Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up.

BACKGROUND: Quantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system's functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories. METHODS: A protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry. RESULTS: The high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope- when coupled with appropriate software for image processing- might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described. CONCLUSIONS: The experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).

Manual stage acquisition and interactive display of digital slides in histopathology.

More powerful PC architectures, high-resolution cameras working at increasing frame rates, and more and more accurate motorized microscopes have boosted new applications in the field of biomedicine and medical imaging. In histopathology, the use of digital slides (DSs) imaging through dedicated hardware for digital pathology is increasing for several reasons: digital annotation of suspicious lesions, recorded clinical history, and telepathology as a collaborative environment. In this paper, we propose the first method known in the literature for real-time whole slide acquisition and displaying conceived for conventional nonautomated microscopes. Differently from DS scanner, our software enables biologists and histopathologists to build and view the DS in real time while inspecting the sample, as they are accustomed to. In addition, since our approach is compliant with existing common microscope positions, provided with camera and PC, this could contribute to disseminate the whole slide technology in the majority of small labs not endowed with DS hardware facilities. Experiments performed with different histologic specimens (referring to tumor tissues of different body parts as well as to tumor cells), acquired under different setup conditions and devices, prove the effectiveness of our approach both in terms of quality and speed performances.

Real-time whole slide mosaicing for non-automated microscopes in histopathology analysis.

CONTEXT: Mosaics of Whole Slides (WS) are a valuable resource for pathologists to have the whole sample available at high resolution. The WS mosaic provides pathologists with an overview of the whole sample at a glance, helping them to make a reliable diagnosis. Despite recent solutions exist for creating WS mosaics based, for instance, on automated microscopes with motorized stages or WS scanner, most of the histopathology analysis are still performed in laboratories endowed with standard manual stage microscopes. Nowadays, there are lots of dedicated devices and hardware to achieve WS automatically and in batch, but only few of them are conceived to work tightly connected with a microscope and none of them is capable of working in real-time with common light microscopes. However, there is a need of having low-cost yet effective mosaicing applications even in small laboratories to improve routine histopathological analyses or to perform remote diagnoses. AIMS: The purpose of this work is to study and develop a real-time mosaicing algorithm working even using non-automated microscopes, to enable pathologists to achieve WS while moving the holder manually, without exploiting any dedicated device. This choice enables pathologists to build WS in real-time, while browsing the sample as they are accustomed to, helping them to identify, locate, and digitally annotate lesions fast. MATERIALS AND METHODS: Our method exploits fast feature tracker and frame to frame registration that we implemented on common graphics processing unit cards. The system work with common light microscopes endowed with a digital camera and connected to a commodity personal computer. RESULT AND CONCLUSION: The system has been tested on several histological samples to test the effectiveness of the algorithm to work with mosaicing having different appearances as far as brightness, contrast, texture, and detail levels are concerned, attaining sub-pixel registration accuracy at real-time interactive rates.

Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells.

This study was designed to investigate the migratory behavior of adult human mesenchymal stem cells (MSC) and the underlying mechanism. Cell migration was assessed by transwell, wound healing and time-lapse in vivo motility assays. Pharmacological inhibitors were used to determine the potential mechanism responsible for cell migration and invasion. The tests that were implemented revealed that MSC were fairly migratory. Protein kinase B (AKT) was strongly activated at the basal level. Through our analyses we demonstrated that pharmacological inactivation of AKT2 but not AKT1 significantly decreased cell migration and invasion. Although preliminary, collectively our results indicate that AKT2 activation plays a critical role in enabling MSC migration.

Vignetting correction by exploiting an optical microscopy image sequence.

Vignetting is one of the most common problem that may affect digital imaging. The effect becomes particularly evident when images are stitched together to increase the camera's field of view (e.g., when building a mosaic), where it can lead to errors in automatic analyses. To correct the effect, the most common approach is to acquire an empty field image in advance that is used later to perform a flat field correction on every subsequently acquired image. However, in several cases, such as when dealing with off-line images or with real time acquisitions, this is not a viable option. The method we propose relies on a non parametric model to characterize in real time the vignetting function from the specimen itself, by using our foreground/background segmentation algorithm. The function is computed over a background built incrementally, detecting regions free of objects of interest. The experiments carried out using cell cultures and histological samples prove that our method yields results at least comparable to those achieved by using empty field.