Biomedical subjects
Publications and source records attributed to M A A Neil.
We demonstrate an optically sectioned fluorescence lifetime imaging microscope with a wide-field detector, using a convenient, continuously tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated in a microstructured fiber.
Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.
We report the development of a high-speed wide-field fluorescence-lifetime imaging (FLIM) system that provides fluorescence-lifetime images at rates of as many as 29 frames/s. A FLIM multiwell plate reader and a potentially portable FLIM endoscopic system operating at 355-nm excitation have been demonstrated.
Confocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.
We describe a simple modification to a confocal microscope, which analyses the state of polarization of light emerging from the specimen so as to permit quantitative polarized light microscopy to be performed. The system uses a novel form of rotating analyser which, together with lock-in detection, permits images to be obtained where the image contrast corresponds to both specimen retardance and orientation (e.g. in the case of a birefringent specimen). Images are presented from a wide range of specimens and the origin of the contrast observed from simple point scatterers is investigated both theoretically and experimentally.
Rigid endoscopes provide high quality optical images of reasonably accessible regions of the inner body, especially regions such as the aero-digestive and genital tracts. In order to enhance the versatility of these instruments we describe a development that permits confocal endoscopic images to be obtained - along with traditional endoscopic images - in real-time, from within the living patient. The system is based around a host lenslet-array tandem scanning microscope, which is capable of producing images viewed directly by eye. These types of confocal microscope are configured for fluorescence imaging together with laser illumination. Hard and soft tissues in the mouth were imaged using this combined system.
High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.