Optimization of Cell Morphology Measurement via Single-Molecule Tracking PALM

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments

Accurate determination of direction depends on localization precision.

<p><b>A</b>. Monte Carlo simulations were used to determine the distance of motion required to accurately determine the direction of a moving molecule localized with precision of σ_loc. 100 pairs of single points were generated in normal distributions with standard deviation σ_loc centered around two points separated by increasing distance. <b>B</b>. Pairs were plotted as a compass plot with the initial point (green) in the center connected to the second point (red) by a blue line. The net distance traveled parallel to the real translation of the distribution is marked by the black dot. <b>C</b>. The percent of vectors pointing toward the correct quadrant (within 45 degrees of the correct direction). <b>D</b>. The measured distance between localized points (blue) and the standard deviation of θ for the accompanying vectors (black). Each dot represents mean of 100 paired measurements.</p

Effect of motion during image acquisition on single molecule photon distribution and localization precision.

<p><b>A</b>. Examples of random walks taken by a molecule over various timescales. Blue lines depict the path taken by the molecules, with green and red dots denoting the starting and ending position, respectively. <b>B</b>. Examples of photon distributions emitted from moving molecules of the indicated D over integration times ranging from 0 to 50 ms. <b>C</b>. The mean value of the brightest pixel (N = 1000 molecules) is plotted against integration time for molecules emitting 100, 250, or 1000 photons over the course of the integration time. <b>D</b>. Histogram of calculated precisions for molecules with D = 1.0 µm²/sec. <b>E</b>. The mean calculated precision for molecules with D = 0.1 µm²/sec (<b>red line</b>) and D = 1.0 µm²/sec (<b>black line</b>). Points represent mean of 1000 molecules. <b>F</b>. To examine the interaction of movement-induced error with photon-dependent precision, the mean error of molecules emitting 100 photons (<b>black line</b>), 250 photons (<b>red line</b>), and 1000 photons (<b>blue line</b>) were plotted as a function of exposure duration.</p

More accurate morphology of living neurons using short, pulsed excitation during acquisition.

<p><b>A</b>. Cultured hippocampal neurons grown 10 days in vitro (DIV) expressing membrane-mEos2 were imaged at 50 Hz using excitation pulses of two durations (t_e = 2 ms and 10 ms) delivered in random order. The distribution of localized positions was plotted (A, enlarged in B), demonstrating a thinner appearance of neuronal processes imaged with longer t_e. <b>C</b>. Intensity profile of line scans drawn perpendicular to the neuronal process as in B. <b>D</b>. Cumulative frequency plot of the line scan full width at half maximum intensity. <b>E</b>. Paired comparison showed that the width of the processes was consistently diminished in the longer exposure. <b>F</b>. Measured spine neck widths (red) and spine lengths (blue) in neurons grown 11 to 12 DIV. Neurons were imaged at 50 Hz for 10,000 frames with t_e = 4 ms.</p

Measurement of morphology is degraded by molecular motion during prolonged integration times.

<p>To determine the effect of diffusion on the measurement of cell structure based on the position of localized molecules, we simulated molecules diffusing within a bounded space analogous to a filopodium. <b>A</b>. Random walks of molecules with D = 1.0 µm²/s were generated within a rectangle 100 nm wide (<b>left, center</b>). The localized position of the molecules is displayed for simulated acquisition using integration times of 0 (i.e., a fixed particle) and 10 ms (<b>right</b>). <b>B</b>. Molecules with D = 1 µm²/s (<b>left</b>) or 0.1 µm²/s (<b>right</b>) began their walks at random initial points within the bounded rectangle as in A. The density of localized positions across rectangles 150 nm in width (<b>Top</b>) or 75 nm (<b>Bottom</b>) plotted as histograms for exposures ranging from 0 to 50 ms are shown. <b>C</b>. The half-width of the bounded regions is quantified for D = 1.0 µm²/s (<b>Left</b>) or 0.1 µm²/s (<b>Right</b>). <b>D</b>. The effect of motion on the distribution of localized positions within a spine was modeled using a region consisting of a 500 nm square spine head and a neck that was 1000 nm long and 100 nm wide connected to a dendrite that was 500 nm wide and 1500 nm long (<b>left</b>). Plots of the paths taken by individual molecules with D = 1 um²/s and an exposure duration of 1 ms are shown (<b>second panel</b>). <b>E</b>. Localized positions of simulated imaged acquired using integration times of 0 (fixed particle), 5, and 50 ms are shown. Note the degradation of morphological accuracy with long exposure times.</p

Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dyneindependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABAB