Building on the swept-field confocal (SFC) technology of Bruker’s first-generation Opterra, the Opterra II system is the latest advancement in high-speed fluorescence microscopy designed specifically for live-cell studies. It utilizes proprietary one-dimensional pinhole array technology to combine the resolution of traditional confocal systems with the speed typically associated with wide-field imaging. With specialized input optics that produce a highly uniform field of view and a sensitive CCD camera for detection, the system produces quantitative data in all dimensions while ensuring cell viability. The user-selectable aperture sizes provide flexibility to achieve the optimal balance of speed, resolution, and fluorescence intensity in real time.
Bruker designed Opterra II to flexibly adjust speed, resolution, and fluorescence intensity based on experiment requirements. The Opterra II’s unique scanner design features a motorized aperture plate that contains pinholes of three different sizes, and slits of four different widths. The use of one-dimensional pinhole arrays offers significant advantages over systems based on two-dimensional arrays, such as spinning disk confocal microscopes. The Opterra array produces half, or less, of the crosstalk of a two-dimensional array, producing sharper images with superior optical sectioning and greater depth. Pinholes provide maximum resolution, while slits allow for higher speed acquisitions, and since aperture selection is controlled by software, optimally matching a selected aperture to each objective is very easy.
The Opterra II has been specifically designed to evenly illuminate the field of view. When measured by a beam profiler, beyond the pinhole aperture plate, the illumination beam shows 3% to 4% deviation across the field. The fluorescence that is emitted from the specimen must also be effectively transmitted though the imaging system to the camera. The Opterra II’s excellent and guaranteed field uniformity is a standard feature, works with multiple dichroics, and provides unparalleled quantitative performance right out of the box. For example, when fluorescence emission of a concentrated dye solution is measured at the camera, the deviation across the field is guaranteed to be less than 10%.
The Opterra II is designed to control the dose of excitation light by minimizing the excitation light exposure time and delivering light to specific locations only when needed. The size of the confocal aperture and the exposure time can be adjusted within the software to minimize the excitation light appropriately for each sample. The optical path also has been optimized to ensure that emitted photons are efficiently transmitted to and collected by the detector. The Opterra II allows researchers to perform studies on highly sensitive samples not possible with other instruments, such as standard confocal microscopes, not only because specimens are kept alive, but because cellular function is maintained as close to biological conditions as possible.
Capture rapid physiological processes in real-time without missing fast transient events. The enhanced swept-field confocal scanning technology of the standard Opterra II allows for high-speed imaging at hundreds of frames per second. With an appropriate camera, over a thousand frames per second can be achieved.
High-Speed Live-Cell Imaging
Opterra II's gentle imaging at full camera rate facilitates high-speed acquisitions over extended periods of time, enabling quantitative measurement of molecular and ionic concentrations using fluorescent reporter molecules.
Opterra II's unique optical design allows FRAP, photoconversion, photoablation, and uncaging to occur simultaneously with imaging. The wide variety of scan methods provides a tool for any photomanipulation protocol.
High-Speed Volumetic Imaging
Opterra II's piezo stage facilitates the acquisition of 3D stacks at camera frame rates, enabling entire multicolor stacks to be acquired in less than a second for monolayer cells, and in a few seconds for small organisms.
Prairie View software incorporates more than 18 years of life sciences application experience into a rich array of tools to give researchers the ability to design detailed and accurate protocols for improved experiment results. Full-access controls allow users to optimize all aspects of image acquisition for speed, resolution, and signal intensity. An intuitive user interface supports streamlined definitions for both simple and complex experiments in multiple dimensions. Triggers and analog signals generated by the system enable seamless interaction with external devices for sensory stimulation, electrical recording, and other experimental paradigms.
The Opterra system gives us fast confocal imaging with higher signal to noise than other available solutions. Our use of a multiphoton laser with the photoactivation module is providing us with a unique opportunity to produce ablations and photobleaching at precise 3 dimensional locations simultaneously with imaging.