Groundbreaking Approaches To Microscopy

Open-source microscopy supports cutting-edge discoveries, explains Craig Goodman

Open-source microscopy projects are providing groundbreaking imaging capabilities to researchers, particularly those using 3D and 4D imaging or looking at single molecules. The developers of these complex systems must think about many different components, including lasers, motion control and positioning of objectives and stages, as well as software for image acquisition and analysis. Knowledge of the options available – and expertise in their application – is key to successfully creating a system with the desired resolution.

Open-source microscopy provides a forum to share new applications and innovative microscope set-ups, which can then be built in several locations around the world to accelerate deployment of novel techniques and progress scientific understanding. When building a microscope, it’s incredibly important that all components within the system work together – regardless of manufacturer – and correct positioning makes all the difference. Piezoelectric actuators have long been considered the gold standard for the fine positioning of both sample stages and objectives across a broad range of microscopy techniques and are currently used for three main applications in super resolution microscopy (SRM): Z-positioning of objectives for autofocussing; manipulation of illumination systems (such as in light sheet and 4Pi techniques); and positioning of sample stages. Each of these applications has unique requirements in terms of range of travel, reproducibility and dynamics but, in general terms, system integrators want piezo-controlled movements to be as rapid, precise and stable as possible.

High speed and high resolution scanners

Many companies have developed advanced objective scanners in response to evolving microscopy capabilities. Physik Instrumente’s (PI) PIFOC range, for example, essentially changes the focal position of a microscope, moving the objective along the Z plane to adjust the focus depth within a sample. This allows researchers to perform ‘layer-by-layer’ analysis for a range of experiments that use fluorescence techniques, stepping through a sample to build up a 3D image. This approach has become increasingly popular for life sciences applications, and the stages have been refined and updated to better serve the market, offering finer resolution and longer travel ranges. For example, the newest PIFOC positioner – the P 725.XCD2 – can travel as far as 800 μm, and offers highly dynamic focus displacement in the Z direction. Imaging is performed while the objective is stationary, then the entire stack of images is digitally stitched together to create a 3D representation. Accurate and consistent objective positioning is vital to allow the images to be perfectly stitched together without any overlap. This approach can be used to understand much more about the structure of cells, tumours, viruses and other biological samples.

Remote access for optimal performance

Optimising the performance of a high-resolution microscope requires all of the individual components to work in harmony, and this is best achieved by assembling the complete instrument before tuning. Some stages have been developed with this in mind and can be supplied with a controller to enable remote optimisation via unique set-up software. Built-in tools allow features such as dynamic stepping and settling to be fine-tuned at full working capacity. The optimisation process can be performed entirely remotely – from a different room or even another site – via an internet connection, allowing application engineers to work side-by-side with customers anywhere in the world to maximise performance and troubleshoot unexpected installation issues. This saves researchers precious time and ensures that they have a robust system that can achieve the most accurate results possible. It gives scientists confidence in their results and is especially helpful for labs developing and optimising new techniques, which is often the case for open-source microscopy projects. Essentially, this helps to take the guesswork out of the development process, ensuring set up problems are not hindering image quality.

The new generation

The latest generation of SRM systems are achieving spatial resolutions of around 20nm, and the sub-nanometre positioning precision offered by current piezo-based drives will help to increase this resolution even further. Microscope integrators have been seeking longer travel ranges, greater stiffness and higher speeds since the construction of the very first SRM systems, and the development of customised, open-source solutions to help meet this growing demand will be vital as fluorescence microscopy pushes further into the nanodimension. These open-source platforms allow scientists from anywhere in the world to reproduce exactly the same expertly devised hardware solutions, to recreate other researchers’ experiments, and to confirm the validity of their work much more quickly and easily, rapidly driving advances in our understanding of many molecular structures and interactions.

Craig Goodman is with Physik Instrumente

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