SubscribeThe world of Microscopy is changing at a pace, with more and more techniques being developed and a seemingly endless increase in new add-ons for your microscope.
This article aims to guide you through the most common of the add-ons and explain the technologies behind them and the benefits to you the user.
Introduction to Automation.
As computers control more features on research microscopes, and experiments take longer and longer, there is a greater need to automate manual functions. Most microscope manufacturers, offer instruments that are fully automated, including stages, filter wheels and focus drives.
However, in many cases these are very expensive research level microscopes, and often do not offer the flexibility of an upgraded manual microscope. Upgrading also provides a more economical solution to automation.
Specifications of motorised stages explained.
The basic specification of any motor driven stage is derived from the motor, ball (or lead) screw, and the quality of the machined bearing system.
The motor provides the power for the system and generally is a 200 step per revolution motor with 250 microsteps per full step.
The ball or lead screw translates the rotational movement of the motor to a linear motion of the stage.
These are generally available in 1, 2 or 5 mm pitch providing 1, 2, or 5 mm of travel per full revolution of the motor. The “accuracy” of the stage will depend upon the quality of its component parts.
The term “accuracy” is often quoted as bi-directional repeatability, or backlash. However, it can be confused with uni-directional repeatability and metric accuracy. Bi-directional repeatability is a measure of the ability of the stage to return to the same point when approaching from any direction.
Uni-directional repeatability is a measure the ability of the stage to return to the same point when approaching from the same direction. The metric accuracy is the error in the stage to move a set distance; this is usually quoted over the entire movement range of the stage.
What improves my “accuracy”?
Prior installs several features in the ProScan range of stages to improve bi- and uni- direction repeatability and metric accuracy. To improve repeatability Prior uses only quality controlled high precision ball screws in the ProScan range of stages and connect the motors to the ball screws via anti-backlash couplings.
Bi-directional repeatability, due to the stage approaching a point from two directions can be simplified by forcing the stage to always approach from the same direction, hence it becomes uni-directional repeatability. This feature is called backlash correction and can be activated in Prior controllers.
A high level of effort goes into machining a flat, square and straight bearing system for the stages, as this is the area of manufacturing which most effects the metric accuracy. To further improve metric accuracy ProScan stages are fitted with Intelligent Scanning Technology, (IST).
IST is a system where the stage movement is analysed during the production process and errors in metric accuracy recorded. Metric errors in stages tend to be linear in form, so the metric error is mapped to a straight line. The description of the straight line, the gradient and offset is stored on a chip on the stage itself. This is done for both axes creating four numbers, hence the common description 4 point mapping.
Prior products are Plug and Play, i.e., any product can be attached to any controller and the information about the stage is loaded into the controller. This makes Prior systems very flexible, and easy to set up.
Choosing your Motor and Ball screws sizes are a trade off between minimum steps size, i.e. smooth movement and high speed. 200 step, or 1.8o motors are supplied as standard on ProScan II stages, combined with 2 mm ball screws these provide a good compromise of speed 24mm/s and step size 0.04um. However, for faster speed 5mm balls screws are available to provide 60mm/s. Similarly by choosing the 1mm ball screw and 400 step 0.9o motors the minimum step size can be reduced to 0.01um, providing very smooth movement under high magnification and improvements in repeatability.
Adding encoders to a stage, although considered expensive is a good method of increasing longevity of specification and improving repeatability. It is generally accepted that encoders do not enhance the Metric Accuracy of a stage above that of IST.
Will my stage be compatible with my Heating inserts and incubator?
ProScan Stages are designed to integrate with most known brands of Incubator and many companies offer heating plates which insert directly into the ProScan range. This makes integration easier for you, as the systems are designed to work together.
Controllers for stages.
The advanced ProScan II controller can control up to one stage, one focus, 3 shutters, 3 filter wheels, 4 TTL input and outputs, and 4 encoded axis.
There are two options, the stand alone unit, which can be upgraded as and when required, or the PCI II controller, which is installed directly into the computer.
The PCI II comes fully loaded and capable of controlling all that the stand alone ProScan unit does and a 0-10V output for control of Piezo focus units, such as the Prior NanoScanZ.
The important check to make when choosing between the two is compatibility with your selected software. Most third party software will control Prior ProScan II stand alone controllers.
