Optical Workstation Research Articles

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567-582, 2016. © 2016 Wiley Periodicals, Inc.

Real time imaging of femtosecond laser induced nano-neurosurgery dynamics in C elegans

In this study we present for the first time the use of confocal microscopy and laser scanning brightfield microscopy (LSBF) for real time imaging of femtosecond laser nanosurgery and its dynamics in C. elegans. A single multimodal optical workstation that provides the ability to perform femtosecond laser nanosurgery and simultaneous confocal and LSBF imaging was used for the purpose. With this tool several dynamic phenomena concomitant with laser nanosurgery in C. elegans were observed and imaged. Some of these dynamic phenomena, like muscular contraction and single muscle cell stimulation, have been imaged for the first time during nano-neurosurgery of C. elegans.

Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: upgrading a commercial confocal inverted microscope.

In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

Analysis of molecular diffusion in ftsK cell-division mutants using laser surgery.

Escherichia coli cells that lack the carboxy-terminal part of FtsK fail to segregate their chromosomes properly during cytokinesis and tend to form chains. These chains are possibly formed as a result of DNA being trapped in the division planes or a failure to fuse the membrane during septum formation. If so, small molecules might diffuse between the apparent cell compartments. To investigate this theory, we developed an optical workstation that allows simultaneous imaging of and surgical operations on cellular objects in the sub-micrometre range. By surgical incisions of E. coli cell poles, diffusion of propidium iodide (PI) can be followed in real time. This analysis showed that PI was unable to diffuse from one cell equivalent to another in chain-forming ftsK mutants. Thus, the cytoplasm of the cell compartments in the chains seems to be fully separated.

Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities

Experimental laser microbeam techniques have become established tools for studying living specimens. A steerable, focused laser beam may be used for a variety of experimental manipulations such as laser microsurgery, optical trapping, localized photolysis of caged bioactive probes, and patterned photobleaching. Typically, purpose-designed experimental systems have been constructed for each of these applications. In order to assess the consequences of such experimental optical interventions, long-term, microscopic observation of the specimen is often required. Multiphoton excitation, because of its ability to obtain high-contrast images from deep within a specimen with minimal phototoxic effects, is a preferred technique for in vivo imaging. An optical workstation is described that combines the functionality of an experimental optical microbeam apparatus with a sensitive multiphoton imaging system designed for use with living specimens. Design considerations are discussed and examples of ongoing biological applications are presented. The integrated optical workstation concept offers advantages in terms of flexibility and versatility relative to systems implemented with separate imaging and experimental components.