Introduction
Applications of precision positioning equipment have gone hand-in-hand with microscopy since the dawn of its inception.
Understanding the requirements that each microscopy system has is fundamental to choosing the correct precision positioning device. This document, while far from an exhaustive list, aims to provide a summary of some of the most common microscopy applications, and discusses the most beneficial positioning products that can be used for them.
Confocal Microscopy
Confocal microscopy is most commonly used to study structures within living cells. By capturing a series of multiple 2D images at differing sample depths, a full reconstruction of 3D microstructures can be made, allowing for further inspection and analysis.
In order to change a 2D image into a 3D representation, individual stacks of XY images are taken. This process, known as optical sectioning, requires a fast moving but precise change in the focal length during a continuous scan in the XY plane. These separate “stacks” of images can then be stitched together to give a full image of the biomaterial.
In order to achieve a fast and precise scanning of the focal planes, a highly precise and fast vertical motion of the objective lens is required, with minimal crosstalk in the X or Y directions. Closed loop, flexure amplified piezoelectric actuators are ideal for this as they can provide position repeatability in the sub-nanometer range over travel ranges in excess of 500μm, and with typical angular errors of less than 20μrad. The high stiffness design of such products allows for high speed and high dynamic range performance, meaning that 3D reconstructions can be realised much more quickly.
Super-Resolution Microscopy Techniques – XY Positioning
New super-resolution techniques, such as stimulated emission depletion microscopy (STED), photo-activated localisation microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), have been developed in order to break the Abbe diffraction limitation of conventional microscopy techniques, bringing the imaging resolution down from 200 nm to between 20 and 30 nm.
Due to this, super resolution microscopes demand highly accurate positioning equipment and particularly demand long term stability so that these highly resolved images can be realised, as the resolution of the positioning stages will directly affect the resolution of the scanned images that you can get from the microscope.
In general, piezo positioning stages are perfectly suited to such applications as they can enable extremely fast scanning applications while maintaining a sub-nm precision and theoretically unlimited resolution in two or three axes, with minimal cross-talk. When paired with a suitably accurate feedback system, such as a strain-gauge or capacitive sensor, the position stability of such devices is in the region of ≤10 nm, provided vibrations and other sources of noise are suitably isolated.
Light Sheet Microscopy
Light sheet microscopy was first developed in the mid-90s in order to scan and section relatively large biological samples (of a few mm thickness) in a fast process, while still retaining a good level of optical resolution. As the name suggests, incident laser light is focussed down in to a thin planar “sheet” in order to illuminate the area of interest of a sample, which will in turn cause it to fluoresce in order to be imaged.
For a light sheet microscope to take a 2D or 3D image either one, or two, or both things must happen. Either the sample itself needs to be scanned in two axes with respect to the incident laser beam, or the sample can be scanned in one axis with the beam also scanning dynamically over the sample to move the light sheet. Increased resolution can be obtained through combining multiple images of the same part of the sample from different angles, meaning an additional rotation in one or two axes can be beneficial. Finally, in order to perform optical sectioning and get 3 dimensional images, the focal plane also needs to be adjusted between each 2D plane.
As such, combining multiple multi-axis positioners is required for a light sheet setup, however customised four or five axis stages on the sample end can also be an option. As this is a high speed technique that is used on larger biological samples, piezo motor stages are the obvious fit here due to their combination of long travel ranges and piezo levels of precision. The wide dynamic range that is made possible by this technology allows for fast scanning at hundreds of mm per second, as well as precise velocity control at the level of nm per second.
Electron Microscopy
Electron microscopy uses a beam of electrons rather than photons of light in order to generate images far exceeding the resolution of a light-based microscope. Due to the much lower (de Broglie) wavelength of an electron versus visible light in the electromagnetic spectrum, resolutions of down to 0.1 nm can be realised to produce high quality images of sub atomic structures.
Although there are many different types of electron microscopy, the two most common examples are scanning electron microscopy (SEM), and transmission electron microscopy (TEM). SEM creates an image by detecting electrons that reflect off the sample object, whereas TEM requires the electrons to fully pass through the material and creates an image from these transmitted electrons. This requires more specific sample preparation and much higher electron beam voltages, but also produces a more highly magnified image.
For both methods, it is typical for the incoming electron beam to be focussed to a tight spot and then raster scanned in X and Y directions over the sample. In order to realise an XY scan with such precision, piezo stages are once again the standout option, this time fitted with high linearity capacitive sensors to ensure for the most accurate and repeatable positioning. Due to the shallow depth of focus, a capacitive sensor would also be required for the Z axis motion. It is also necessary to optimise the control loop through a highly sophisticated controller to achieve the necessary results.
Endoscopy
Endoscopy is a minimally invasive procedure designed to look inside your body. Typically, a long, thin tube complete with a small camera enters the body through a natural orifice and passes through your body to reach the intended area. Endoscopies can be used for a wide range of medical uses such as for observation, diagnoses, imaging, and surgery.
Endoscopes are not single-use and might need to be used for different uses throughout their lifetime. Motorising the lens allows for a much larger range of focal lengths for the different use cases of an endoscope. Given its high precision, small size, and low voltage and current requirements, piezo motor technology is perfect for use in motorised endoscope cameras.
By utilising this technology effectively, autofocussing modules with sub-micron precision and travel ranges of up to 8 mm can be miniaturised into modules with dimensions of less than 6 mm diameter. It is important to allow the patient as much comfort as possible – it is after all only a minimally invasive procedure – while also providing the endoscope itself with sufficient versatility.
