Fig 1 - uploaded by Kersti Alm
Content may be subject to copyright.
A cell culture captured using digital holographic microscopy (A) and phase contrast microscopy (B). The cells are BN7005-H1D2 mouse fibroblasts which are approximately 20 μ m in diameter. image calculation is then performed using a Fresnel approximation (Cuche et al., 1999) Varieties of the setup exist (Popescu et al., 2006; Gustafsson et al., 2004) and it is also sometimes given different names (Ikeda et al., 2005). There are also variations in the calculation of the actual recognizable image. The outcome of the reconstruction of a digital hologram is two images, one representing the amplitude of the light and one representing the phase changes of the light. The amplitude image is similar to an image of the sample captured in ordinary white light. The resolution in the direction of the incident light is very high, down to the order of nanometers (Cuche et al., 1999). By combining the phase imaging technique with physical rotation of the sample, a high resolution, label-free tomographic image can be obtained (Popescu et al., 2008). One of the advantages of DHM is that the object that is studied will always be in focus, and the image can be recalculated many times if necessary to find the best focus. Images can be captured of both single cells and populations, and the images can be presented as traditional cell images as well as 3-D representations. DHM can most likely compete with many of the other methods used today within the area of cell biology and microscopy. The technique is easy to learn and simple to use, it is cheap and gives both qualitative and quantitative results. Several research groups have used DHM for cell biology studies. 

A cell culture captured using digital holographic microscopy (A) and phase contrast microscopy (B). The cells are BN7005-H1D2 mouse fibroblasts which are approximately 20 μ m in diameter. image calculation is then performed using a Fresnel approximation (Cuche et al., 1999) Varieties of the setup exist (Popescu et al., 2006; Gustafsson et al., 2004) and it is also sometimes given different names (Ikeda et al., 2005). There are also variations in the calculation of the actual recognizable image. The outcome of the reconstruction of a digital hologram is two images, one representing the amplitude of the light and one representing the phase changes of the light. The amplitude image is similar to an image of the sample captured in ordinary white light. The resolution in the direction of the incident light is very high, down to the order of nanometers (Cuche et al., 1999). By combining the phase imaging technique with physical rotation of the sample, a high resolution, label-free tomographic image can be obtained (Popescu et al., 2008). One of the advantages of DHM is that the object that is studied will always be in focus, and the image can be recalculated many times if necessary to find the best focus. Images can be captured of both single cells and populations, and the images can be presented as traditional cell images as well as 3-D representations. DHM can most likely compete with many of the other methods used today within the area of cell biology and microscopy. The technique is easy to learn and simple to use, it is cheap and gives both qualitative and quantitative results. Several research groups have used DHM for cell biology studies. 

Context in source publication

Context 1
... Living, dying or dead cells as well as fixed cells can be studied. The first DHM images showing living cells were published in 2003 and 2004 (You et al., 2003; Carl et al., 2004), making this field of research rather new. Two of the most interesting functions of DHM is 3-D imaging of objects and to make in-focus measurements over time. Digital holography has been used to study a wide range of cells, e.g . protozoa, bacteria and plant cells as well as several types of mammalian cells such as nerve cells and tumor cells (Emery et al., 2007; Kemper et al., 2006; Moon and Javidi 2007). It has also been applied for studies of cell proliferation, cell movement and cell morphology (Kemper et al., 2009; Yu et al., 2009). Movement in both 2-D and 3-D has been studied (Langehanenberg et al., 2009; Persson et al., in press). In addition, cell viability status can be determined using DHM (Kemper et al., 2006; Kemmler et al., 2007). Interestingly, it is possible to study both single cells and entire populations simultaneously, allowing for very detailed studies. In this chapter we will compare DHM with previously used techniques and discuss the benefits and drawbacks of digital holography cell measurements. We will also present cell studies made possible by DHM. Cell imaging plays a crucial role in the understanding of cell biology. Cells are almost invisible in standard light microscopes as they do not absorb light. Cells shift the phase of the light and different light microscopy methods, such as phase contrast (Zernike, 1942) and Nomarski's differential interference contrast (DIC) (Nomarski, 1955), have been developed to transform phase information into amplitude or intensity information. Some advantages of DIC are better contrast