Measuring tubulin content in Toxoplasma gondii : A comparison of laser-scanning confocal and wide-field fluorescence microscopy

Abstract

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-␣-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-␣-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for Ϸ10 microtubule segments of 0.5-m length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains Ϸ1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

Brain-Derived Neurotrophic Factor Promotes Long-Term Potentiation-Related Cytoskeletal Changes in Adult Hippocampus

“…From preliminary studies (supplemental Fig. 2, available at www.jneurosci.org as supplemental material), we determined that the superior detection efficiency achieved by a charge-coupled device (CCD) camera compared with laser-scanning confocal microscopy made wide-field microscopy coupled with parametric estimation signal processing the favorable choice for quantifying a broad range of label intensities (Swedlow et al, 2002). Rhodamine-phalloidin labeling was examined using epifluorescence illumination and a Zeiss (Oberkochen, Germany) Axioscope microscope equipped with an Axiocam CCD camera and AxioVision 3.1 software.…”

Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

“…Also, misalignment of the lamp can result in significant changes in the excitation intensity. Commonly used laser sources in confocal microscopy show short-and long-term variations (Swedlow et al, 2002). Periodic alignment of the excitation source and calibration of the excitation intensity is therefore important.…”

“…Especially for objects that are much larger than the diffraction-limit along the optical axis, it is important to use either a confocal microscope or a wide-field microscope with suitable deconvolution to assign the outof-focus light to the correct plane. This has been described previously in Swedlow et al (2002) using 6 µm fluorescent beads. Alternatively, confocal microscopy may also be useful as discussed later.…”

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

“…Wide-field microscopy, on the other hand, uses full field illumination and all the light passing through the objective is sent to the detector. This coupled with a high quantum efficiency cooled coupled device (CCD) camera ensures imaging with a high signal to noise ratio compared to confocal microscopy 9 , with very fast exposure times. In our application, we must image for long periods, record 3D stacks and ultimately resolve small-near-diffraction limited structures.…”