ProScan II controllers provide the ability to change almost any part of the controller functions, from reversing axis direction for the joystick, for right and left handed people, to changing the drive current for user specific applications. These functions can be accessed via RS232 commands or via the handy free demonstration software supplied with the controller.
What focusing options do I have?
A standard ProScan II focus motor can be attached to almost all modern microscopes and provides a focusing repeatability of around 1um depending on the microscope focus mechanism. This can be improved by using a linear encoder to around 0.05um for metric focusing accuracy and repeatability.
For most microscopes one rotation of the fine focus corresponds to 100um, hence a standard focus motor is capable of a step size of 2nm, however it is unlikely to achieve this with slippage in the microscope focus mechanisms.
The Piezo focus system NanoScanZ can provide step size and repeatability of around 1nm. The full travel range of the stage can be selected from 100um, 200um or 500um and any movement made, however large or small, is completed in 10ms compared to a standard motor which will take closer to a second. The NanoScanZ stage insert is compatible with many manual and motorised stages, and is controllable via most third party imaging software systems.
There are two methods of auto-focus, software and hardware. The software technique usually chooses focus at the best contrast for the image, the speed of this is based on computing speed and streamlining of software code. Hardware auto-focus mostly use low power lasers to reflect off the focusing surface, these systems are quicker to auto-focus as there is no image analysis included.
Which Shutters and Filter Wheels do I need?
The speed at which filter positions can be changed is an important characteristic along with vibration of the filter wheel or shutter. Modern filter wheel systems should be able to move from one position to the next adjacent position in approximately 55ms. The acceleration and deceleration profiles are important as considerable work has recently been done to give minimum vibration transfer to the microscope.
Many specimens are photosensitive which leads to filter wheels often being used in conjunction with a shutter system. Shutter systems typically are able to open and close in as little as 14ms, protecting valuable specimens from over exposure.
Microscope Illumination:- on or off the microscope?
New systems such as the Lumen200 light source and Lumen 200 Pro light source with filter wheel and shutter, now allow users to choose to keep vibrations and heat sources off the microscope.
These light sources are as powerful as the mercury lamp and are more than capable of providing adequate lighting for the majority of applications.
Live cell imaging and time lapse experiments are two fluorescence applications which benefit from the reduced focus drift which can be caused by the heating effect of the mercury lamp.
During automated time lapse experiments, time is added to each step to allow any vibration of the microscope to dissipate, using Lumen 200 this time is not required and the experiment can be completed quicker reducing the exposure time on the fluorophor.
Photo-bleaching of fluorophors can be further reduced using the light attenuator to supply the correct amount of light and reduce overexposure. This is often done using manual Neutral Density filters.
The Lumen200 Pro is supplied with a light attenuator capable of attenuating the light from 100%- 0% in 1% increments.
The choice of automated accessories now available is extensive but which one should I choose?
Live cell imaging.
One of the most demanding techniques live cell imaging requires a high precision stage and fast accurate focus. For this application an encoded 400 step 1mm ball screw ProScan stage and a NanoScanZ focus as repeatability is important in both XY and Z axis.
As Time Lapse is often used in live cell imaging, encoders are a good way of ensuring any thermal drift is compensated for. The Lumen200 Pro would be applicable to provide illumination for fluorescence as the filter wheel and heat source (from the bulb) is kept away from the microscope reducing the possibility of thermal drift and vibrations.
Digital slides/ Virtual slides.
This technique uses a motorised stage and focus to take sequential (overlapping) images of a sample area. These are then tiled (Stitched) together in software to produce an image of the entire slide. In this technique the metric accuracy of the motorised stage is important as when tiling the images each image needs to be taken at exactly the correct position. In this case a 400 step stage with 1mm ball screw would be advised.
Hardness testing.
Hardness testing stages designed to fit onto hardness testing microscopes are best suited to accept the loads required for this technique. For Micro-hardness testing the ProScan and OptiScan Upright stages can be used as they offer greater flexibility.
Cytology
This technique requires a low accuracy scan of overlapping images in a set pattern analysed continuously by a user. In some cases the images are analysed via computer and interesting features recorded for users to recall after the initial automatic scan is complete. In both cases there is little requirement of high precision or high speed stage. An economical OptiScan stage of around 1mm repeatability is often ideal for these types of applications.