and sharpness of the images. These light microscopy methods can only provide qualitative information as quantitative information cannot be calculated. In a recent publication, Petibois has made a review of imaging methods for cells (Petibois, 2010). Another method that is used for measurements of unlabeled adherent cells in real-time is electric impedance. The instrument xCELLigence was developed by Roche Diagnostics (Basel, Switzerland). It is a real-time cell analyzer that measures living cells without labeling. Electric impedance allows measurements of cellular processes such as proliferation, cytotoxicity, invasion, migration and cell viability (Atienza et al., 2006; Boyd et al., 2008; Ge et al., 2009) and give results comparable to imaging methods in some ways. Different staining methods have been developed that enable cells to absorb light. Dyes such as methylene blue which stains e.g. nucleic acids, haematoxylin which stains cell nuclei, eosin which stains cytoplasm and silver stains which stain proteins and DNA have been used since the mid 19 th century, while fluorescent stains, such as rhodamine which stains mitochondria, neutral red which stains lysosomes, acridine orange and DAPI, which stain nucleic acids were developed and have been widely used since the beginning of the 20 th century (Kricka & Fortina, 2009). New dyes that stain specific parts of the cells and new labels that fluoresce at certain wavelengths are developed continuously. The method to label cells with green fluorescence protein (GFP) was a major breakthrough in the 1990 ́s. GFP is less toxic than most commonly used dyes and the DNA code of GFP can be transfected into the cell's genome (Chalfie et al., 1994). When the gene contains the GFP DNA is activated, GFP will be produced by the transfected cells. As GFP affects cells less than traditional stains it gives more accurate results. However, GFP is a rather large molecule and the size probably causes steric problems. Fluorescence microscopy as well as traditional light microscopy may cause phototoxicity, and several researchers have attempted to develop non-damaging microscopy methods (Hoebe et al., 2007; Frigault et al., 2009; Logg et al., 2009). The search for a method to study cells accurately without labeling or staining them has resulted in several interferometric quantitative microscopy techniques utilizing the phase properties of coherent light to image a sample. One of them is digital holography. In 1948, Dennis Gabor invented a way to encode the phase of the light both as information and as a record containing all the information in a single recording, i.e. the hologram (Gabor, 1948). Holograms are commonly used as pieces of art and are displayed as illuminated 3-D images. Gabor's findings were the base for the development of digital holography during the 1990s (Schnars & Jueptner 1994; Cuche et al., 1999), where the information is collected on a digital sensor and then fed to a computer. Using DHM it is possible to measure cell shape, volume and dry mass without any labeling and with a very low intensity light source (Rappaz et al., 2005; Mölder et al., 2008; Rappaz et al., 2008) where the intensity is well below what is considered photo-toxic. In Fig. 1, an L929 mouse fibroblast cell culture is captured using both phase contrast microscopy and DHM. The DHM images look similar to the phase contrast images. DHM is a full field imaging of the phase of the light incident on the sensor. It can be used to image solid samples in reflection, or, which is most common in the case of biological samples, to image transparent samples in transmittance. The most commonly used system for DHM today uses an optical setup common to that of a Mach-Zender interferometer, but with the reference light at a slight angle to the light passing through the sample, and the Cells shift light, and can therefore be detect with DHM. The magnitude of the phase shift depends on the refractive index of the cell and the cell thickness as well as the difference in refractive index between the cells and their surroundings. For the DHM system to be able to measure the phase shift of the cells, their refractive index must differ from that of the background. Some cell types have a refractive index that is very close to the index of the cell culturing medium and the signal from thin parts of those cells will disappear into the background noise. This can be improved e.g . by using a double laser system, but there will always be thin cells that disappear into the background. As the DHM imaging is done using coherent light, the setup is very sensitive to refraction, reflection or changes in the polarization of the light, and large amounts of the effort of constructing a digital holographic setup is aimed at reducing background noise, introduced by optical components in the light path. One drawback is also that as of today, no known contrast agents exist that selectively increase the refractive index of different cells or parts of a cell. As with all label-free techniques, the sample is “as it is”. Also, the phase shift is modular, and to measure absolute values, a base line must be set to identify the