The collection of three-dimensional image data is an essential part of automated microscopy. Imaging cells in a single plane in biological sciences is not possible due to the inherent inequality of the sample or the thickness of the cell greater than the depth of field of the microscope objective or the imaging method.

Image Credit: Prior Scientific Instruments Ltd.

As it provides a more realistic representation than traditional 2D cultures, imaging cells grown on 3D substrates is becoming more and more popular.

To image and render multiple layers of cells or tissues, organs or even whole animals need even greater penetration of the sample. Live events occurring in these samples must also be tracked in three-dimensional space.

The acquisition of images in 3D space is also required by material microscopy. Large samples, for example 12-inch semiconductor wafers, generally cannot be positioned so flat that areas of interest are always sharp when mapping the sample.

Extended focus, where small Z stacks (series of images taken at the same XY coordinates) are put together at each frame, assembles a tile scan image in focus when the scan routine is complete using the best image of each stack.

To image all relevant materials, multilayer samples, for example LCD displays, also require Z stacking. Automated microscopy is required for this type of analysis, and taking 3D images by hand takes a considerable amount of time. and puts a strain on the user of the microscope.

On the other hand, the motorized acquisition takes a few seconds and is repeatable. To make 3D image data acquisition as efficient as possible, Prior Scientific offers three technologies to upgrade an existing microscope or create a stand-alone focusing system.

3 technologies used to collect 3D image data

Image Credit: Prior Scientific Instruments Ltd.

Simple and easy to install motorized focus drive – Earlier PS3H122R microscope add-on

Entry-level 3D imaging often begins with the motorization of a manual microscope frame. Prior’s PS3H122R focus drive attaches directly to the coarse focus button and drives the fine focus mechanism.

The PS3H122R has a much better mechanical resolution than the manual focusing system of the microscope, so the same axial imaging resolution can be obtained but in a faster and more reliable manner.

Direct coupling between the microscope’s fine focus mechanism and the focus drive can be used to further improve focus repeatability, which is useful for autofocus or time-lapse applications. .

No disassembly of the microscope is necessary when installing the PS3H122R on one of the many compatible microscopes from Leica, Nikon, Zeiss, Olympus and others. This will take only few minutes.

A common cause of repetitive strain injuries in long-term microscope users is manual rotation of the focus controls. Adding Prior’s PS3H122R can eliminate this via the use of a lightweight joystick or digipot.

Prior recently fitted a UK university hospital service with a motorized focus upgrade for this purpose. Through the use of programmable buttons on its joystick, quicker, longer-range movements were enabled without the need to use rotational hand movements when focusing.

3 technologies used to collect 3D image data

Image Credit: Prior Scientific Instruments Ltd.

Self-Contained Motorized Z-Axis Focus Brackets – Front FB Series Focus Blocks

Sometimes, the use of a conventional microscope can place constraints on the research or analysis performed at universities, production lines, or industrial design companies. Custom-made breadboard-based optical arrangements and microscopes are commonly used to solve this problem, opening the door to more efficient motorized focusing.

of the prior FB Series Focus Blocks feature ball screws instead of a rack-and-pinion design, allowing for improved precision, linearity and pitch resolution. It also allows them to provide up to five times the travel range and double the load capacity of traditional microscope focusing mechanisms.

Finally, unlike a conventional microscope, where the focusing mechanism is coupled to the PS3H122R via a focus knob, the ball screws are also coupled directly to a motor to create a compact linear axis that can easily be fitted with ‘linear encoders to improve repeatability and resolution.

To create stand-alone XYZ devices for research and industry, Prior has regularly used the FB series in combination with its XY stages. The Mesolens microscope has a microscope lens that offers the unique combination of a large field of view with high resolution.

The unique nature of optics required a high-performance XY focus and translation system, which could be decoupled from the overall microscope frame.

In combination with many Prior steps over the past ten years of research, Prior’s FB204 has always been up to par and is currently being used in several Mesolens research microscopes.

Openstand – Prior’s imaging system development platform – also features a focus block-style Z-axis. This means that all the advantages of the FB series can be combined with a complete optical system and an XY stage as part of a fully motorized and customized microscope.

3 technologies used to collect 3D image data

Image Credit: Prior Scientific Instruments Ltd.

Piezo nano-positioning steps for rapid acquisition of high-resolution Z-stacks

Ultimately, however, depending on the resolution of the encoder, the focus blocks are limited to microscopy with axial resolution of around 100nm and Z-axis movements at longer range at slower speeds. The stop-start nature of Z-stack acquisition means that focusing mechanisms with rapid acceleration and deceleration are required to capture dynamic events or optimize image collection speeds.

Prior’s Queensgate nano-positioning stage brand offers industry-leading performance from microscopy requiring axial resolution at confocal microscopy level to high-end super-resolution methods or applications that require rapid Z-cell acquisition, like 3D time-lapse imagery.

These stages can be used as part of a custom imaging solution, or they can be added to existing microscopes.

Piezoelectric motors respond to increased voltage by increasing by an extremely precise amount at the nanoscale, allowing a dramatic increase in performance over stepper motor devices.

Queensgate’s SP Series nanostroke stages and the OP400 objective positioner provide sub-nanometer resolution and stabilization times in 7 millisecond steps over displacement ranges up to 600 microns. Regarding the potential this offers for 3D image acquisition, 400 images can be collected in less than 30 seconds when stacking in Z.

This can be reduced to four seconds by using a position-based hardware trigger, where camera acquisition occurs following a positional trigger directly from the Queensgate device to acquire each frame, allowing the Queensgate device to stop at each Z position independently of the software. shooting by camera.

The wide range of Queensgate nano-positioning devices means that the user’s Z-axis hardware is able to match the resolution and penetration depth of the user’s imaging method (eg, multiphoton imaging).

The stability and speed of the Queensgate SP400 has enabled UK-based imaging instrument company Visitech International to achieve a unique time-saver for a system designed around their super-resolution imaging system. high-speed structured lighting (VT-iSIM).

Using the SP400, movements between adjacent positions in each Z-stack can be fully performed without oscillation in five milliseconds, allowing each axial movement in a Z-stack to be performed within the camera’s playback time. This meant no loss of time between frames caused by the hardware, allowing the system to run as quickly as possible.

In addition, the SP400 provided the perfect match in terms of precision and accuracy, as the VT-iSIM provides an axial image resolution of 300nm, twice as good as conventional wide-field microscopy.

Choose the right focusing device for your application

It can be difficult to find the right component to automate Z-axis movement. Prior Scientific has the expertise to guide the user to a product that meets their technical requirements or can recommend one that could sustain a system or take it to the next level.

The three options presented here can add low, medium, or high precision automated focusing to many systems, from custom systems underpinning research or new product design to microscopes from world-class manufacturers.

This information has been obtained, reviewed and adapted from documents provided by Prior Scientific Instruments Ltd.

For more information on this source, please visit Previous Scientific Instruments Ltd.