method for determining the refractive index of a spheroid cell based on orthogonal phase microscopy

Refractive index imaging of a cell that reflects its morphological and structural feature has attracted increasing attention because of its important applications in life science, bioengineering and clinical applications. Based on the analysis of two orthogonal phase micrographs, we present a decoupling method for solving the refraction index distribution. In this method, a cell with one nuclei is take as a sample. At first, the radii of the nucleus and cytoplasm are determined by analyzing the gradient distributions of the associated phases. Then, the refractive index of each medium of the cell is decoupled from the measured phases. Lastly, the whole refractive index of the cell can be obtained according to the spatial position of each medium. Simulation and experimental results of a spheroid lymphocyte demonstrate the effectiveness and accuracy of this method. This method provides a new technical idea for 3D sub-structure imaging of the spheroid cells.

Effect of intermittent high glucose on oxygen-glucose deprivation/refurnish neuronal survival

To investigate the effect of intermittent high glucose on oxygen-glucose deprivation/refurnish (OGD/R) neuronal survival. The primary cultured hippocampal neurons of mice were sub-cultured when the cell fusion reached about 80%. Cells in logarithmic growth phase were placed in a hypoxic incubator (37 centigrade, 5% CO2, 95% N2) to simulate cell hypoxia. The culture medium was replaced by glucose-free Hank equilibrium salt solution (HBSS) to simulate cell hypoglycemia. The normal glucose and oxygen control group was set up. Cell morphology was observed under inverted phase contrast microscope after 6 hours of hypoxia and hypoglycemia treatment, and cell viability was detected by CCK-8 cell proliferation assay kit, and then grouping experiment was carried out. The cells were randomly divided into four groups. The cells were cultured in different concentration glucose medium under normal oxygen, 5% CO2 and 37 centigrade for 72 hours to prepare OGD/R model of cell ischemia/reperfusion. The low-glucose control group was cultured in medium containing 5.5 mmol/L glucose. The constant high-glucose group was cultured in medium containing 33.0 mmol/L glucose. The intermittent high-glucose group was cultured in medium containing 33.0 mmol/L glucose for 3 hours then in medium containing 5.5 mmol/L glucose for 2 hours alternately for 3 times during the day, and overnight in medium containing 33.0 mmol/L glucose at night. The hyperosmotic control group was made up of 5.5 mmol/L glucose medium and mannitol. The osmotic pressure was the same as that of the constant high-glucose group, and the effective glucose concentration was the same as that of the normal glucose and oxygen group, so as to eliminate the effect of osmotic pressure changes caused by the high-glucose medium on the results. Cell morphology was observed under inverted phase contrast microscope after 72 hours of cell culture in each group. Cell viability was measured by CCK-8 kit, and apoptotic rate was measured by flow cytometry. The inverted phase contrast microscope showed that the cells in the normal glucose and oxygen control group were plump and refractive, and had obvious nucleus, clear processes and high cell activity. After 6 hours of hypoxia and hypoglycemia treatment, the cells were shrunk, refractive index was poor, the nucleus was unclear, the processes were not clear, and the cell activity was significantly lower than that of normal glucose and oxygen control group (A value: 0.34±0.06 vs. 1.09±0.06, P < 0.01), which indicated that the model of oxygen-glucose deprivation (OGD) was successfully prepared. After 72 hours of culture with different concentrations of glucose, the cells in the low-glucose control group were shrunk, the cell membrane was incomplete, the nucleus was unclear, and number of necrotic cells were more. In the constant high-glucose group, the refractive index of cells was poor, a large number of cells floated, and the nucleus was not obvious. In the intermittent high-glucose group, the cell morphology was normal, the refractive rate of cells was decreased slightly, and the necrotic cells were less. In the hypertonic control group, the cell status was close to that in the constant high-glucose group. Compared with the low-glucose control group or constant high-glucose group,the cell viability in the intermittent high-glucose group was significantly increased (A value: 2.04±0.15 vs. 0.64±0.18, 1.16±0.16, both P < 0.01), the apoptotic rate was significantly decreased [(59.60±2.55)% vs. (78.15±15.77)%, (95.60±0.14)%, both P < 0.05]. There was no significant difference in cell activity or apoptotic rate between the hypertonic control group and the constant high-glucose group [cell activity (A value): 1.07±0.07 vs. 1.16±0.16, apoptotic rate: (87.80±4.53)% vs. (95.60±0.14)%, both P > 0.05]. Intermittent high glucose within a certain range had protective effect on OGD/R neuronal survival.