background. In some samples background identification is difficult, and this affects the image quality. Another drawback is the time required for reconstructing an actual image from the captured hologram, a time that is however reduced as computers successively grow more powerful. The reconstruction can be separated from the actual image capture, thus allowing image capture that is faster than the reconstruction time. Studies of cell morphology can show how the cells have been affected by different treatments or by environmental factors such as temperature or pH. These studies are usually performed using different microscopy techniques, and staining or labeling of the cells is often needed. Several researchers have studied cell morphology using DHM in different contexts using neither staining nor labeling (Rappaz et al., 2005; Mölder et al., 2008; Rappaz et al., 2008). Among others, Kemper and coworkers have shown that erythrocyte shape can be clearly visualized by DHM (Kemper et al., 2007). Kemmler and colleagues studied the morphological changes during trypsinization using oligodendrocytes from rat (Kemmler et al., 2007) and Emery and colleagues detected cell swelling and shrinking in primary mouse cortical neurons (Emery et al., 2007). Schnekenburger et al (2007) have applied DHM to study the dynamics of cytoskeleton changes in the human pancreatic tumor cell lines PaTu 8988S and PaTu 8988T. They showed that the cell shape changed visibly after Latrunculin B treatment. DHM has been used to sense, monitor and recognize microorganisms (Moon & Javidi, 2008) and to study drug-induced morphological changes in pancreatic cells (Kemper & von Bally, 2008). We have used DHM to compare cell morphology during proliferation of four different adherent cell lines (Mölder et al., 2008). Nerve cells are intensely studied in order to elucidate their growth and signaling mechanisms. Nerve cells have long, almost invisible protrusions which change shape and grow. An early study showed that it is possible to measure the thickness of the neuronal processes as well as the nerve cell body with DHM (Marquet et al., 2005). In Fig. 2, the protrusions of the nerve cell can clearly be seen. Rappaz and collegues showed that swelling of nerve cell bodies caused by hypotonic shock could be studied non-invasively using DHM (Rappaz et al., 2005). The mechanisms of action The differentiation process makes cells more specialized, both in shape and performance. The process is usually studied using microscopy and western blot, and different cell labels are often used. We have studied adherent 3T3L1 fibroblasts which differentiate into adipocytes after 3 days of treatment with 0.5 mM IBMX, 10 μ g/ml insulin and 1 μ M dexamethasone. The differentiation process is easy to monitor using DHM as the cells remain undisturbed. As is clearly seen in Fig. 3, the differentiated adipocyte cells display lipid droplets which are clearly seen in DHM, but which are not so obvious when using phase contrast microscopy. In the 3-D renderings, ...

Similar publications

Article
Full-text available
An optical cloud droplet and ice crystal measurement system ICEMET (icing condition evaluation method), designed for present icing condition monitoring in field conditions, is presented. The aim in this work has been to develop a simple but precise imaging technique to measure the two often missing parameters needed in icing rate calculations cause...
Article
Full-text available
We present a method to investigate large object by digital holography with effective spectrum multiplexing under single-exposure approach. This method splits the original reference beam and redirects one of its branches as a second object beam. Through the modified Mach-Zehnder interferometer, the two object beams can illuminate different parts of...
Article
Full-text available
The variety of laser systems available to industrial laser users is growing and the choice of the correct laser for a material target application is often based on an empirical assessment. Industrial master oscillator power amplifier systems with tuneable temporal pulse shapes have now entered the market, providing enormous pulse parameter flexibil...
Article
Full-text available
Digital holography is the best tool for measuring object information data. Parallel phase-shift digital holography (PPSDH) is particularly suitable for measuring moving objects. This method generates images of objects, but the image quality is diminished because they contain abundant speckle noise. In this paper, we propose an interpolation-based a...

Citations

... Digital holographic microscopy (DHM) is a specific application of digital holography used to study microscopic scale samples. DHM has shown to be a powerful technique with high potential to analyze important cell characteristics [1,2] such as refractive index [3] and morphology, and to determine the refractive index and dry mass density of 3T3 fibroblasts and HaCaT keratinocytes during their culture growth [4], among others. The information gathered with this technique can be conveniently transformed and used in biomedical fields to gain more knowledge about cell functions. ...
