Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell.

Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.

Imaging protein kinase Calpha activation in cells.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy.

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.

Femoral pseudoaneurysm following nonpenetrating trauma in a patient with aortic insufficiency.

Although not uncommon after penetrating vascular trauma, arterial pseudoaneurysms rarely develop following blunt trauma. A patient is described in whom indirect trauma led to pseudoaneurysm of the profunda femoris artery, and persistent bleeding required surgical intervention. In this case, the coexistence of significant aortic valvular regurgitation suggests that wide pulse pressure may predispose to this arterial complication.

Long-term antiarrhythmic therapy. Problem of low drug levels and patient noncompliance.

Maintenance of adequate serum blood levels is crucial to successful antiarrhythmic therapy. Serum levels of four antiarrhythmic agents (long-acting procainamide, quinidine sulfate, quinidine gluconate, and disopyramide) were determined in 98 consecutive ambulatory patients receiving long-term oral therapy. Medication dosages, dosing intervals, and time elapsed from last dosage until blood sampling were determined. Seventy-five patients (76.5 percent) had subtherapeutic blood levels (with mean levels less than 50 percent of the suggested minimum), and only 22 patients (22.5 percent) had therapeutic levels. Even among the 61 patients who claimed to have taken their medications within the six hours prior to blood sampling, 43 (70 percent) had subtherapeutic levels. These ratios held among all subgroups studied. Physicians should be aware of the high proportion of patients receiving long-term oral antiarrhythmic therapy with inadequate serum blood levels when planning therapeutic regimens.

Sharpness overconstancy in peripheral vision.

Although much has been learned about the spatial sampling and filtering properties of peripheral vision, little attention has been paid to the remarkably clear appearance of the peripheral visual field. To study the apparent sharpness of stimuli presented in the periphery, we presented Gaussian blurred horizontal edges at 8.3, 16.6, 24, 32, and 40 deg eccentricity. Observers adjusted the sharpness of a similar edge, viewed foveally, to match the appearance of the peripheral stimulus. All observers matched blurred peripheral stimuli with sharper foveal stimuli. We have called this effect "sharpness overconstancy". For field sizes of 4 deg, there was greater overconstancy at larger eccentricities. Scaling the field size of the peripheral stimuli by a cortical magnification factor produced sharpness overconstancy which was independent of eccentricity. In both cases, there was a slight sharpness underconstancy for peripherally presented edges blurred only slightly. We consider various explanations of peripheral sharpness overconstancy.

Sharpness overconstancy: the roles of visibility and current context.

In a previous study we found that blurred edges presented in peripheral vision look sharper than when they are looked at directly, a phenomenon we have called peripheral sharpness overconstancy (Galvin et al. (1997). Vision Research, 37, 2035-2039). In the current study we show that when visibility of the stimulus edges is compromised by very brief presentations, we can demonstrate sharpness overconstancy for static, foveal viewing. We also test whether the degree of sharpening is a function of the current visual context, but find no difference between the peripheral sharpness overconstancy (at 24 degrees eccentricity) of edges measured in a blurred context and that measured in a sharp context. We conclude that if the visual system does carry a template for sharp edges which contributes to edge appearance when visibility is poor, then that template is resistant to changes in context.

Community nurses are not GPs' attendants.

The Government announced in May 1994 that it had agreed changes to the terms of service of GPs and that they can be flexible in the way they meet their out-of-hours responsibilities. GPs must accept their duty to arrange 24-hour medical care. All nurses need to promote nursing services, not medical services. Community nurses need to look at 24-hour nursing/health visiting care, not medical care. Consumers need to be asked what kind of 24-hour service they need.

Nurses' perceptions of good nursing care.

Nursing has been described as 'caring for patients'. By telling stories about their experiences nurses may define what they understand to be good nursing care. Nurses' narratives emphasised the importance of building up a rapport with a patient to overcome fears, provide assistance and diffuse difficult situations.

Paediatric surveillance--a calendar for change.

A course was held for health visitors to evaluate their present practice in paediatric surveillance. Problem areas were highlighted and a set of recommendations produced which sought to improve practice and bring about a calendar for change.

Three dimensional image restoration in fluorescence lifetime imaging microscopy.

A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov-Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.

Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data.

The deconvolution of fluorescence lifetime imaging microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.

Multiple frequency fluorescence lifetime imaging microscopy.

The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set-up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto-optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons 'on' and 'off' as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the 'on' state of the intensifier relative to its 'off' state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square-pulse modulation. A phase-dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel-by-pixel basis using a non-linear fit to the dispersion relationships. The fitting algorithms were tested on a simulated data set and were successful in disentangling two populations having 1 ns and 4 ns fluorescence lifetimes. Spatial invariance of the lifetimes was exploited to improve the accuracy significantly. Multiple frequency fluorescence lifetime imaging microscopy was then successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co-expressed in live cells.

Global analysis of fluorescence lifetime imaging microscopy data.

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.

Atrial premature beats with variable return cycle lengths--a clue to sinus node re-entry?

A patient with atrial premature beats (APBs) with variable return cycle lengths is reported. Two rare responses of the sinus node were observed: APBs in which the return and sinus cycle lengths were equal and APBs which were interpolated. Several possible mechanisms for these unusual effects of APBs on the sinus node are discussed. One mechanism which can explain both of these observations is sinus node re-entry. This possibility is indirectly supported by documentation of sinus node re-entry during electrophysiologic studies.

Wide-field optically sectioning fluorescence microscopy with laser illumination.

We describe an extremely simple method by which optically sectioned fluorescence images may be obtained with conventional microscopes using laser illumination. A one-dimensional grid pattern is introduced into the illumination system, together with a rotating ground glass diffuser. This causes an image of the grid pattern to be projected into the specimen. Images taken at three spatial positions of the grid are processed in a simple manner to provide optically sectioned images of fluorescent specimens.