A variable temperature, U.V. luminescence spectrograph for small samples.

We have developed an assay system for endo-beta-N-acetylglucosaminidase and glycoamidase (PNGase), using Eu(3+)-labeled Man(9)GlcNAc(2) glycopeptides as substrates in combination with lectin capture. Two glycopeptides of different peptide lengths, derived from soybean agglutinin, were labeled with Eu(3+) via a diethylenetriaminepentaacetate (DTPA) chelating linker and served as substrates for two types of enzymes: one with (Man(9)GlcNAc(2))Asn for endo-beta-N-acetylglucosaminidase and the other with Ala-Ser-Phe-(Man(9)GlcNAc(2))Asn-Phe-Thr for glycoamidase activities. Following enzymatic hydrolysis, concanavalin A, immobilized or soluble, was added to the mixture to bind unreacted substrate and unlabeled hydrolysis product. The labeled peptide product could then be separated from the lectin-bound complexes by filtration for quantification by dissociation-enhanced lanthanide fluorescence immunoassay. Activities as low as 2 fmol min(-1) could be rapidly quantified for both types of enzymes, and enzymological parameters could be determined within minutes. Applicability of the assay was tested for identification of a glycoamidase activity peak in the fractionation of sweet almond emulsin, a classic example. This assay offers sensitivity, ease of use, and high throughput. In addition, it is versatile and should be applicable to other glycobiology enzyme systems.

Fluorescence↗

PubMed2000

Background suppression in frequency-domain fluorometry.

Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry. The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and instrumental conditions, the data can be analyzed as usual for frequency-domain data without further processing. We show by simulations that this method can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence.

Fluorescence↗

PubMed2000

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.

Fluorescence↗