Anna Mölder

Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography

Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label‐free and completely non‐invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor™ (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non‐invasive automatic cell counting as precisely as conventional manual cell counting

Digital Holography and Cell Studies

Digital holographic microscopy (DHM) is a novel high-resolution imaging technique that offers real-time imaging and quantitative measurements of physiological parameters. It has developed into a broad field, and one of many interesting applications is to study cells without staining or labeling them and without affecting them in any way. Digital holography makes it possible to easily measure cell properties that previously have been very difficult to study in living cells, such as cell thickness, volume, and cell refractive index (Marquet et al., 2005; Rappaz et al. 2005; Molder et al., 2008; El-Schish et al., in press; Persson et al., in press). 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.

Semiautomated analysis of embryoscope images: Using localized variance of image intensity to detect embryo developmental stages.

Embryo selection in in vitro fertilization (IVF) treatment has traditionally been done manually using microscopy at intermittent time points during embryo development. Novel technique has made it possible to monitor embryos using time lapse for long periods of time and together with the reduced cost of data storage, this has opened the door to long‐term time‐lapse monitoring, and large amounts of image material is now routinely gathered. However, the analysis is still to a large extent performed manually, and images are mostly used as qualitative reference. To make full use of the increased amount of microscopic image material, (semi)automated computer‐aided tools are needed. An additional benefit of automation is the establishment of standardization tools for embryo selection and transfer, making decisions more transparent and less subjective. Another is the possibility to gather and analyze data in a high‐throughput manner, gathering data from multiple clinics and increasing our knowledge of early human embryo development. In this study, the extraction of data to automatically select and track spatio‐temporal events and features from sets of embryo images has been achieved using localized variance based on the distribution of image grey scale levels. A retrospective cohort study was performed using time‐lapse imaging data derived from 39 human embryos from seven couples, covering the time from fertilization up to 6.3 days. The profile of localized variance has been used to characterize syngamy, mitotic division and stages of cleavage, compaction, and blastocoel formation. Prior to analysis, focal plane and embryo location were automatically detected, limiting precomputational user interaction to a calibration step and usable for automatic detection of region of interest (ROI) regardless of the method of analysis. The results were validated against the opinion of clinical experts.

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds.

Supervised classification of etoposide-treated in vitro adherent cells based on noninvasive imaging morphology

Abstract. Single-cell studies using noninvasive imaging is a challenging, yet appealing way to study cellular characteristics over extended periods of time, for instance to follow cell interactions and the behavior of different cell types within the same sample. In some cases, e.g., transplantation culturing, real-time cellular monitoring, stem cell studies, in vivo studies, and embryo growth studies, it is also crucial to keep the sample intact and invasive imaging using fluorophores or dyes is not an option. Computerized methods are needed to improve throughput of image-based analysis and for use with noninvasive microscopy such methods are poorly developed. By combining a set of well-documented image analysis and classification tools with noninvasive microscopy, we demonstrate the ability for long-term image-based analysis of morphological changes in single cells as induced by a toxin, and show how these changes can be used to indicate changes in biological function. In this study, adherent cell cultures of DU-145 treated with low-concentration (LC) etoposide were imaged during 3 days. Single cells were identified by image segmentation and subsequently classified on image features, extracted for each cell. In parallel with image analysis, an MTS assay was performed to allow comparison between metabolic activity and morphological changes after long-term low-level drug response. Results show a decrease in proliferation rate for LC etoposide, accompanied by changes in cell morphology, primarily leading to an increase in cell area and textural changes. It is shown that changes detected by image analysis are already visible on day 1 for 0.25-μM etoposide, whereas effects on MTS and viability are detected only on day 3 for 5-μM etoposide concentration, leading to the conclusion that the morphological changes observed occur before and at lower concentrations than a reduction in cell metabolic activity or viability. Three classifiers are compared and we report a best case sensitivity of 88% and specificity of 94% for classification of cells as treated/untreated.

Three Dimensional Visualisation of Microscope Imaging to Improve Understanding of Human Embryo Development

The analysis of processes on a cellular and sub-cellular level plays a crucial role in life sciences. Commonly microscopic assays make use of stains and cellular markers in order to enhance image contrast, but in many cases, cell imaging requires the sample to be undisturbed during the imaging process, making staining, dying and fixing impractical. Non-destructive techniques are especially useful in long term imaging or in the study of sensitive cell types, such as stem cells, embryos or nerve cells. Novel advances in computation, imaging and incubator technology have recently made it possible to prolong the imaging time, reduced the cost of storing data and opened a door to the development of new computer aided analytical tools based on microscopic image data. Here we illustrate how Hoffman Modulation Contrast imaging and Confocal Microscopy can be combined with visual computing and present results from determination of cell number, volume, spatial location and blastomere connectivity, using examples from embryos grown for in vitro fertilisation. We give examples of how knowledge of the imaging technique can be used to further improve the computer analysis and also how visually guided tools may aid in the diagnostic interpretation of image data and improve the result. Finally we discuss how the use of microscopic data as a basis for embryo modelling may help in both research and educational purposes. The aim of this chapter is to give an example of how microscopic imaging can be combined with standard computer vision techniques to aid in the interpretation of microscopic data, and demonstrate how visual computing techniques can make an essential difference in terms of scientific output and understanding.

Automatic Detection of Embryo Location in Medical Imaging Using Trigonometric Rotation for Noise Reduction

The assessment of embryos in vitro is an important tool both in In Vitro Fertilization (IVF) treatment and for research purposes. Traditionally, such assessment has been done manually and demands extensive training and expertise. Interobserver variability limits the use of evaluation, and the manual labor increases the cost of treatment and research. To this end, feature extraction and automatic annotation using computer aided tools would both improve objectivity and save time. Due to the sensitivity of embryos, any evaluation must be performed using non-invasive imaging techniques, which sets a limit for the ability to artificially enhance image contrast, and computer aided assessment of microscopic images under clinical conditions also poses a number of difficulties due to the high level of noise and the unpredictable imaging environment. To overcome this, we propose a framework using a Hough Transform (HT) for the extraction of circular shapes in microscopic embryo images, and also use the algorithm for successful automatic profiling of the pronuclear breakdown (PNB) at syngami and detection of its timing. The best results are achieved when using a HT for embryo outline detection in combination with image variance of the embryo interior. Using our method, we achieve an overall accuracy of 95.0% detecting the position of the embryo outline, and 83.0% detecting the timing of the PNB.