In microscopy systems, alignment of the sample and achieving the highest image quality are common challenges that can vary depending on the sample, i.e., what is to be imaged and at what length scale. In modern cell analysis instruments, a custom lens design is often deployed to achieve optimal imaging performance and to control costs. When considering a custom lens design understanding the application, use case, and image resolution needed are key aspects to consider in achieving the required performance. This article covers the key considerations involved in specifying and developing optical imaging objectives and considers two common use cases.

Microscopes for cell imaging (e.g., spatial genomics, high-content screening, digital pathology, as examples of “microscopes in a box”) operate under the principle of optical magnification, often referred to as optical power. Prior to the advent of digital imaging, most microscopes were built for general purpose use, with reconfigurable objective lenses and spectral filters, providing different degrees of magnification. Applications requiring additional contrast using fluorescence dyes add more complexity to the microscope system. Today, the high performance offered by general purpose instruments can now be achieved in a compact low-cost format suitable for new product introductions.

Fig. 2 - Example of a point-to-point macro lens design. The object plane is on the left and image plane on the right and the magnification is 0.75x, meaning the image is smaller than the object.

Understanding the Application

Often new life science and medical diagnostic products or techniques are first established using high-end laboratory grade microscopes. To address the market or application needs, the final product must meet specific imaging performance, size, and cost requirements. Digital imaging technology has enabled more compact and lower cost microscope instruments in many markets. In microscopy, low-cost CMOS cameras and lower power, efficient light sources have enabled the development of high-volume commercial products that incorporate sophisticated scientific techniques. The following questions should be evaluated at the start of the product development process.

  • If a general-purpose microscope is being used today, can the core functionality be implemented in a simplified design?

  • What existing techniques are used to make the measurement currently?

  • What technologies (photonic, mechanical) can be implemented to reduce cost or complexity?

It is critical to ensure a successful start to the product development process to understand the use case.

Defining the Use Case

An understanding of what is being imaged or detected (100s or 1000s of single cells over a large area (i.e., field-of-view or sub-micron features or molecules within a single cell) is critical early in the design process. Next, it is important that the engineer or designer understands how the image data will be used. Will it be processed or analyzed by a computer algorithm (i.e., machine learning/artificial intelligence), or will a human be interacting with the image (i.e., zooming or panning to certain areas across the entire image)? Understanding the use case and how image quality meets the user’s needs is a crucial first step in developing the optical imaging performance requirements of the new microscope system and has a significant impact on how to design the imaging optic and other critical optical assemblies (the tube lens, for example). How the product will be used informs the final image quality needed. In turn, this information is used to develop the specifications of the image sensor and optical system. When a custom microscopy system is being developed, achieving the imaging performance (i.e., optical resolution) required to meet the use case and no more is critical and helps ensure that the complete concept to product commercialization can be conducted on time and on budget and leads to a product that is successful commercially.

Optical Resolution vs. Camera Resolution

In modern digital imaging instruments, the final image quality is determined by the combination of four primary components: display, image processing, image sensor, and optical assemblies or optical subsystem, composed of the imaging lens and illumination.

Finding the right balance of specifications for each subsystem is key to avoid overdesigning the product. It is also critical to achieving product cost targets when manufactured in volume. Properly matching the optical resolution to the image sensor is an important step in the design process. The optimal match is determined through a complete understanding of how the final image(s) will be used. Typically, when the imaging objective has greater resolution than the image sensor, a digital sampling artifact called aliasing can occur. When edges or patterns are imaged, artifacts appear in the final image. To avoid this, the most optimal combination of objective lens and image sensor is when the resolution of the image sensor is at least 2X greater than that of the objective lens or configured for Nyquist sampling. That is, the sampling frequency f = d /2, where d is the image space spatial frequency of smallest feature size to be detected. For most imaging systems, no greater image sensor resolution is needed. When the final image needs to be zoomed or manipulated, greater than 2X sampling is beneficial. Finally, the optical resolution of a lens is often compared to the diffraction limit, or the physical resolution limit that is defined by the diameter of the lens aperture size and focal length of the lens. How sharp the image is, depends on how close to the diffraction limit the lens performs at.

Fig. 3 - Lens sharpness measurement. The dotted black line represents diffraction limit for a high magnification lens, blue represents the nominal design and orange represents the measured performance.

Custom vs. Off-the-Shelf

Most existing off-the-shelf microscopy components and optics are interchangeable on larger bench top systems. They are made to reasonably high precision and good consistency of quality. Off-the-shelf components are best suited in the following situations:

  • For proof of concept and early prototypes.

  • When cost and size is not critical.

  • If product volumes are relatively low (under 200–500 per year).

  • If quality control does not need to be tightly held.

Once product volumes rise above 500 per year or the need for more compact instrument form factors is critical, the cost, size, and performance can be optimized through investing in custom optical design. Through a custom supply chain, including development of suitable metrology and inspection steps, quality and consistency is easily controlled.

Examples

The degree of magnification in cell microscopy systems generally fall into two primary categories, macro imaging, where the magnification is under 4X and high magnification at 10X, or greater. For macro imaging, the resolution is limited to approximately 1–2 mm and individual cells cannot be fully resolved in most microscope configurations. In these applications, only aggregate information about the cell sample is needed to perform a measurement. In high magnification optical microscopy, where submicron resolution can reach the optical resolution limit of 200 nm, individual cells, or even sub-cellular features can be resolved. High magnification microscopy can be used for cell counting, cell morphology measurements and cell differentiation and editing using lasers. Furthermore, in some advanced techniques such as super-resolution imaging, the physical limits of optical resolution can be overcome, enabling sub-50 nm resolution, which is ideal for next-generation spatial sequencing within cells.

Conclusion

Developing the right product for the use case and achieving the required image quality requires understanding and balance of the design parameters. Often, an off-the-shelf lens is a suitable option for product development and initial product release. Once product volumes increase or unique performance is required, a custom lens design should be considered. When engaging with a design firm for a custom lens design, providing documentation of the requirements and commercial aspects of the product help to streamline the process and reduce the risks of arriving at a design that does not suit the application.

This article was written by Neil Anderson, Vice President of Sales and Marketing for Gray Optics, Portland, ME. For more information, visit here .


Medical Design Briefs Magazine

This article first appeared in the August, 2021 issue of Medical Design Briefs Magazine.

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