Focus on Focus: Three Technologies for Collecting 3D Image Data

Posted in Microscope automation

Sep 2nd 2021

Collection of 3-dimensional image data is a staple of automated microscopy. In biological science, cells often cannot be imaged in a single plane, either due to the thickness of the cell being greater than the depth of field of the microscope objective or imaging technique or to inherent unevenness across the sample. Imaging cells grown on 3D substrates is increasingly prevalent as it provides a more true-to-life representation than traditional 2D cultures. Organs or even whole animals require even greater penetration of the sample to image and render multiple cell layers or tissues. Live events happening in these samples also need to be tracked in three-dimensional space.

Materials microscopy also requires image acquisition in 3D space. Large samples, such as 12-inch semiconductor wafers, often cannot be positioned so flat that the areas of interest are always in focus when mapping the sample. Extended focus, where small Z-stacks (series of images taken at the same XY coordinates) are collected at each frame, allows an in-focus tile scan image to be stitched together at the end of the scanning routine using the best image from each stack. Multilayer samples, such as LCD screens, also require Z-stacking to image all the relevant materials therein.

This type of analysis requires the use of automated microscopy – taking 3D images by hand not only takes considerable time but also places strain on the microscope user. In contrast, motorized acquisition is repeatable and takes a matter of seconds. Prior Scientific offers three technologies to upgrade an existing microscope, or create a standalone focusing system, to allow the acquisition of 3D image data as efficiently as possible.

Simple, Easy to Fit Motorized Focus Control – Prior PS3H122R Microscope Add-on

Entry-level 3D imaging often begins by motorizing a manual microscope frame. Prior’s PS3H122R focus drive clamps directly onto the coarse focus knob and drives the fine focus mechanism. The PS3H122R has far superior mechanical resolution than the manual focusing system of the microscope, so that the same axial imaging resolution can be achieved but in a faster and more reliable way. A direct coupling between the focus drive and the microscope fine focus mechanism can be employed to further enhance focusing repeatability, which is useful for time lapse or autofocusing applications. Fitting the PS3H122R to any of the vast array of compatible microscopes from Nikon, Olympus, Zeiss, Leica, and others requires no microscope disassembly and takes only a matter of minutes.

Manual rotation of focus drives is a common cause of repetitive strain injuries in long-term microscope users. The addition of Prior’s PS3H122R can eliminate this via use of a lightweight joystick or digipot. Recently Prior equipped a UK university hospital department with a motorized focus upgrade for exactly this purpose and allowed longer range fast movements through the use of programmable buttons on its joystick, avoiding the need to use rotational hand movements when focusing altogether.

Standalone Z Axis Motorized Focusing Mounts – Prior FB-series Focus Blocks

In universities, industrial design companies or production lines sometimes the use of a conventional microscope places too many constraints on the research or analysis being undertaken. Bespoke microscopes and breadboard-based optical arrangements are common, which opens the door to higher performance motorized focusing. Prior’s FB-series of focus blocks feature ballscrews rather than a rack-and-pinion design, enabling improved linearity, accuracy, and step resolution. This also allows them to give to 5 times the travel range and double the load capacity of traditional microscope focusing mechanisms. Finally, the ballscrews are coupled directly to a motor, in contrast to a conventional microscope where the focus mechanism is coupled to the PS3H122R via the focus knob, creating a compact linear axis that can easily be fitted with linear encoders to enhance resolution and repeatability further.

Prior has routinely used the FB-series in combination with its XY stages to create standalone XYZ devices for research and industry. The Mesolens microscope features a microscope lens that offers the unique combination of a large field of view with high resolution. The unique nature of the optics required a high performance focusing and XY translation system that could be decoupled from the overall microscope frame; Prior’s FB204, in combination with various Prior stages over the past 10 years of research, has always been up to the task, and is currently used in multiple Mesolens research microscopes.

Prior’s imaging system development platform, Openstand, also features a focus block-style style Z-axis. This means all the benefits of the FB-series can be combined with a complete optical system in addition to an XY stage as part of a completely motorized, customized microscope.

Piezo-based Nanopositioning Stages for Rapid Acquisition of High Resolution Z-stacks

Ultimately, however, focus blocks are limited to microscopy with an axial resolution of approximately 100 nm, depending the resolution of the encoder, and to longer-range Z-axis movements at slower speeds. The stop-start nature of acquiring Z-stacks leads to the requirement for focusing mechanisms with fast acceleration and deceleration. Prior’s Queensgate brand of nanopositioning stages offer world-leading performance for microscopy requiring axial resolution at the level of confocal microscopy through to high-end super-resolution techniques, or applications that require rapid acquisition of Z-stacks, such as 3D timelapse imaging. These stages can be added to existing microscopes or be used as part of a customized imaging solution.

Piezo motors respond to an increased voltage by expanding by an extremely precise amount, which allows for a vast increase in performance compared to stepper motor devices. Queensgate’s SP-series nanopositioning stages, and OP400 objective positioner, offer sub-nanometre resolution and 7-millisecond step settle times over up to 600-micron travel ranges. Regarding the potential this offers for 3D image acquisition, 400 images can be acquired in less than 30 seconds when Z-stacking. Using a position-based hardware trigger, where camera acquisition occurs following a positional trigger directly from the Queensgate device to acquire each image, allowing the Queensgate device to stop at each Z-position independently from software-based camera acquisition, can reduce this to 4 seconds. The extended range of Queensgate nanopositioning devices means that your Z-axis hardware can match your imaging technique’s penetration depth and resolution (e.g. multiphoton imaging).

The speed and stability of the Queensgate SP400 allowed Visitech International, the UK-based imaging instrumentation company, to achieve a unique time saving for a system designed around their high speed structured illumination (VT-iSIM) super-resolution imaging system. Movements between adjacent positions in each Z -stack can be fully completed in five milliseconds using the SP400 with no oscillation, which allowed every axial movement in a Z-stack to be completed within the readout time of the camera. This meant that there was no time loss between images caused by the hardware, allowing the system to run as fast as possible. Furthermore, as the VT-iSIM provides an axial image resolution of 300 nm, two times better than regular widefield microscopy, the SP400 provided the perfect match in terms of precision and accuracy.

Choosing the right focusing device for your application

Finding the right component for automating your Z-axis movement can be challenging. Prior Scientific has the expertise to guide you to a product that meets your technical requirements or recommend one that could future-proof your system or take it to the next level. The three options presented here can add low, moderate, or high precision automated focus to a variety of systems, from microscopes from world-class manufacturers to customized systems underpinning research or new product design.