Automated Microscope Focus Stacking using the WeMacro Micromate

In microscopy, capturing images with every detail in sharp focus can be challenging due to the limited depth of field, especially at high magnifications. Automated microscope focus stacking, a technique that combines multiple images taken at different focus depths, addresses this issue. However, manually achieving precise focus adjustments can be tedious and error-prone. The WeMacro Micromate offers an innovative solution to this problem, automating the focus stacking process for microscope photography.

Build Quality and Compatibility

The Micromate boasts solid construction and is designed to work with virtually any microscope equipped with a fine focus knob. Whether you’re using an Olympus, Nikon, Zeiss, or other brands, the Micromate can be attached as long as your microscope has a standard fine focus control. The unit features an adjustable motor height ranging from 2 cm to 18 cm, ensuring compatibility with various microscope models for automated focus stacking.

How Automated Microscope Focus Stacking Works

At the core of the Micromate is a stepper motor that couples directly with the microscope’s fine focus knob. This motor automates the incremental adjustments needed for focus stacking, controlled via the WeMacro app. The app allows you to set the step size, which determines how much the focus shifts between each captured image.

In the WeMacro app, when using the MicroMate, the step length is set in arbitrary “steps” rather than microns. While these ‘steps’ don’t directly correspond to physical units, they provide a precise way to control the motor’s movement. For instance, when the step length is set to 1 um in the app, the stepper motor takes 2132 steps to complete a full rotation of the microscope’s fine focus knob.

Setting Up the Micromate for Automated Focus Stacking: Key Parameters

To effectively use the Micromate for automated microscope focus stacking, it’s crucial to understand your microscope’s specifications. One of the most important pieces of information is the resolution of your microscope’s fine focus knob. This information is typically found in the microscope’s user manual. 

For example, let’s consider this data from the manual for Olympus CX43 clinical microscope:

– Movement distance per full rotation of the fine focus knob: 0.3 mm (300 μm)

This means that with every full rotation of the fine focus knob, the microscope’s stage moves vertically by 0.3 mm. The depth of field (DOF) change per rotation is linked to both this movement and the specific objective lens you’re using.

Understanding Depth of Field (DOF) and Step Size in Automated Focus Stacking

Depth of Field (DOF) refers to the distance within which objects appear acceptably sharp in an image. In microscope photography, the DOF becomes progressively smaller as the magnification increases. For high-magnification objectives, the DOF might only be a few microns.

The DOF for a microscope objective is calculated using the following approximation:

DOF = 0.55 μm / (NA²)

Where NA is the Numerical Aperture of the objective lens.

For focus stacking, the recommended step size is usually 75% – 80% of the calculated Depth of Field to ensure overlap between slices. For example, an objective lens with a Numerical Aperture (NA) of 0.25 has a DOF of approximately 8.8 μm and the safe step size is about 7 μm. To capture an image that covers a microscopic scene, the microscope stage has to be moved by this step size and an image is captured for each focal plane.

Translating Steps to Real-World Movement in Automated Focus Stacking

To determine the appropriate step size for the WeMacro app, we need to translate the microscope’s movement into the app’s “steps”. Here’s how:

1. Calculate the smallest step size achievable by the Micromate:

   – If the fine focus knob moves the stage by 300 μm per full rotation (as in the Olympus CX43 example)

   – And the Micromate motor completes 2132 steps per rotation

   – Then the smallest step size = 300 μm / 2132 ≈ 0.14 μm per step

2. Determine the required step size for your objective:

   – For an objective with DOF of 7 μm (NA = 0.25):

   – Step size in the app = DOF / Minimum step size

   – 8.8 μm / 0.14 μm ≈ 50

In this case, you would set the step length in the WeMacro app to 50 for the NA 0.25 objective. For higher-magnification lenses with finer DOF, you’ll need to program smaller steps for precise automated focus stacking.

Benefits of Automated Microscope Focus Stacking

The WeMacro Micromate’s value lies in its ability to automate this intricate process. It ensures each step is exact and consistent, reducing the margin for human error. Instead of manually turning the focus knob for each image and risking slight inconsistencies, the Micromate executes the process flawlessly with precision, regardless of microscope brand.

Considerations When Using Automated Focus Stacking

While the WeMacro Micromate is a powerful tool for automated microscope focus stacking, there are a few points to consider:

– Attachment Mechanism: The method used to attach the Micromate to the microscope’s fine focus knob can be tricky. Over time, the screws may loosen, potentially causing slippage and inaccuracies in focus adjustments. Regular checks and tightening of the screws can help mitigate this issue.

– Stability: The Micromate needs to be fixed to the table with a gel pad. The effectiveness of this pad can vary depending on the table material. A potential solution is to place both the microscope and the Micromate on a glass surface for improved stability during the automated focus stacking process.

– Step Length Setting: CAUTION: Always use the “slow” setting for the Step Length when performing microscopy with the WeMacro Micromate. The “fast” setting, if used inadvertently, could potentially cause severe damage to the objective lens by causing it to crash into the microscope stage.

– Initial Setup and Testing: Before beginning any stacking workflow or leaving the Micromate to operate unattended, always perform a test run with careful observation. This allows you to verify that all settings are correct and that the movement of the focus mechanism is smooth and within safe limits. Take shots are various focal planes to make sure the lighting is as you expect it to be.

Some MicroMate Step Sizes for the Olympus CX43

Objective	Numerical Aperture	Micromate Step Size
4x	0.1	312
10x	0.25	50
20x	0.4	19
40x	0.65	7
60x	0.8	4

MicroMate Step Size Calculator

To simplify this process, I have developed a calculator that computes the MicroMate step sizes based on the Numerical Aperture and the movement distance of a rotation of the fine focus knob. This calculator can be accessed at this page. Using this tool can streamline the setup process and ensure accurate step sizes for your specific microscope and objective combination.

Example Images Using the Olympus CX43

Conclusion

The WeMacro Micromate is an essential tool for simplifying automated microscope focus stacking, offering greater precision, consistency, and ease of use. Whether you’re a researcher, photographer, or hobbyist, the Micromate streamlines the complex process of manual focus stacking, allowing you to focus on the creativity and quality of your final images. 

By understanding your microscope’s specifications and the principles of depth of field, you can optimize the Micromate’s settings for your specific needs. This combination of automated precision and informed setup enables the capture of stunningly detailed microscopic images that were previously challenging to achieve. With the WeMacro Micromate, automated microscope focus stacking becomes an accessible and powerful technique for producing high-quality microscope imagery.

Disclaimer: I am not affiliated with WeMacro, nor have I received any compensation for this review. All opinions shared are my own, based on my personal experience using the WeMacro rails.