Article
Digital holographic microscopy (DHM) is a technique that has high potential for analyzing biological samples and has been successfully applied to the study of cells and cell lines providing information about important parameters such as refractive index, morphology, and dry mass, among others; it has also found applicability to study the effects of therapeutic treatments. Finding the size and shape of cells is important since they tend to change in the presence of some pathologies. In this research work, we obtain the morphology thickness and refractive index of the A375 melanoma cell line through a slight tilting of the cell in a DHM setup. Further, the development of a novel mathematical expression based on this tilt and in the optical phase difference is presented. We show images of melanoma cells with the refractive index information included, and their morphology thickness as rendered from the holographic phase maps recorded with DHM.
... This knowledge can help to protect the human skin against undesirable side effects of various substances (including some siloxanes) used in skin products. DHM has also already been used to study human tissues, blood cells, and the pathophysiology of live biological cells, including nerve cells [17][18][19][20][21][22][23][24], however this microscopic technique has not yet been used in skin studies. Resources, K.M.-P.; Data curation, K.M.-P. ...
Article
Full-text available
Cyclic siloxanes (D4, D5, D6) are widely used in skin products. They improve skin sensory properties and alleviate dry skin, but there is still one report (published 2019), which regards their effects on the destruction of the skin barrier, by using fluorescence microscopy and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). A new skin-imaging technique, digital holographic microscopy (DHM), was used for the first time to investigate the impact of D4, D5, and D6 on the skin barrier. We observed irreversible damage of the stratum corneum due to the interaction with cyclic siloxanes. These substances changed: (a) the first level of the skin barrier through destabilization of the intercellular lipid lamellae and destruction of the corneocyte structure (measured with axial nanometer resolution), (b) the second level by collapse of not only corneocytes but also of a significant part of the clusters, leading to the loss of the stratum corneum integrity and formation of the lacunae, (c) the third level as an effect of the change in the surface geometrical topography of the stratum corneum and disruption of the integrity of this skin layer, measured with lateral micrometer resolution. DHM allowed also to identify an important pathway for substances to penetrate into the skin through canyons surrounding the clusters. Our investigations provide advanced information for understanding the mechanisms by which various substances pass the skin barrier, including uncontrolled diffusion into the skin.
... Further, as DHM-derived features are dependent on technical, computational, and biological variables, interpretation of these metrics must be conducted with great care. For example, optical volume has been correlated with actual cell volume, cell detachment, cell flattening, calcium fluctuations, cell cycle, cell death, cell differentiation, and protein content 8,10,[23][24][25][26][27][28][29] . Other features are of completely unknown biological meaning. ...
Article
Full-text available
Digital holographic cytometry (DHC) permits label-free visualization of adherent cells. Dozens of cellular features can be derived from segmentation of hologram-derived images. However, the accuracy of single cell classification by these features remains limited for most applications, and lack of standardization metrics has hindered independent experimental comparison and validation. Here we identify twenty-six DHC-derived features that provide biologically independent information across a variety of mammalian cell state transitions. When trained on these features, machine-learning algorithms achieve blind single cell classification with up to 95% accuracy. Using classification accuracy to guide platform optimization, we develop methods to standardize holograms for the purpose of kinetic single cell cytometry. Applying our approach to human melanoma cells treated with a panel of cancer therapeutics, we track dynamic changes in cellular behavior and cell state over time. We provide the methods and computational tools for optimizing DHC for kinetic single adherent cell classification.
... Digital holographic (DH) microscopy is a digital high-resolution holographic imaging technique with the capacity of quantification of cellular status without any staining or labeling of cells[1][2][3]. Various cellular parameters can be visualized and calculated from the particular hologram, including individual cell area, thickness, volume, and population confluence and cell counts[4][5][6][7][8][9][10]. One of the advantages of studying cells with DH microscopy is that they can be grown and analyzed in their normal growth medium during the entire study. ...
