ABSTRACT
In modern 3D microscopy, holding and orienting arbitrary biological objects with optical forces instead of using coverslips and gel cylinders is still a vision. Although optical trapping forces are strong enough and related photodamage is acceptable, the precise (re-) orientation of large specimen with multiple optical traps is difficult, since they grab blindly at the object and often slip off. Here, we present an approach to localize and track regions with increased refractive index using several holographic optical traps with a single camera in an off-focus position. We estimate the 3D grabbing positions around several trapping foci in parallel through analysis of the beam deformations, which are continuously measured by defocused camera images of cellular structures inside cell clusters. Although non-blind optical trapping is still a vision, this is an important step towards fully computer-controlled orientation and feature-optimized laser scanning of sub-mm sized biological specimen for future 3D light microscopy.
Subject(s)
Interferometry/methods , Microscopy/methods , Refractometry/methods , Cell Line, Tumor , Humans , Interferometry/instrumentation , Microscopy/instrumentation , Models, Theoretical , Optical Tweezers , Refractometry/instrumentationABSTRACT
Optical gradient forces generated by fast steerable optical tweezers are highly effective for sorting small populations of cells in a lab-on-a-chip environment. The presented system can sort a broad range of different biological specimens by an automated optimisation of the tweezer path and velocity profile. The optimal grab positions for subsequent trap and cell displacements are estimated from the intensity of the bright field image, which is derived theoretically and proven experimentally. We exhibit rapid displacements of 2 µm small mitochondria, yeast cells, rod-shaped bacteria and 30 µm large protoplasts. Reliable sorting of yeast cells in a microfluidic chamber by both morphological criteria and by fluorescence emission is demonstrated.
Subject(s)
Cell Separation/methods , Microfluidic Analytical Techniques/instrumentation , Optical Tweezers , Animals , Bacteria/isolation & purification , Cell Separation/instrumentation , Mice , Microfluidic Analytical Techniques/methods , Mitochondria/physiology , Protoplasts/cytology , Saccharomyces cerevisiae/isolation & purificationABSTRACT
To investigate the set of mtDNA molecules contained in small biological structures, powerful techniques for separation are required. We tested flow cytometry (FCM(1)), laser capture microdissection (LCM(2)) and a method using optical tweezers (OT(3)) in combination with a 1µ-Ibidi-Slide with regard to their ability to deposit single mitochondrial particles. The success of separation was determined by real-time quantitative PCR (qPCR(4)) and sequencing analysis. OT revealed the highest potential for the separation and deposition of single mitochondrial particles. The study presents a novel setup for effective separation of single mitochondrial particles, which is crucial for the analysis of single mitochondria.