Statistical Analysis of Photonic Crystal Spectra for the Independent Determination of the Size and Refractive Index of Cells.

Photonic crystal flow cytometry is a very attractive platform due to its great sensitivity in combination with a very compact design. Previous studies have demonstrated the possibility to use spectral processing for the measurement of a wide range of parameters, from simple object counting to independent analysis of the buffer solution and immersed microscale objects. Here we propose to go to the next level and simultaneously determine the shape and the refractive index of the cells.

Determination of the complex refractive index of cell cultures by reflectance spectrometry

In this paper we propose a new approach to using reflectance spectrometry in connection with the Kramers-Kronig analysis for the determination of the complex refractive index of biological cells. Applying this procedure, the real and imaginary parts of the refractive index (refractive index and extinction coefficient) can be simultaneously determined. The accuracy of this procedure in the determination of the refractive index and extinction coefficient of culture media proved to be comparable with spectroscopic ellipsometry. Applying this procedure on the human umbilical vein endothelial cells (HUVEC), the results obtained from time-series measurements showed significant changes in the complex refractive index of cell cultures within 72h, the most important increases for both real and imaginary parts of the refractive index being recorded in the first 24h, when synthesis processes are happening. Thus, the analysis of the time-dependent changes in the complex refractive index provides information about the frequencies of the modifications that occur on both organizational structure and cells composition during the cell cycle. In conclusion, the combination of reflectance spectrometry with the Kramers-Kronig analysis is a feasible way to determine the complex refractive index of biological cells and to assess the events taking place during the cell cycle.

On the birefringence of healthy and malaria-infected red blood cells

The birefringence of a red blood cell (RBC) is quantitatively monitored as it becomes infected by a malarial parasite. Large changes occur in the cell's refractive index at different stages of malarial infection. The observed rotation of an optically trapped, malaria-infected RBC is not a simple function of shape distortion: the malarial parasite is found to itself exercise a profound influence on the rotational dynamics by inducing stage-specific birefringence. Our measurements shed new light on the competition between shape- and form-birefringence in RBCs. We demonstrate the possibility of using birefringence to establish very early stages of infected parasites and of assessing various factors that contribute to birefringence in normal and infected cells. Our results have implications for the development and use of noninvasive techniques of quantifying changes in cell properties induced by malaria disease pathology.

Cell separation by the combination of microfluidics and optical trapping force on a microchip

We investigated properties of cells affecting their optical trapping force and successfully established a novel cell separation method based on the combined use of optical trapping force and microfluidics on a microchip. Our investigations reveal that the morphology, size, light absorption, and refractive index of cells are important factors affecting their optical trapping force. A sheath flow of sample solutions created in a microchip made sample cells flow in a narrow linear stream and an optical trap created by a highly focused laser beam captured only target cells and altered their trajectory, resulting in high-efficiency cell separation. An optimum balance between optical trapping force and sample flow rate was essential to achieve high cell separation efficiency. Our investigations clearly indicate that the on-chip optical trapping method allows high-efficiency cell separation without cumbersome and time-consuming cell pretreatments. In addition, our on-chip optical trapping method requires small amounts of sample and may permit high-throughput cell separation and integration of other functions on microchips.

Scattering of He–Ne laser light by an average-sized red blood cell

The scattering of He-Ne laser light by an average-sized human red blood cell (RBC) is investigated numerically. The RBC is modeled as an axisymmetric, low-contrast dielectric, biconcave disk. The interaction problem is treated numerically by means of a boundary-element methodology. The differential scattering cross sections (DSCS's) corresponding to various cell orientations are calculated. The numerical results obtained for the exact RBC geometry are compared with those corresponding to a scattering problem in which the cell is assumed to be either a volume-equivalent sphere or an oblate spheroid. A parametric study demonstrating the dependence of the DSCS on the wavelength of the incident wave and the cell's refractive index is presented.

