Combination of fluorescence microscopy and nanomotion detection to characterize bacteria

Antibiotic-resistant pathogens are a major health concern in everyday clinical practice. Because their detection by conventional microbial techniques requires minimally 24h, some of us have recently introduced a nanomechanical sensor, which can reveal motion at the nanoscale. By monitoring the fluctuations of the sensor, this technique can evidence the presence of bacteria and their susceptibility to antibiotics in less than 1h. Their amplitude correlates to the metabolism of the bacteria and is a powerful tool to characterize these microorganisms at low densities. This technique is new and calls for an effort to optimize its protocol and determine its limits. Indeed, many questions remain unanswered, such as the detection limits or the correlation between the bacterial distribution on the sensor and the detection's output. In this work, we couple fluorescence microscopy to the nanomotion investigation to determine the optimal experimental protocols and to highlight the effect of the different bacterial distributions on the sensor. Copyright (c) 2013 John Wiley & Sons, Ltd.

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

EPFL2004

Interdigitated electrode DNA biosensor for detection of food-borne pathogens by impedance spectroscopy

Daniel Berdat

The commercial application of biosensors has had a significant impact in a number of areas, particularly in the field of medical diagnostics. But in spite of the great number of publications, only a few systems are commercially available in food analysis. We propose an innovative approach to achieve safer food products by creating a user-oriented device for the detection of food-borne pathogens. Our aim is to perform quality control along the food-chain distribution to protect the consumer's health against contaminations; the measurement is based on the detection by impedance spectroscopy of DNA extracted from bacteria. Our work focuses on the conception of a DNA biosensor able to detect the presence of specific DNA sequences from Salmonella bacteria. Within the European project GoodFood, the choice was made to use dairy products and seafood as starting materials, from which cell growth and DNA amplification were performed by our partners, to finally be measured by impedance spectroscopy with the DNA biosensor. The major advantages of this approach are the portability, ease-of-use and rapidity of the system. The sensor fabrication was performed in several steps: At first, the design and the clean-room fabrication was optimized for platinum interdigitated structures with 5 µm spacing. Once the production of the metal electrodes was standardized, the immobilization of the probe DNA (capturing the target DNA) on the sensor surface was performed by silanization. The selectivity of the sensor towards the target DNA was evaluated by fluorescence microscopy using fluorescently-labelled DNA target sequences. The biochip, consisting of 4 interdigitated electrodes, was connected to an impedance analyzer and a first series of measurements was done without DNA, to characterize the sensor in high ionic strength solutions. A next series was performed with probe DNA on the sensor surface, with decreasing concentrations of synthetic target DNA to reach the nanomolar detection limit of the system. Finally, a series of measurements was carried out with DNA target sequences extracted from food samples spiked with salmonella, to apply the biosensor within a "real" biological DNA sample.

EPFL2009

Bacterial cell cycle dynamics: size regulation during exponential growth and role of polyphosphate during starvation response

Aster Louis K Vanhecke

Bacteria are the most diverse and abundant kingdom of life and have adapted to survive and thrive in habitats around the globe. When provided with ample nutrients they grow and divide at staggering rates, increasing their population exponentially. Upon nutrient depletion on the other hand, they quickly adapt by drastically altering their metabolism, halting growth to survive for a very long time. Since bacteria are tiny -about a few micrometers-, visualizing these processes requires microscopy. To measure the dynamics of their shape and inner structures precisely, one needs to choose a technique that balances spatial resolution, temporal resolution and photo-toxicity. In this thesis, I present two projects using advanced dynamic microscopy, first to study cell size regulation during exponential growth in the abundance of nutrients and then to elucidate the role and positioning of polyphosphate granules during cell cycle exit in response to nutrient starvation. During exponential growth, bacteria balance growth and division to regulate their size, resulting in a narrow size distribution, referred to as cell size homeostasis. Recent work tried to uncover what cells sense to decide to divide in order to achieve size homeostasis: time, size, growth or a combination of those. Control of cell division is often equated to control of constriction onset; however, the constriction period still accounts for up to 40% of cell growth and could thus contribute significantly to cell size regulation. We used SIM microscopy to measure constriction kinetics and their impact on cell size regulation in Caulobacter crescentus. We found that constriction rate regulation can determine cell size. Moreover, constriction rate modulation compensates for variability in elongation before constriction, allowing a higher fidelity cell size homeostasis. We suggest a parsimonious model where excess cell wall precursors accumulate proportionally to elongation before constriction and set the rate of constriction. This is the first direct demonstration that constriction rate can contribute to cell size control and homeostasis in bacteria. Upon nutrient starvation, bacteria exit their cell cycle to preserve energy and nutrients. In many bacteria, such as Pseudomonas aeruginosa, this is associated with the accumulation of polyphosphate (polyP) in intracellular granules. PolyP is created by polyphosphate kinases (ppkâs), which are required for successful cell cycle exit and survival of and recovery from long-term starvation. Interestingly, these polyP granules are regularly spaced within the nucleoid. To date, it is not known during which stage polyP is required for cell cycle exit, and what causes the spacing of the granules. Here, we use fluorescence microscopy to probe the cell cycle stage of Îppk cells arrested during nutrient starvation as well as the localization and dynamics of ppkâs. We show that a majority of Îppk cells are arrested with open replication forks. Furthermore, we find that ppkâs localize in distinct patterns, already prior to starvation and polyP granule production, which could be responsible for the positioning of polyP granules. To this end, we developed a background subtraction algorithm to remove cytoplasmic fluorescence, improving accuracy of spot detection and localization.

EPFL2019