Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Swedlow et al. (February 5, 2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.022554999.

Supporting Methods
Preparation of Fluorescent Beads.
Seven types of fluorescent 2.5-µm polystyrene beads (InSpeck microspheres, Molecular Probes) with different fluorophore densities were mixed in equal proportion and spread onto the surface of a coverslip. After drying, the beads were either mounted in 90% glycerol/0.1% phenyl propylene diamine (Sigma)/10 mM Tris (pH 8.0) (wide-field microscopy, WFM) or an aqueous enzymatic oxygen-scavenging mixture (laser-scanning confocal microscopy, LSCM; 0.1 mg/ml catalase/0.03 mg/ml glucose oxidase/10 mM glucose/15mM b-mercaptoethanol/50 mM potassium acetate/4 mM MgSO₄/1mM EGTA/10 mM Tris acetate, pH 7.5). Beads were imaged immediately. To obtain an independent measure of bead fluorescence, both sets of beads were analyzed with a FACS on a Becton-Dickson FACSort using the FL2-H detector.

For WFM, beads with excitation maximum 580 nm and emission maximum 605 nm were used. For LSCM, beads with excitation maximum 540 nm and emission maximum 560 nm were used. Different beads were chosen for each system to maximize fluorescence excitation and emission in each system. Because of batch variations, the relative intensities of the two bead populations differ (compare positions of arrows showing results from FACS analysis in Fig. 2 B and C). In Fig. 2 C and D, we note that the WFM result for the weakest beads matches the expected intensity relative to the other beads in the same set, whereas the FACS is slightly too low. Both results were reproducible, and thus they may reflect a systematic error in very low-intensity measurements in our FACS.

LSCM of Fluorescent Beads.
LSCM images of beads were collected through the ´63 C Apochromat water immersion lens by using the 543-nm line from a helium-neon laser for excitation and collecting fluorescence through an LP560 emission filter. The pinhole was opened to a diameter corresponding to 1.0 Airy disk. The signal-to-noise ratio changes slowly as pinhole diameter is adjusted, and thus our experiments are not affected by any error in this adjustment. Typically, 25 optical sections spaced at intervals of 0.3 µm vertically, each section being 90 ´ 90 pixels (0.1 µm per pixel), were collected for single beads well separated from their neighbors. The dwell time per pixel was 4.5 ms. The laser intensity was controlled by an acousto-optical deflector. The intensity was chosen to be as bright as possible subject to the condition that ground-state depletion was insignificant, ensured by confirming that doubling the laser power resulted in a 2-fold increase in emitted fluorescence. Having thus set the illumination intensity, the photomultiplier gain and offset were adjusted to give the maximum signal intensity while ensuring that no pixels of zero intensity were in the background and no saturated pixels were in the images of the brightest beads. With these imaging conditions, photobleaching of the beads after collecting an entire three-dimensional (3D) data stack was insignificant compared to the noise level in our measurements. WFM of Fluorescent Beads.
3D images of beads were recorded by using an ´100/1.4 numerical aperture oil immersion objective lens on a DeltaVision restoration microscope built on a Nikon TE200 platform using the same camera as described above. 3D images (165 sets, 12 optical sections per set, 0.5 µm per optical section) were collected to cover a field encompassing 290 beads. All images were collected by binning the images from the charge-coupled device 2 ´ 2 to give an effective pixel size of 0.102 µm in the image plane. Each exposure lasted 0.1 s. Images were corrected and restored as described above. No further processing was performed. Removing the nonnegativity constraint from the deconvolution algorithm produced no effect on the results.

In all cases, the choice of lens in the bead experiments was based on the best performing lens on each workstation. We independently characterized the degree of aberrations, transmittance, and axial resolution for the lenses available on each microscope. The lens that gave the best performance in these tests was used for these analyses. Thus, our analysis of signal response in WFM and LSCM does not compare specific types of lenses but the whole systems working as optimally as we could achieve.

Quantitative Measurements of Bead Fluorescence.
To determine the fluorophore density in each bead, we first manually identified the center of mass of each bead from both the LSCM and WFM. We used this manual method because methods based on edge detection of fluorescence intensity thresholds or gradients returned different edge positions on beads of different fluorophore densities. We then summed the fluorescence signal within a cylinder that described a subvolume of 70% of the sphere. Identical subvolumes were used for all beads. The summed intensities then were corrected by subtraction of a background (taken to be the median minus 2 s of the weakest bead population) and expressed as a fraction of (median + 2 s) of the brightest bead population. Data are presented as the logarithm of this normalized fluorescence. We estimated the mean intensity of each group of beads by using the central 80% of the populations to prevent skewing by extreme outliers and estimated the standard error of this mean by bootstrapping. Measurement of Yellow Fluorescent Protein (YFP)-a-Tubulin Fluorescence.
Deconvolved images (DeltaVision, Applied Precision) stored as 16-bit TIFF files were imported into Adobe photoshop. Subsequent manipulations were carried out within the photoshop shell by using the Fovea Pro (Reindeer Games, Asheville, NC) 16-bit plugins. Contrast-enhanced copies of the images were used to draw by hand regions enclosing individual subpellicular microtubules in all layers of the 3D stack in which it could be seen. These regions then were transferred to the unenhanced images, and all included pixel intensities were summed. At the same time, the total length of the microtubule and the total area within the hand-drawn regions were recorded. Background cytoplasmic fluorescence was measured in regions of the cytoplasm containing no discernible structure. After subtracting this background from the summed microtubule intensity, the residual was divided by the microtubule length to give an estimate of the net YFP fluorescence per micrometer of microtubule. Background-corrected integrated intensities for the conoid, spindle poles, and centrioles were determined similarly and then divided by the fluorescence per unit length of microtubules to give a measure of tubulin polymer content for each organelle in units of "micrometers of microtubule."