Colloids and Surfaces B: Biointerfaces 88 (2011) 407–412 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces jou rn al h om epage: www.elsevier.com/locate/colsurfb Total internal reflection imaging of microorganism adhesion using an oil immersion objective Tzvetan Velinov a,∗ , Yana Asenovska a , Dessislava Marinkova b , Lyubov Yotova b , Stoyanka Stoitsova c , Maria Bivolarska a , Lyuba Stavitskaya d a Department of Solid State Physics and Microelectronics, Sofia University, 5 blvd J. Bourchier, 1164 Sofia, Bulgaria b Department of Biotechnology, University of Chemical Technology and Metallurgy, 8 Kl. Ohridski blvd, 1756 Sofia, Bulgaria c Department of Morphology of Microorganisms and Electron Microscopy, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria d Department of BCV, Lausitz University of Applied Science, Großenhainer Str. 57, 01968 Senftenberg, Germany a r t i c l e i n f o Article history: Received 31 March 2011 Received in revised form 29 June 2011 Accepted 6 July 2011 Available online 18 July 2011 Key words: Total internal reflection microscopy Evanescent waves Escherichia coli Saccharomyces cerevisiae Biofilms a b s t r a c t In this paper, we report the results of total internal reflection microscopy investigations of the interaction of two types of microorganisms: Saccharomyces cerevisiae and Escherichia coli with substrates. It is shown that with this method qualitative and quantitative information about cells–substrate interaction can be obtained. One can easily make a difference between attached and non-attached as well as between dead and alive cells, and more generally can follow the dynamics of the process of cells’ attachment to substrates. Quantitative information about the cell size and cell–substrate distance is obtained by using a model in which yeast cells and bacteria are approximated by ellipsoids, and multiple reflections of the evanescent waves between the cells and the substrate are neglected. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Biofilms are formed by microorganisms attached to a surface and enveloped in an extracellular matrix. While the structure of a mature biofilm is complex, many of its properties depend on the early stage of its formation when individual cells attach to the sur- face and then form colonies. The rate and the extent of the cells’ attachment to the surface determine the onset of biofilm growth and the force that needs to be applied to remove the biofilm. Light microscopy, especially confocal microscopy, is the most often used method for investigating biofilms [1,2]. However, this and other common optical techniques are unable to visualize the contact between cells and supports. Often, when optical microscopy is used to examine attached bacteria, for example dark field microscopy [3] or phase contrast microscopy [3,4], it is combined with a flow cell in order for the liquid flow to remove the non-attached cells. This method does not guarantee that all remaining bacteria are attached to the surface since a stagnant fluid layer forms near the surface during liquid flow. Its thickness depends on the flow rate and is typically a few microns. A sufficiently fast flow needs to be used to reduce the thickness of this layer but faster flows apply stronger ∗ Corresponding author. Tel.: +359 2 8161727; fax: +359 2 9625276. E-mail address: tvel@phys.uni-sofia.bg (T. Velinov). shear forces to the attached cells and there is a chance that some of them are also removed. Also, in many cases the growth of a biofilm in still or slow-moving fluids is of interest and in this case this technique cannot be applied. Two optical techniques exist that can be used to visualize the contact between cells and supports without labeling the cells: interference reflection microscopy [5] and total internal reflection microscopy (TIRM). TIRM is based on evanescent waves created when optical waves are incident on a surface at an angle larger than the critical angle [6]. A closely related method is TIRFM (total inter- nal reflection fluorescence microscopy) which detects the emission of fluorescent labels excited by evanescent waves. While in recent years TIRFM has become an increasingly popular method [7,8], TIRM remains a relatively rarely used imaging method in inves- tigating cells and cell–substrate interactions [9,10]. Byrne et al. [9] investigated the contacts (or focal points) between large fibro- blast cells and supports using TIRM and TIRFM and the authors concluded that the two techniques provided similar information. Robertson and Bike [10] used TIRM to study the nonspecific inter- actions between model cells consisting of 5–10 m polystyrene spheres coated with phospholipids and a glass plate. In this paper, we report the results of TIRM investigation of the interaction of two types of microorganisms: baker’s yeast (Saccha- romyces cerevisiae) and Escherichia coli with substrates. It is shown that with this method qualitative and quantitative information 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.07.022