The traditional microscope that you know and love is operated manually. Picture the scene: the microscopist chooses the light source, gently places the sample the moveable stage, selects the objective lens, and scans to select the field of view. This process is perfect for processing and analyzing a small number of samples per day.
But nowadays, automated microscope systems can turn this time-tested process into an over powering force—providing higher throughput, more systematic analysis, and automatic workflows. This automation is valuable when many repeated observations are required, either in the context of live cell imaging or in high throughput analysis.
This article takes you through the rudiments of an automated microscopy, and sets you on the path to exploring this powerful tool.
Modern day microscopes have a wide array of components that can be automated. Shutters, filter wheels, stages, light sources, and focus control can all be replaced with versions that are electronically controlled. One option, of course, is to retrofit your existing microscope setup with these components, hook them up to a computer, and control them with any of a number of commercially available image acquisition software packages.
However, assembling a fully automated optical imaging system that performs optimally is an extremely complex task that is not for the faint of heart. Getting the setup right requires expertise, experience in optics and electronics, and a big investment of time.
For most of us, purchasing an off-the-shelf automated system is, by far, the most efficient way to enter this new world of experimental possibility. Whether you build your own, or buy readymade, a basic understanding of the individual components of a fully automated microscopy system will be useful in helping you to decide which system to choose and how to best use its capabilities for your research.
So, here are some of the microscope components that can be automatically controlled in your shiny new (or retrofitted old) system:
Focus and Stage Control
Focus motors are connected to the fine focus transmission gear set of a microscope stage to enable automated focus control through the image acquisition software. This setup can be controlled by software for autofocusing and/or z-stack collection. These motors perform manual focusing in many advanced microscopes, too, so there is no mechanical coupling between the user control and the stage.
Motors can also control the stage in the x- and y-axes. Just as in the case of focusing, these can be used either to transmit user commands without mechanical coupling between the controls and the stage, or to enable the software to directly control x-y positions.
Electromechanical shutters block the light sources from illuminating the specimen between camera exposures, and they are especially essential when imaging fluorescent or live specimens to minimize photobleaching and phototoxicity. They are also used to select between multiple light sources and pathways, in some cases in rapid mode, for example to gather two images per time point in time-lapse sequences. Automated high-performance shutters are microprocessor controlled and are interfaced with a computer to coordinate their operation with other parts of the microscope.
Wavelength selection can be effected through various devices, such as multiple filters, beam splitting units that direct light through several pathways, monochromators, or acousto-optic tunable filters (AOTFs). While manual switching is slow and requires constant user intervention, automatic switching enables the user to achieve rapid switching between the desired wavelengths and schedule those experiments in the user’s absence.
There are several practical mechanisms for automatically interchanging fluorescence filters, the most common using rotating filter wheels controlled by motor units. Among the advantages of these wheels is the flexibility of rapidly changing filter sets and the numerous configuration possibilities for both excitation and emission light. One disadvantage of such systems is that their switching speed, although high, has limitations. Also, vibration introduced by the mechanism that may be a problem in some cases.
Alternative wavelength selection systems involve multichannel and spectral imaging systems that enable simultaneous multi-color imaging or spectral separation and linear unmixing without changing filters, using a single digital camera detector. Such setups offer freedom from motion-related artifacts and avoid registration errors between images.
Wavelength selection can be done manually, either by direct selection or by actuation of a control mechanism, or it can be done by the software in a pre-programmed sequence.
Light sources for widefield microscopy include tungsten-halogen sources for transmitted light, arc gas lamps for fluorescence excitation, while various kinds of lasers are used for confocal applications. The metal-halide lamp and LED sources are often used as a replacement for the mercury arc-discharge lamp. Light source selection can be done by the user through direct manual intervention or using controls available by the software. In advanced systems, the software can switch between light sources in pre-determined sequences.
The user can control the operation of the camera or detector (for example in confocal microscopes) through the use of the software. Software-control can be applied for image collection in the absence of the user.
Carbon Dioxide Sensor and Regulator Plus Temperature Control
Carbon dioxide and temperature must be controlled during live imaging. They can be adjusted independently or through the automated microscope software.
An automated microscope system can be pre-set to record images in time-lapse mode, in the absence of the user. The desired spectral profiles of the fluorophores, the exposure time, gain and threshold can be selected, and the time interval between shots adjusted.
In confocal or deconvolution microscopy, Z-stack settings can be stored in memory and applied to the desired fields. Images for each field can be acquired with the settings selected for the particular field, and these settings can be different from one field to another. Storage of coordinates allows return of the motorized stage to the individual fields at the appropriate time intervals (and allows the user to return to them later at will) while autofocusing protects from focus drift over time, something especially important for live specimens.
When both large fields of view and high resolution is required, the solution is given by tiling. The software guides the system to acquire the desired number of neighboring fields that will then be automatically stitched to one large field of view. Axio Imager, Axio Observer, Axio Scan.Z1 and Celldiscoverer 7 employing ZEN imaging software, are examples of systems with such capabilities. Meta-data and label storage allows proper archiving of the data, with replication of exact imaging conditions, when desired.
Examples of Automated Microscopy in the Lab
Microscope automation is an important tool for experimental biology. Its utility is expanding as fast as we can invent new applications to harness it. To learn more about automation of microscopy, and about some real life examples of how it is being harnessed in the lab, take a look at the white paper series “Automation in Microscopy.”