Atomic force microscopy (AFM)-based nanomotion detection is a label-free technique that has been used to monitor the response of microorganisms to antibiotics in a time frame of minutes. The method consists of attaching living organisms onto an AFM cantilever and in monitoring its nanometric scale oscillations as a function of different physical-chemical stimuli. Up to now, we only used the cantilever oscillations variance signal to assess the viability of the attached organisms. In this contribution, we demonstrate that a more precise analysis of the motion pattern of the cantilever can unveil relevant medical information about bacterial phenotype. We used B. pertussis as the model organism, it is a slowly growing Gram-negative bacteria which is the agent of whooping cough. It was previously demonstrated that B. pertussis can expresses different phenotypes as a function of the physical-chemical properties of the environment. In this contribution, we highlight that B. pertussis generates a cantilever movement pattern that depends on its phenotype. More precisely, we noticed that nanometric scale oscillations of B. pertussis can be correlated with the virulence state of the bacteria. The results indicate a correlation between metabolic/virulent bacterial states and bacterial nanomotion pattern and paves the way to novel rapid and label-free pathogenic microorganism detection assays.

Candida albicans is an emerging multidrug-resistant opportunistic pathogen representing an important source of invasive disease in humans and generating high healthcare costs worldwide. The development of a rapid and simple antifungal susceptibility test (AFST) could limit the spread of this disease, increase the efficiency of treatment, and lower the risk of developing resistant strains. We developed a microfluidic chip containing an array of microwells that were designed to trap the cells and perform rapid antifungal susceptibility tests using optical nanomotion detection (ONMD). Yeast cell entrapment in a microwell allows for a very rapid exchange of growth medium with the antifungal, which enables performing single-cell ONMD measurements on the same cell before and after antifungal treatment. The exposure to a low concentration of the antifungal caspofungin or flucanozole induced a significant decrease in the nanomotion signal, demonstrating the high sensitivity of this technique. We used this chip to quantify the real-time response of individual C. albicans cells to the antifungal treatment in as fast as 10 min. This simple and label-free technique could be further developed into a simple-to-use device that allows the performance of fast AFST as part of a routine hospital procedure in developed and also eventually developing world countries.

Cells of two molecular genetic types of breast cancer—hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)—were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we revealed the unique patterns of the dependence of Young’s modulus on the indentation depth for two cancer cell lines that correlate with the features of the spatial organization of the actin cytoskeleton. Within a 200–300 nm layer just under the cell membrane, BT-20 cells are stiffer than ZR-75 cells, whereas in deeper cell regions, Young’s modulus of ZR-75 cells exceeds that of BT-20 cells. Two cancer cell lines also displayed a difference in cell nanomotion dynamics upon exposure to cytochalasin D, a potent actin polymerization inhibitor. The drug strongly modified the nanomotion pattern of BT-20 cells, whereas it had almost no effect on the ZR-75 cells. We are confident that nanomotion monitoring and measurement of the stiffness of cancer cells at various indentation depths deserve further studies to obtain effective predictive parameters for use in clinical practice.

Nanometric scale size oscillations seem to be a fundamental feature of all living organisms on Earth. Their detection usually requires complex and very sensitive devices. However, some recent studies demonstrated that very simple optical microscopes and dedicated image processing software can also fulfill this task. This novel technique, termed as optical nanomotion detection (ONMD), was recently successfully used on yeast cells to conduct rapid antifungal sensitivity tests. In this study, we demonstrate that the ONMD method can monitor motile sub-cellular organelles, such as mitochondria. Here, mitochondrial isolates (from HEK 293 T and Jurkat cells) undergo predictable motility when viewed by ONMD and triggered by mitochondrial toxins, citric acid intermediates, and dietary and bacterial fermentation products (short-chain fatty acids) at various doses and durations. The technique has superior advantages compared to classical methods since it is rapid, possesses a single organelle sensitivity, and is label- and attachment-free.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method’s applicability.

The fast emergence of multi-resistant pathogenic yeasts is caused by the extensive—and sometimes unnecessary—use of broad-spectrum antimicrobial drugs. To rationalise the use of broad-spectrum antifungals, it is essential to have a rapid and sensitive system to identify the most appropriate drug. Here, we developed a microfluidic chip to apply the recently developed optical nanomotion detection (ONMD) method as a rapid antifungal susceptibility test. The microfluidic chip contains no-flow yeast imaging chambers in which the growth medium can be replaced by an antifungal solution without disturbing the nanomotion of the cells in the imaging chamber. This allows for recording the cellular nanomotion of the same cells at regular time intervals of a few minutes before and throughout the treatment with an antifungal. Hence, the real-time response of individual cells to a killing compound can be quantified. In this way, this killing rate provides a new measure to rapidly assess the susceptibility of a specific antifungal. It also permits the determination of the ratio of antifungal resistant versus sensitive cells in a population.