Refractive index of planktonic cells as a measure of cellular carbon and chlorophyll a content

Current methods for determining carbon content in individual planktonic cells from particle volume alone may involve large errors, and no routine technique exists for determining chlorophyll a content in individual phytoplankters. In this study the concept of using the refractive index of cells as a measure of intracellular concentrations of carbon and chlorophyll a is discussed. Specifically, the real part of the refractive index n (at light wavelengths where absorption is negligible or very small) is shown to correlate well with the intracellular concentration of carbon, C i. The imaginary part of the refractive index n′ (in the red band of chlorophyll a) correlates well with the intracellular chlorophyll concentration, Chl i. These relationships were found to be nearly identical for two species, a cyanobacterium Synechococcus and a diatom Thalassiosira pseudonana, over a two-fold range in C i and Chl i. This range was associated with interspecies differences and intraspecies variations in the cell properties over a day–night cycle. These observations and the underlying theoretical considerations suggest that the relationships C i vs. n and Chl i vs. n′ may be robust and hold for a variety of planktonic species regardless of interspecies and intraspecies variability in cellular carbon content, Chl a content, and cell size. In addition, these relationships may be applicable to single-particle analysis of natural water samples, which promises a unique capability to acquire information about the distribution of carbon and chlorophyll a among individual cells, different size classes, and taxonomic groups of planktonic microorganisms in the ocean. Further research with various planktonic species is needed to examine the generality of the relationships C i vs. n and Chl i vs. n′ before the approach can be implemented in field studies.

The effect of nitrogren limitation on the absorption and scattering properties of the marine diatom Thalassiosira pseudonana

Optical properties of the marine diatom Thalassiosira pseudonana were examined in nitrate‐limited semicontinuous cultures for growth rates (µN) varying from 0.22 to 1.32 d−1. Two experiments were conducted at each growth rate in which sampling of the culture was done at either the dark‐to‐light (D‐L) or light‐to‐dark (L‐D) transition of a 12‐h photoperiod. Optical cross sections and efficiency factors for absorption, scattering, and attenuation were calculated from measurements of the spectral absorption and beam attenuation coefficients, cell concentration, and the size distribution of cells in suspension. The refractive index of cells, relative to seawater, was calculated from these data using an inverse method. Nitrogen limitation strongly influences the optical properties of this species. Absorption cross sections increased more than twofold with increasing µN, predominantly caused by increases in the imaginary part of the refractive index, n′. At the red absorption peak, n′(673) increased 140% and was strongly correlated with intracellular Chl a concentration. For most visible light wavelengths, scattering cross sections were not a strong function of growth rate but were consistently largest in the L‐D transition experiments. The difference between D‐L and L‐D experiments at any given µN increased with increasing growth rate; for example, scattering cross sections at 660 nm in the L‐D experiments were 25% (µN = 0.22 d−1) and 90% (µN = 1.32 d−1) larger than in the respective D‐L experiments. The real part of the refractive index at λ = 660 nm varied from 1.024 to 1.039 among all experiments and was positively correlated with intracellular carbon concentration. The Chl a‐specific absorption coefficient decreased with increasing µN (e.g. 43% decrease in the blue Soret band) in response to decreases in the carotenoid‐to‐Chl a ratio and increased pigment packaging. The Chl a‐specific scattering coefficient at 660 nm decreased from 0.55 to 0.09 m2 (mg Chl a)−1 with increases in µN. Our results suggest that the optical properties of phytoplankton populations in nitrogen‐limited surface waters may be significantly altered from those in deeper portions of the water column.

Optical characterization of the oceanic unicellular cyanobacterium Synechococcus grown under a day‐night cycle in natural irradiance

The optical properties of the oceanic cyanobacterium Synechococcus (clone WH8103) were examined in a nutrient‐replete laboratory culture grown under a day‐night cycle in natural irradiance. Measurements of the spectral absorption and beam attenuation coefficients, the size distribution of cells in suspension, and microscopic analysis of samples were made at intervals of 2–4 hours for 2 days. These measurements were used to calculate the optical properties at the level of a single “mean” cell representative of the actual population, specifically, the optical cross sections for spectral absorption , scattering , and attenuation . In addition, concurrent determinations of chlorophyll a and particulate organic carbon allowed calculation of the Chl a‐ and C‐specific optical coefficients. The refractive index of cells was derived from the observed data using a theory of light absorption and scattering by homogeneous spheres. Low irradiance because of cloudy skies resulted in slow division rates of cells in the culture. The percentage of dividing cells was unusually high (&gt;30%) throughout the experiment. The optical cross sections varied greatly over a day‐night cycle, with a minimum near dawn or midmorning and maximum near dusk. During daylight hours, and can increase more than twofold and by as much as 45%. The real part of the refractive index n increased during the day; changes in n had equal or greater effect than the varying size distribution on changes in and . The contribution of changes in n to the increase of during daylight hours was 65.7% and 45.1% on day 1 and 2, respectively. During the dark period, when decreased by a factor of 2.9, the effect of decreasing n was dominant (86.3%). With the exception of a few hours during the second light period, the imaginary part of the refractive index n′ showed little variation over a day‐night cycle, and was largely controlled by variations in cell size. The real part of the refractive index at λ = 660 nm was correlated with the intracellular C concentration and the imaginary part at λ = 678 nm with the intracellular Chl a concentration. The C‐specific attenuation coefficient showed significant diel variability, which has implications for the estimation of oceanic primary production from measurements of diel variability in beam attenuation. This study provides strong evidence that diel variability is an important component of the optical characterization of marine phytoplankton.

Diel variations in the optical properties of a marine diatom

Diel variations in the optical properties of the marine diatom Thalassiosira p̀seudonana were examined in a nutrient‐replete laboratory culture exposed to a day‐night cycle in natural irradiance which simulated the light regime in upper layers of the euphotic zone (~10 m). Measurements of the beam attenuation coefficient, absorption coefficient, and size distribution of cells in suspension were used to determine optical cross‐sections and refractive index of cells. The optical cross‐sections varied greatly over a diel cycle; the absorption cross‐section (Xa) increased by as much as 35% and the attenuation (σ̄c) and scattering (σ̄b) cross‐sections by 80% between minima near sunrise and maxima near sunset. These changes were associated with significant variations in refractive index and cell size. Diel changes in the imaginary part of the refractive index (n'), driven by variation in intracellular pigment concentration, accounted for 58– 72% of the difference between the daily maxima and minima in σ̄a. Variations in the real part of the refractive index (n) contributed substantially (up to 85%) to the daily range in σ̄c and σ̄b. Increases in cell size were important during the morning, increasing the attenuation cross‐section to a similar extent as the refractive index. Variability in n correlated strongly with changes in intracellular C concentration. The normalization of attenuation and scattering cross‐sections to cellular C content resulted in a threefold reduction in the magnitude of diel variations. If such patterns are general features of planktonic microorganisms in the natural environment, it would suggest that varying particle concentration need not be the only (or even major) factor responsible for diel variability in the bulk optical properties of seawater. As a consequence, estimating particle production rates from diel variability in the beam attenuation coefficient may not be justified in some oceanic situations; however, using this variability to estimate production in terms of particulate organic C may be appropriate.

Factors affecting the viability of air-borne bacteria. III. The role of bonded water and protein structure in the death of air-borne cells.

The relationship between cellular water and the death of air-borne cells, and the effect of added substances on these relationships, has been studied. The rate of death of air-stored cells has been correlated with the amount of water bonded to the cellular proteins. Refractive indices of cells, measured by light scatter and interferometry, have suggested that cells suspended in solutions of various compounds contain less water. With the exception of urea, the more able a compound is in displacing water, the less its protectiveness to air-borne cells. Inositol produced the smallest change in the water content and refractive index of cells. This is taken to indicate that, in the absence of water, this compound can form hydrogen bonds with cellular protein which are reversible with water. There appears also to be some relationship between the Gram stain reaction and aerosol stability of the cells. Ribonuclease and lysozyme treatment of air-stable cells rendered them air sensitive, whereas resistance to some antibiotics, and suspension in some antibiotics, afforded stability to air-sensitive cells. It is suggested that the site of damage is the cellular "membranes" which break down on desiccation and result in a loss of differentiation within the cell.