Visualizing Synaptic Multi-Protein Patterns of Neuronal Tissue With DNA-Assisted Single-Molecule Localization Microscopy.

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.

The effect of air, SF6 and C3F8 on immortalized human corneal endothelial cells.

PURPOSE: While anterior chamber air bubbles aid attachment during posterior lamellar surgery only for few days, these periods can be prolonged with gases in non-expanding concentrations. To test the effects of different gas compositions on immortalized human corneal endothelial cells (HCEC-12), we utilized Transwell inserts with semipermeable membranes as an artificial anterior chamber model. METHODS: Human corneal endothelial cells (HCEC-12) were cultured on Transwell inserts for 24 hr, then flipped, burdened and sunk with titanium rings in medium (M1), as well as filled with 2 ml of air (A), 20% sulphur hexafluoride (SF6) (S), or 12% C3F8 (C). After gas exposition for 24, 48 and 120 hr, cells were evaluated by live/dead staining, cell viability assay and Ki67 immunohistochemistry. RESULTS: Proliferation was significantly reduced (Ki67-positive fraction; M1, 14.8 ± 2.0%; A, 7.9 ± 1.4%; S, 8.1 ± 1.3%; C, 9.9 ± 2.3%; p-values; A, S, C versus M1 < 0.01), the total cell number decreased and the percentage of dead cells increased under gas exposition, independently of the type of gas (120 hr cell count/2.25 cm2 : M1 = 660.8 ± 57.0 cells; A = 125.5 ± 17.4 cells, S = 123.5 ± 17.0 cells, C = 118.8 ± 16.6 cells; p-value: M versus A/S/C < 0.001; 120 hr dead cells: M = 2.6 ± 1.0%, A = 8.4 ± 2.7%, S = 9.5 ± 3.2%, C = 11.3 ± 3.1%; p-value: M1 versus A/S/C < 0.01). Medium (M1)-control also proved significantly higher cell viability values in comparison with the gases, which did not differ significantly among them (120 hr luminescence: M1 = 1752.2 ± 91.4, A = 433.0 ± 30.3, S = 507.8 ± 23.3, C = 523.8 ± 20.3; p-value: M1 versus A/S/C < 0.01). CONCLUSIONS: Gas exposition led to a reduction in proliferation and an increase in cell death in HCEC-12, independently of the gas composition.

The effects of thoracic and cervical spinal cord lesions on the circadian rhythm of core body temperature.

Individuals with a spinal cord injury (SCI) have compromised afferent and efferent information below the lesion. Intact afferent information regarding skin temperature and the ability to regulate skin blood flow lead to an altered heat balance, which may impact the circadian variation in core body temperature (Tcore) and sleep-wake cycle. The authors assessed the circadian variation of Tcore in SCI individuals and able-bodied controls matched for the timing of the sleep-wake cycle. The authors examined subjects who had a high (cervical) or a low (thoracic) lesion. Intestinal Tcore (telemetry system) and physical activity (ambulatory activity monitor) levels were measured continuously and simultaneously in 8 tetraplegics, 7 paraplegics, and 8 able-bodied controls during one 24-h period of "normal" living. The regression slope between activity and Tcore was also calculated for each 2-h bin. Circadian rhythm parameters were estimated with partial Fourier time-series analysis, and groups were compared with general linear models, adjusted for the influence of individual wake-time. The (mean ± SD) dominant period length for controls, paraplegics, and tetraplegics were 24.4 ± 5.4 h, 22.5 ± 5.0 h, and 16.5 ± 5.1 h, respectively (p = .02). A significantly more pronounced 8-h harmonic was found for the variation in Tcore of SCI individuals (p = .05). Tetraplegics showed the highest nocturnal mean Tcore (p = .005), a 5-h phase-advanced circadian trough time (p = .04), and more variable relationships between physical activity and Tcore (p = .03). Taken together, tetraplegics demonstrate a pronounced disturbance of the circadian variation of Tcore, whereas the variation of Tcore in paraplegics was comparable to able-bodied controls.