Chapter
Full-text available
Digital holographic (DH) microscopy is a digital high-resolution holographic imaging technique with the capacity of quantification of cellular conditions without any staining or labeling of cells. The unique measurable parameters are the cell number, cell area, thickness, and volume, which can be coupled to proliferation, migration, cell cycle analysis, viability, and cell death. The technique is cell friendly, fast and simple to use and has unique imaging capabilities for time-lapse investigations on both the single cell and the cell-population levels. The interest for analyzing specifically cell volume changes with DH microscopy, resulting from cytotoxic treatments, drug response, or apoptosis events has recently increased in popularity. We and others have used DH microscopy showing that the technique has the sensitivity to distinguish between different cells and treatments. Recently, DH microscopy has been used for cellular diagnosis in the clinic, providing support for using the concept of DH, e.g., screening of malaria infection of red blood cells (RBC), cervix cancer screening, and sperm quality. Because of its quick and label-free sample handling, DH microscopy will be an important tool in the future for personalized medicine investigations, determining the optimal therapeutic concentration for both different cancer types and individual treatments.
... Digital holography (DH) is a novel technique capable of three dimensional (3-D) profiling and tracking of microscale objects [8,9]. DH allows analyzing multiple parameters such as cell number, confluence, shape phase volume, cell thickness, and cell refractive index [10]. Additionally, it is an imaging technique that enables real time detection, and quantification of both single and whole populations of cells without the need of labeling. ...
Article
Atypical antipsychotic drugs induce hepatic toxicity. Thus, it is of importance to eliminate the side effects of these drugs. Herein we describe the preparation of nanoemulsions with a dietary supplement; wheat germ oil (WGO), to ameliorate the liver damage induced by clozapine and olanzapine. THLE-2 cell line was used as a model to investigate the effects of these nanoemulsions on cell viability as well as antioxidative efficiency after antipsychotic insult. In this context, a conventional cell culture method; MTT was used along with a novel cellular imaging technique called digital holography (DH) to evaluate cell viability. Obtained data confirmed that both clozapine and olanzapine induced the liver damage in in vitro model and WGO nanoemulsions were found to be effective on cells and eliminate the cytotoxic effects of these drugs. Briefly, this study has some outputs as follows; it showed that different dietary supplements can be used in such formulations instead of their pristine forms to increase bioavailability. Also, DH was successfully applied for the monitoring of cell viability and it could be a promising approach as the reactive-free cytotoxicity test.
... The cells are BN7005-H1D2 mouse fibroblasts which are approximately 20µm in diameter. [166] Theoretically, the phase measured at each pixels, ϕ i , is proportional to the OPL, which is the sum of the thickness of the different objects the laser encounters along its path times their refractive indexes. In our system the formula below [164] can be applied: ...
... It shows the body of the dendrite section with the details of protruding dendritic spines. Scale is in µm and the color code represents the difference in refractive index (from 0.04 (red) to 0.16 (violet)) between the dendrite and the extracellular medium.[166] ...
Thesis
Full-text available
The thesis deals with the control and the measurement of neuronal shapes, "control" by using adhesive micropatterns allowing to constrain cells shape in two dimensions, "measurement" by using either interferometry or fluorescence measurements in confined spaces to gain knowledge on cell dry mass and volume.The biological question at the heart of this thesis is the regulation by the neuron of its various morphological characteristics such as length, volume, in association with the establishment of the axo-dendritic polarity. These aspects are developed and deepened in the course of this thesis on experimental but also theoretical (coll. Nir Gov, Weizmann Institute) point of views.This multidisciplinary thesis topic thus builds on biological aspects and physical instrumentation.
... The importance of RI measurements for biology is well underlined in a work that it is one of the first introducing Quantitative Phase Microscopy (QPM) [14], presenting results for the thickness and RI of airway smooth muscle (ASM) cells. Due to the difficulties of QPM to decouple evaluation of thickness and RI, both involved in the evaluated optical path difference (OPD), a further procedure is presented based on digital holographic microscopy (DHM) [15] founded on a dual wavelength observation, as for other successive works [16,17]. Here, the KK equation is part of the needed formalism. ...
... As an example, we can use the reflectance data to show some parameters' extraction. We can use the procedure described in the Equation (18) and the solutions defined in Equations (16), (19) and (20). In a future paper, extending the procedures to include full numerical approach, it will be possible to fit the whole curve and compute also the imaginary part of the local RI. ...
