Biomedical subjects

L Sacconi

Publications and source records attributed to L Sacconi.

6 recordsLinked to original sources

PubMed2006

New techniques in linear and non-linear laser optics in muscle research.

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

Animals↗

PubMed2006

Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials.

Second-harmonic generation (SHG) has proven essential for the highest-resolution optical recording of membrane potential (Vm) in intact specimens. Here, we demonstrate single-trial SHG recordings of neuronal somatic action potentials and quantitative recordings of their decay with averaging at multiple sites during propagation along branched neurites at distances up to 350 mum from the soma. We realized these advances by quantifying, analyzing, and thereby minimizing the dynamics of photodamage (PD), a frequent limiting factor in the optical imaging of biological preparations. The optical signal and the PD during SHG imaging of stained cultured Aplysia neurons were examined with intracellular electrode recordings monitoring the resting Vm variations induced by laser-scanning illumination. We found that the PD increased linearly with the dye concentration but grew with the cube of illumination intensity, leading to unanticipated optimization procedures to minimize PD. The addition of appropriate antioxidants in conjunction with an observed Vm recovery after termination of laser scanning further refined the imaging criteria for minimization and control of PD during SHG recording of action potentials. With these advances, the potential of SHG as an effective optical tool for neuroscience investigations is being realized.

Action Potentials↗

PubMed2006

Cell imaging and manipulation by nonlinear optical microscopy.

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

Animals↗

PubMed2004

Exploring molecular motors and switches at the single-molecule level.

Single-molecule techniques have propelled an impressive number of biophysical studies during the last decade. From relatively simple video-microscopy techniques, to sophisticated manipulation and detection apparata, single-molecule techniques are capable of tracking the movements and the reaction trajectories of single enzymatic units. By observing microspheres attached to biomolecules it is possible to follow the motion of molecular motors, or to detect conformational "switching" induced by regulatory proteins. Micromanipulation tools like optical tweezers have been widely applied to understand the mechanisms of linear molecular motors, and have allowed the measurement of the elementary steps and the forces produced by several motor proteins, including myosin, kinesin, and dynein. New experimental assays based on magnetic or optical "wrenches," which are able to apply and detect torques on rotary motors and biopolymers, are opening new possibilities in this field. Here, established and emerging magneto-optical manipulation and video-tracking techniques are reviewed, in the perspective of single molecular motors and regulatory proteins studies.

Adenosine Triphosphate↗

PubMed2003

Multiphoton multifocal microscopy exploiting a diffractive optical element.

Multiphoton multifocal microscopy (MMM) usually has been achieved through a combination of galvo scanners with microlens arrays, with rotating disks of microlens arrays, and cascaded beam splitters with asynchronous rastering of scanning mirrors. Here we describe the achievement of a neat and compact MMM by use of a high-diffraction-efficiency diffractive-optic element that generates a multiple-spot grid of uniform intensity to achieve higher fidelity in imaging of live cells at adequate speeds.

Journal Article↗

PubMedDate not supplied

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.

Animals↗