Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the ...context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip.
Fluorescence labeling is the prevailing imaging technique in cell biology research. When they involve statistical investigations on a large number of cells, experimental studies require both low ...magnification to get a reliable statistical population and high contrast to achieve accurate diagnosis on the nature of the cells’ perturbation. Because microscope objectives of low magnification generally yield low collection efficiency, such studies are limited by the fluorescence signal weakness. To overcome this technological bottleneck, we proposed a new method based on metal-coated substrates that enhance the fluorescence process and improve collection efficiency in epifluorescence observation and that can be directly used with a common microscope setup. We developed a model based on the dipole approximation with the aim of simulating the optical behavior of a fluorophore on such a substrate and revealing the different mechanisms responsible for fluorescence enhancement. The presence of a reflective surface modifies both excitation and emission processes and additionally reshapes fluorescence emission lobes. From both theoretical and experimental results, we found the fluorescence signal emitted by a molecular cyanine 3 dye layer to be amplified by a factor ∼30 when fluorophores are separated by a proper distance from the substrate. We then adapted our model to the case of homogeneously stained micrometer-sized objects and demonstrated mean signal amplification by a factor ∼4. Finally, we applied our method to fluorescence imaging of dog kidney cells and verified experimentally the simulated results.
Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous ...biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.
We report a wide-field fluorescence lifetime imaging microscope based on a low-repetition-rate (3.7-MHz) passively mode-locked diode-pumped laser source. This inexpensive and compact laser source ...operating in the visible and UV range can excite a wide range of fluorophores of biological interest. We demonstrate that the power of this laser source is highly sufficient for studying biological systems with low quantum yields (autofluorescence of tissues and stained living cells). The maximum measurable lifetime is also strongly increased with this laser source, as fluorescence intensity measurement can occur 250 ns after the excitation pulse.
Two-photon microscopy is a powerful tool for imaging of cells or tissues. However, it presents the drawback of being a laser-scanning technique that involves a long acquisition time for ...fluorescence-lifetime imaging. Thus it is commonly limited to intensity images that give only indications of the location of fluorophores but do not identify the physicochemical properties and interactions between cells' components. To protect biological samples from experiments that are too long and to provide a more comprehensive spectroscopic tool we have developed a time-resolved multifocal multiphoton microscope. This setup allows us to speed up the acquisition while retaining the possibility of measuring both intensity and lifetime images of the sample.
Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the ...point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.
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