Microscopy techniques offer unprecedented detail

Novel microscopy technologies are enabling scientists to examine samples in new levels of detail. Sean Ottewell reports.

Scientists and engineers in Scotland are developing a powerful camera that will enable living cells to be examined in unprecedented detail.

The fingernail-sized device, mounted within a microscope, will enable close-ups of the movement of molecules within cells. It should give scientists fresh understanding of how healthy – and diseased – cells behave.

Engineers are designing the camera to be sensitive enough to capture single photons of light. Conventional cameras typically capture thousands of photons at once on each of their pixels. The camera will combine thousands of light detectors that are fast and sensitive enough to capture short-lived photons. These detectors, housed on a microchip, should enable a detailed study of many molecules in a single frame.

Cells can be studied by marking them with fluorescent dye, and capturing microscopic emissions of the light as small changes occur in the cells – typically at a rate of billions of photons per second.

Researchers at the University of Edinburgh and Heriot-Watt University are working together on the five-year, €2.3 m project.

Dr Robert Henderson of the University of Edinburgh’s School of Engineering, who is leading the project, said: “This camera will take digital imaging technology to the next level. It will allow us to look at what goes on in living cells, which until now has eluded scientists. This device could be the key to understanding on a molecular level exactly how our cells function, and what happens when this goes wrong.”

Once the first generation of the camera is developed, Dr Colin Rickman at Heriot-Watt University will use the camera to study insulin secretion and how this can change in diabetes. This will test the camera and its capabilities to provide feedback for the next phase of camera development.

Dr Rickman commented: “For the first time, this unique camera will allow us to examine in real time, protein interactions in live cells. Initially, we’ll use the camera to study the release of insulin in diabetes, however ultimately it will be used in diverse areas of biomedical research to help scientists gain a deeper understanding of how diseased cells behave in patients living with other long term conditions such as cancer.”

Multiphoton excitation

In April, Olympus used Analytica 2014 in Munich for the launch of its new multiphoton excitation system the FluoView FVMPE-RS.

The system enables high-precision, ultra-fast scanning and stimulation, allowing researchers to see deep within specimens, take measurements at the highest speeds, and capture images, even when working under the most demanding conditions.

A high-speed scanner provides 438 frames/second at 512 × 32 scan performance. The scanner unit combines a newly developed high-speed resonant scanner with a conventional galvanometer scanner to provide high-speed and high-definition imaging in a single system. It is possible, for example, to capture rapid calcium channel reactions and membrane potential-sensitive dyes in action.

In addition, a proprietary silver coating Improves excitation efficiency by 50 per cent when compared with a traditional aluminium coating.

Silver-coated scanner mirrors achieve extremely high reflectance characteristics across a broad wavelength range from visible to near infrared. Total reflectance for the XY scanner is enhanced by more than 25 per cent in the near-infrared range compared to conventional aluminium coating mirrors, and this increased reflectance provides more than a 50 per cent improvement when converted to multi-photon excitation efficiency. The result, says the company, is a highly effective apparatus that delivers the superior power needed for deep in vivo probing. Nikon has launched a new laser application called Ti-LAPP for its ECLIPSE Ti range of inverted research microscopes.

With more than 32 different configurations spanning a wide variety of applications, Nikon says its new system provides researchers with unparalleled flexibility and modularity.

The Ti-LAPP system provides modular illumination modalities ranging from simple epi-fluorescence, to total internal reflection fluorescence (TIRF), photobleaching and digital micromirror device-mediated photoactivation. This modularity enables users the freedom to custom combine different imaging modalities to suit their needs. The system works seamlessly with Nikon’s NIS-Elements software, as well. By providing multiple modes of imaging on the same microscope, the Ti-LAPP system offers ease of experimentation as well as time and money saved, since users will no longer need to purchase multiple microscopes or systems to support different modes.

“Optical imaging techniques have evolved rapidly beyond just qualitative visualisation.  The Ti-LAPP system responds to those advances in imaging technologies and probes that further enable researchers to quantitatively address biological questions,” said Stephen Ross, general manager of product and marketing for Nikon Instruments. “This is the first system providing researchers with the ability to combine multiple imaging techniques with ease on the same imaging platform, as well as the freedom to make additional modifications to those configurations as research and experiments evolve and change.”

Launched in San Diego, USA, late last year, Ti-LAPP is expected to be available commercially shortly.

Fully confocal Raman microscopy

New from HORIBA Scientific is the XploRA PLUS fully confocal Raman microscope. Incorporating powerful research functions in a compact bench footprint, HORIBA says the new microscope does not compromise data quality or image resolution.

“XploRA PLUS is a fully confocal and high performance Raman microscope, offering an unmatched and enhanced range of options such as multiple laser wavelengths, complete automation, EMCCD detection, Raman polarisation and even AFM coupling. Our SWIFT Fast Raman imaging offers the best and most detailed Raman images, typically ten times faster than conventional mapping methods,” notes the company.

New LabSpec 6.3 software is aimed at multiple user environments such as HORIBA’s One-Click Raman. It will automatically adapt and optimise data acquisition parameters, producing quality Raman spectra time after time. Security of the system and hardware is assured by user-specific LabSpec software login accounts, while LabSpec sample methods provide an ideal way to standardise or automate Raman analysis at the touch of a button.

”Sensitivity and speed is unparalleled utilising deep-cooled CCD detector technology,” said Dr Andrew Whitley, vice president of sales for HORIBA Scientific. “This enables us to run the system faster, or reduce the amount of laser energy required for each analysis, and offer true non-destructive sample testing, preserving your sample no matter how delicate.”

He adds that XplorRA PLUS is the full Raman microscope package, offering ‘exceptional price/performance metrics’ with no compromise on performance.

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