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.

An introduction to optical super-resolution microscopy for the adventurous biologist.

Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist's toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

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.

Updated feasibility trial experience with the Viaderm percutaneous access device.

The percutaneous access device (PAD) is used to connect an external drive unit to the Kantrowitz CardioVad (KCV), a cardiac assist device for the treatment of chronic heart failure. The PAD conveys pneumatic power from a drive unit to the implanted blood pump and an electrocardiogram signal from the myocardium to the drive unit. The device-tissue interface of the PAD is precoated with autologous fibroblasts cultured from a skin sample of the intended recipient. This preparation demonstrated long-term stability in animals and was adopted for use in patients receiving the KCV. The KCV is activated intermittently, and the drive unit may be connected and disconnected by the patient, which subjects the PAD to frequent manipulation. To date, the PAD has been implanted in nine patients ranging in age from 41 to 74 years. Implant times ranged from 2 to 458 days, for a total of 1082 days, of which 557 days were in an outpatient setting. Two patients experienced episodes of infection that did not originate from the PAD-tissue interface. This feasibility study demonstrated that (1) the PAD is stable and infection resistant in long-term ambulatory patients, (2) the PAD withstood the challenge of daily manipulation (the KCV is turned on and off electively), and (3) PADs can be replaced, if necessary.

A mechanical auxiliary ventricle. Histologic responses to long-term, intermittent pumping in calves.

The mechanical auxiliary ventricle (MAV) is an avalvular, inseries left ventricular assist device (LVAD) comprised of a 60 cc Biomer blood pump implanted in the descending thoracic aorta, a percutaneous access device (PAD), and an external pneumatic drive. In four calves the MAV was implanted and activated intermittently for 192, 249, 423, and 785 days, respectively; no anticoagulants were administered. When the animals were killed, the implants were retrieved, and autopsies, including gross, light microscopic, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) examinations, were performed. The MAV was securely attached to the aorta in every animal. A thin, even smooth, pseudoneointimal layer (PNI) contiguous with the aorta at the level of the suture line covered the MAV's blood-contacting surface. Areas of calcification beneath the PNI, expected in growing animals with implanted pumping devices, were noted. At autopsy there was no evidence of thrombosis or infection in the regions of the blood pump or the PAD. These findings confirm the calves' benign histologic response to the MAV, and together with the results of hematologic and biochemiccal studies, and bench tests of system components being reported elsewhere, indicate the system's readiness for limited clinical investigation in selected patients with chronic congestive failure.