Fluorophore unmixing based on bleaching and recovery kinetics using MCR-ALS.

Fluorescence microscopy is a key technology in the life sciences, though its performance is constrained by the number of labels that can be recorded. We propose to use the kinetics of fluorophore photodestruction and subsequent fluorescence recovery to distinguish multiple spectrally-overlapping emitters in fixed cells, thus enhancing the information that can be obtained from a single measurement. We show that the data can be directly processed using multivariate curve resolution - alternating least squares (MCR-ALS) to deliver distinct images for each fluorophore in their local environment, and apply this methodology to membrane imaging using DiBAC4(3) and concanavalin A - Alexa Fluor 488 as the fluorophores. We find that the DiBAC4(3) displays two distinct degradation/recovery kinetics that correspond to two different label distributions, allowing us to simultaneously distinguish three different fluorescence distributions from two spectrally overlapping fluorophores. We expect that our approach will scale to other dynamically-binding dyes, leading to similarly increased multiplexing capability.

Live-cell monochromatic dual-label sub-diffraction microscopy by mt-pcSOFI.

We expand photochromic super-resolution optical fluctuation imaging (pcSOFI) to monochromatic dual-channel sub-diffraction microscopy. Multi-tau (mt-)pcSOFI unmixes spectrally identical reversibly switchable fluorescent proteins (RSFPs) based on their blinking kinetics. We show that mt-pcSOFI can be used to simultaneously image two structures in living cells with existing RSFPs and the newly developed ffDronpa-F.

Second thoracic sympathetic ganglionectomy in sympathetically maintained pain.

Twenty-four individuals with sympathetically maintained pain were treated by posterior paravertebral T2 sympathectomy following transient response to sympathetic nerve blockade. Eight surgical patients (33.4%) had causalgia, and 16 patients (66.4%) suffered with reflex sympathetic dystrophy. Overall, physical evidence of improvement was noted in 87% of surgical patients, with subjective improvement in 71%. Reflex sympathetic dystrophy patients fared better than those with causalgia. Complications were minor. The techniques employed appear safe and effective; a multidisciplinary approach with neurosurgery, physiatry, anesthesiology, psychology, and allied health services is recommended.

Fixed- vs adjusted-dose heparin in the prophylaxis of thromboembolism in spinal cord injury.

Thromboembolism is a common and occasionally catastrophic complication in patients with spinal cord injury and complete motor paralysis. We, therefore, evaluated the efficacy and safety of two prophylactic regimens, using heparin in either fixed doses of 5000 U given subcutaneously twice daily or in doses adjusted to prolong the activated partial thromboplastin time (APTT) to approximately 1 1/2 times control. Patients were monitored with daily clinical examinations, serial impedance plethysmography, and Doppler flow studies. All events suspected to be thromboembolic were confirmed by venography, ventilation-perfusion scans, or pulmonary angiography. Seventy-five patients were randomized, and 58 (29 in each group) either remained in the study for more than seven weeks or experienced a thrombotic or hemorrhagic event. Patients on the adjusted-dose regimen received a mean (+/- SD) of 13,200 +/- 2200 U of heparin per dose and had an APTT 1 1/2 times higher than those on the fixed-dose regimen. Thromboembolism was detected in nine (31%) of 29 patients randomized to the fixed-dose regimen and two (7%) of 29 on the adjusted-dose regimen. While no patient who received the adjusted dose and whose APTT reached the target level had a thrombosis, bleeding occurred in seven patients. No patient on the fixed-dose regimen bled. We conclude that patients with spinal cord injury who can be maintained on doses of heparin sufficient to prolong their APTT to 1 1/2 times control values will be spared thromboembolic complications, but these patients are at high risk of bleeding, especially if they have trauma to other tissues in addition to their spinal cord injury.