Measurement of wheat germ agglutinin binding with a fluorescence microscope

Abstract

Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide-field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield-corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA-Alexa 488 and WGA-TRITC. Consistent results were obtained by comparing microscopy with flow cytometry, comparing imaging through different objectives and comparing different WGA conjugates. Reproducibility of calibration was within 97% when low magnification was used. Fluorescence of free and bound WGA was found to be different, however, and therefore precise measurement of the number of cell-bound molecules was problematic in this particular system. We conclude that the method achieves reliable measurement of cellular staining in the units of soluble fluorophore. For probes whose fluorescent properties are unaffected by binding, quantification of staining in true molecular units should be possible. 1-4). Conceptually, the first step in this type of measurements is to compare on a calibrated spectrofluorimeter the brightness of a bead suspension to the brightness of a standard solution of a fluorochrome. This allows equating the brightness of one bead to the brightness of a certain number of fluorochrome molecules, and that number is designated as the MESF value of a bead. Commercial beads with specified MESF values are available and are used to calibrate flow cytometers for analyzing unknown cells or particles. Measurements of antigen expression (4-6), ligand-receptor interactions, (7-9) and telomere length (10,11) have proved the usefulness of standardized units of fluorescence intensity.Fluorescence microscopy shares many applications with flow cytometry, but in contrast with the latter it lacks convenient standards that would allow quantification in absolute units. Due to the ever increasing importance of digital imaging in medicine and drug discovery, the need for standardization in microscopy has been repeatedly stressed by many authors (12-15). Although a number of different approaches to calibrate fluorescence microscopes have been proposed (15-17), most of them rely on more or less arbitrarily chosen units of intensity.

Intensity Calibration and Flat‐Field Correction for Fluorescence Microscopes

“…For example, in terms of TRITC-labeled wheat germ agglutinin, the values for N st were measured as 56.1 × 10 3 molecules/μm 2 for 20% Rose Bengal, 12.7 × 10 3 molecules/μm 2 for 30% acid fuchsin, and 360,000 × 10 3 molecules/μm 2 for a red plastic slide (Chroma Technology; Model et al, 2009). 4. Moderate brightness.…”

Fluorescence mapping of afferent topography in three dimensions

“…In recent years, several new tracers have proven to be invaluable tools for labeling neurons and have become standard tract tracing approaches. Among these are the various dextran conjugates (Sugihara and Shinoda 2004;Sugihara and Quy 2007;Pakan and Wylie 2008;Pakan et al 2010), Neurobiotin and Biocytin (Lapper and Bolam 1991;Grishkat and Eisenman 1995;Paradies et al 1996), and wheatgerm agglutinin (WGA) alone or conjugated to horseradish peroxidase (WGA-HRP; Mesulam 1982). Here, we compare the efficiency of a new line of WGAAlexa fluorophore conjugates with two well-known anterograde tracers, WGA-HRP and BDA (biotinylated dextran amine), in labeling the spinocerebellar tract and revealing its parasagittal topography.…”

Bright fluorogenic squaraines with tuned cell entry for selective imaging of plasma membrane vs. endoplasmic reticulum

“…4 Fluorescently labelled lectins, notably wheat germ agglutinin (WGA), are among the most popular fluorescent membrane probes. 5 On the other hand, membrane probes based on small organic dyes, being less expensive and more chemically stable, present the unique possibility to be precisely located and oriented in the lipid bilayer, 6 which is important for studies of the lateral lipid organization of biomembranes (lipid rafts), [7][8][9] FRET with membrane proteins 10 and super-resolution imaging. 11,12 However, the wide use of these probes is limited by their poor performance compared to labelled WGA.…”