Article
Full-text available
A hyperspectral reflectance confocal microscope (HSCM) was realized by CNR-ISC (Consiglio Nazionale delle Ricerche-Istituto dei Sistemi Complessi) a few years ago. The instrument and data have been already presented and discussed. The main activity of this HSCM has been within biology, and reflectance data have shown good matching between spectral signatures and the nature or evolution on many types of cells. Such a relationship has been demonstrated mainly with statistical tools like Principal Component Analysis (PCA), or similar concepts, which represent a very common approach for hyperspectral imaging. However, the point is that reflectance data contains much more useful information and, moreover, there is an obvious interest to go from reflectance, bound to the single experiment, to reflectivity, or other physical quantities, related to the sample alone. To accomplish this aim, we can follow well-established analyses and methods used in reflectance spectroscopy. Therefore, we show methods of calculations for index of refraction n, extinction coefficient k and local thicknesses of frequency starting from phase images by fast Kramers-Kronig (KK) algorithms and the Abeles matrix formalism. Details, limitations and problems of the presented calculations as well as alternative procedures are given for an example of HSCM images of red blood cells (RBC).
... The recent emergence of new label-free imaging techniques can overcome some of these limitations. Examples of these include digital holographic microscopy (DHM)234567 and the transport-of-intensity equation (TIE)21. For example, DHM has been used to provide quantitative phase images of live cells2 in vitro, to detect cell division in endothelial cells3, to determine the refractive index using incorporated microspheres5 and to detect early cell death based on cell volume changes6. ...
... There have been some significant advances recently in non-invasive, label-free imaging1. Examples of these include digital holographic microscopy234567 and ptychography8910. ...
Article
Full-text available
Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis.
Article
Patterned microstructures are commonly used for investigating cells proliferation, guiding cell fate and promoting adhesion and differentiation. Analyzing the behavior of cells layered onto functionalized micropatterned substrates ideally requires large Field of View (FoV), depth of focus, and spatial resolution. Managing the trade-off among these features is pretentious with standard microscopy. Furthermore, patterned substrates have often very complex geometries. Thus, optical systems should be able to get rid of artefacts due to light scattering/diffraction from both the cells’ layer and the underneath structure whose pitches typically have dimensions at the visible wavelength scale. Moreover, the layers are seen as coupled by transmission imaging systems. Decoupling them is pivotal to understand their own properties and related dependences and for interpreting how the structure geometry, point-by-point, addresses the behavior and the functions of the living cells. Here we show the successful use of Fourier Ptychographic Microscopy (FPM) for investigating cell-substrate interaction on micropatterned substrates, solving in full the issue of the layers decoupling. In fact, we demonstrate that it is possible to extract paired but separate clear images of both layers, by computationally decoupling them in FPM reconstruction. In order to test the proposed modality, we chose fibroblast cells in adhesion on a complex substrate consisting of irregular micro-wrinkles on a thin layer of Au. We rely on a numerical multi-look approach, which is capable of restoring quantitative phase-contrast maps of the label-free cells unaffected by artefacts, over a 3.3 mm² FoV with 0.5 µm resolution. Moreover, from one single FPM map, we demonstrate separate extraction of cells’ morphometry and the underneath wrinkled patterns. Two parameters characterizing the cell-substrate interaction are defined, showing correlation that paves the way to future exploitations of this processing protocol in the fields of mechanobiology and cells and tissue engineering.
Article
Low-level laser therapy (LLLT) is a therapeutic tool that uses the photobiochemical interaction between light and tissue. Its effectiveness is controversial due to a strong dependence on dosimetric parameters. In this work, we demonstrate that digital holographic microscopy is an effective label-free imaging technique to analyze the effects of LLLT on biological cells, and we propose the full methodology to create correct synthetic aperture phase maps for further extensive, highly accurate statistical analysis. The proposed methodology has been designed to provide a basis for many other biological experiments using quantitative phase imaging. We use SHSY-5Y and HaCaT cells irradiated with different doses of red light for the experiment. The analysis shows quantitative changes in cell dry mass density and the projected cell surface in response to different radiation doses.