Fluorescence Imaging

We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional ...

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.

The feasibility of using in vivo autofluorescence for the diagnosis of skin cancer was evaluated. In vivo fluorescence measurements were performed on healthy human volunteers, and patients with different types of benign and malignant skin tumours. Fluorescence spectra as well as fluorescence images were acquired. The excitation-emission matrix of normal skin (n=3) showed a broad peak at the shortest excitation wavelength (365 nm) and at 440 nm fluorescence wavelength, smoothly decreasing towards longer excitation and fluorescence wavelengths. Non-melanoma skin tumours (n=31) and control skin excited with 375 nm showed a broad fluorescence band from 400 to 700 nm, peaking around 436 nm. No significant differences in measurements between tumours and the corresponding control sites were found. A large spatial variation in the fluorescence intensity was observed both in the tumours and in the control sites. Standard deviations found ranged from 0.15 to 1.5 times the mean fluorescence. Fluorescence images, excited with 375 nm and taken with an image intensified CCD camera, on eight malignant melanomas and eight benign pigmented lesions did not indicate any fluorescence intensity distribution specific to the malignancy of the lesion. Neither the shape of the fluorescence spectra, nor the spatial distribution of the fluorescence intensity showed any signature specific to the histopathological nature of the lesions investigated. Optical diagnostics of skin tumours using the autofluorescence does not seem to be feasible at the present time.

Reflectance and luminescence imaging in the ultraviolet (UV), visible and infrared (IR) regions are relatively straightforward non-invasive, non-contact investigative techniques that can provide spatial information on the presence of original and non-original materials on museum objects and in situ. For most of these so-called multispectral techniques, imaging is normally conducted with the aid of a tripod in a darkened environment and using continuous radiation sources such as tungsten halogen, LED or mercury lamps. In some circumstances, particularly when working on site or in historic buildings, it is not possible to black out the working environment, to use a tripod or to transport (or have access to) the appropriate radiation source. Xenon flashtubes are very compact radiation sources that produce rapid bursts of intense radiation in the UV, visible and IR ranges of the electromagnetic spectrum. If appropriately filtered, it is possible to use xenon flashtubes as a single radiation source for all the commonly encountered types of imaging in the UV, visible and IR portions of the spectrum. In addition, the intense radiation emitted by these sources allows luminescence images to be acquired in the presence of considerable ambient light, under conditions that have previously proved challenging when making luminescence images in situ.

We present tumor hypoxia mapping by diffuse optical fluorescence tomography. A novel 2-nitroimidazole bis-carboxylic acid indocyanine dye conjugate has been developed for tumor-targeted hypoxia fluorescence imaging. The hypoxia probe has been evaluated in-vitro using 4T1 tumor ...

Fluorescent imaging microscopy has been an essential tool for biologists over many years, especially after the discovery of the green fluorescent protein and the possibility of tagging virtually every protein with it. In recent years dramatic enhancement of the level of detail at which a fluorescing structure of interest can be imaged have been achieved. We review classical and new developments in high-resolution microscopy, and describe how these methods have been used in biological research. Classical methods include widefield and confocal microscopy whereas novel approaches range from linear methods such as 4Pi, I(5) and structured illumination microscopy to non-linear schemes such as stimulated emission depletion and saturated structured illumination. Localization based approaches (e.g. PALM and STORM), near-field methods and total internal refraction microscopy are also discussed. As the terms 'resolution', 'sensitivity', 'sampling' and 'precision&#3...

Chemosensors play important roles in cation and anion recognition in biological, industrial, and environmental processes. Although many efforts have been made to develop artificial fluorescent receptors for Cu2+ and S2−, their applications in the detection in bulk solutions are limited. In this work, we report a novel fluorescence chemosensor (NL) based on the 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorophore for the quantification of Cu2+ and S2− in single intact cells. NL specifically binds to Cu2+ in the presence of other competing cations, and evident changes in UV-vis and fluorescence spectra in HEPES buffer are noticed. Based on the displacement approach, the selective sense S2− with the in situ generated NL-Cu2+ ensemble gives a remarkable recovery of fluorescence and UV-vis absorption spectra. The detection limits of NL for Cu2+ and NL-Cu2+ for S2− were estimated to be 1.6 nM and 0.17 μM, respectively. NL and the resultant complex NL-Cu2+ exhibit low cytotoxicity and cell-membrane permeability, which makes them capable of Cu2+ and S2− imaging and quantification in living MDA-MB-231 cells.

In the article is made an overview of the application of different satellite remote sensing methods and technologies in detection of the abiotic stress in coniferous landscapes. The review paper is discussing in short the application of different remote sensing technologies such as: satellite multispectral and infrared (thermal), imaging spectrometry and fluorescence imaging. The studied period spans from the onset of the satellite remote sensing in the 1960s until present day. In conclusion, are drawn requirements for the perspective technologies in satellite remote sensing which should address the fast and reliable detection of the manifestation of abiotic stress in coniferous landscapes.

Following the considerable impact of the application of convenient ultrafast lasers to multiphoton microscopy on biomedical imaging, it seems to us that FLIM and MDFI continue the trend in which advances in instrumentation will facilitate new discoveries — and modes of discovery — in biology and medicine. We hope we have shown the reader that fluorescence lifetime can provide intrinsic molecular contrast in unstained tissue and that the prospects for in vivo application are exciting. We believe that the capability to excite fluorophores at almost any excitation wavelength and the opportunities to extract more information from fluorescence signals by resolving with respect to lifetime, excitation and emission spectrum and also polarisation, will have a major impact on the ability to identify and exploit intrinsic contrast and on investigations of molecular biology. There the combination of new fluorescence probe technology, including genetically-expressed labels and nano-engineered devices, with new modes of interrogation and analysis, will continue to fuel the astounding advances in this field. There is a real prospect that our ability to ask and test biological questions will cease to be limited by the availability of suitable instrumentation. Rather it is likely to be limited by our ability to analyse and comprehend the (rapidly increasing volume of) data that we collect.