Breaking Barriers in Macro-to-Micro Confocal Imaging

Achieving Wide Field of View, High-Resolution, and Deep-Tissue Observation with a Single Objective Lens

In modern life science research, including neurobiology, oncology, and developmental biology, there is a critical need to analyze intracellular structures and functions in correlation with the architecture and functionality of the tissue in which the cells originally existed.

Confocal laser scanning microscopy (CLSM) and spinning disk confocal microscopy (SDCM) are core modalities for high-resolution fluorescence imaging. However, achieving comprehensive macro-to-micro correlation in thick samples requires overcoming optical and workflow constraints, notably limited working distance, aberrations from refracted index (RI) mismatch, and cumbersome objective lens switching.

This white paper addresses these limitations and details a solution that integrates a wide field of view, long working distance, and high numerical aperture (NA) in a single objective lens—paired with automated, depth-aware spherical aberration correction—to streamline macro-to-micro imaging.

Challenges in Macro-to-Micro Imaging with Confocal Microscopy

Conventional confocal imaging using objective lenses with a high magnification (e.g., 60X), high NA, and oil immersion medium enables acquisition of detailed cellular-level information.

However, correlating these data with the global structure of thick tissue has been difficult. A typical workflow requires first capturing the overall tissue using a low-magnification dry objective, followed by switching to a high-magnification oil immersion objective to image localized regions. This process introduces operational complexity and misalignment risk due to repeated switching between dry and oil objectives.

To overcome this limitation, a single objective lens capable of macro-to-micro imaging—simultaneously providing a wide field of view, long working distance, and high NA—has been needed. Previously, objectives meeting these criteria were generally confined to 75 mm parfocal length, limiting application to specialized upright systems and preventing use on inverted microscopes. As a result, these lenses were restricted to dedicated platforms rather than broadly deployable systems.

Furthermore, the refractive indices  of tissue-clearing reagents (Table 1) span approximately 1.38–1.56; which is relatively high. Refractive index mismatches introduce spherical aberration, degrading resolution and contrast and creating a major barrier to deep-tissue observation and quantitative 3D analysis.

^{Table 1. Representative refractive indices of tissue-clearing reagents.}

Innovating the LUPLAPO25XO Oil Immersion Objective Lens

Evident has applied its ultra-thin lens polishing technology built into the high-performance X Line™ objectives series¹ to develop the world’s first oil immersion apochromatic objective with a compact 45 mm parfocal length that achieves an NA of 1.00 and working distance of 1.0 mm (Table 2).

The LUPLAPO25XO objective lens (25X) combines a wide field of view, long working distance, and high NA into a single, universally deployable objective—providing broader, deeper, and more detailed imaging without changing optics (Figure 1, Table 2). The lens incorporates a correction collar that supports specimen refractive indices from 1.45 to 1.56, ensuring compatibility with a range of tissue-clearing reagents and effectively suppressing spherical aberration (Figure 2).

When paired with the FLUOVIEW™ FV5000 confocal and FV5000MPE multiphoton laser scanning microscopes, the TruResolution™ system provides automated correction collar adjustment. This system enables easy and reproducible spherical aberration correction for users at any skill level. During Z‑stack acquisition, the system continuously optimizes the collar position according to depth, maintaining correction throughout the entire 3D volume.²

^{Table 2. LUPLAPO25XO specifications.}

Imaging Applications in Advanced Life Science Research

The LUPLAPO25XO objective lens is ideally suited for deep observation of tissue-cleared specimens and for simultaneous visualization of fine cellular structures and overall tissue architecture. Its performance is maximized when used with the FLUOVIEW FV5000 for confocal three-dimensional imaging.

1. Wide Field of View, High-Resolution 3D Imaging

Using the LUPLAPO25XO objective lens, it is possible to capture a wide area of approximately 0.5 mm × 0.5 mm at a resolution of about 230 nm in a single scan. The image shown in Figure 3 represents a maximum intensity projection (MIP) of a Z-stack acquired from the surface to a depth of 250 µm in a mouse brain slice cleared with the SeeDB2 tissue-clearing reagent (n = 1.52).

Traditionally, capturing an area of this size at equivalent resolution required imaging nine separate regions (3 × 3) using a 60X oil immersion objective, followed by stitching the images together. Moreover, the working distance of a 60X oil immersion lens is only about 0.1 mm, making imaging at 250 µm depth impossible. In contrast, the LUPLAPO25XO objective lens combines a wide field of view and long working distance, enabling efficient deep observation and acquisition of highly detailed 3D images in significantly less time.

2. Capturing Subcellular High-Resolution Images Using Ultra-High Pixel Density Imaging

When capturing the maximum field of view at 8192 × 8192 pixels, the system achieves a pixel pitch of 69 nm, enabling extremely high spatial sampling. By applying TruSight™ deconvolution processing to these datasets, structures that previously required super-resolution microscopy for visualization can now be rendered with exceptional clarity (Figure 4). This advancement enables researchers to observe tissue architecture and fine cellular structures within a single image, providing a unified view that bridges macro-scale organization and micro-scale detail.

3. Acquiring Wide-Area, High-Resolution Images

By continuously capturing adjacent fields of view and stitching them together, it is possible to obtain high-resolution images over an even larger area. Using this approach, researchers can capture the entire structure of a mouse embryo in fine detail (Figure 5).

Conclusion

When the LUPLAPO25XO objective lens—combining a wide field of view, long working distance, and high NA—is used with the FLUOVIEW FV5000, it enables acquisition of a 0.5 mm × 0.5 mm area at approximately 230 nm resolution in a single scan. Moreover, the system performs automated spherical aberration correction according to specimen refractive indices (RI 1.45–1.56), enabling three-dimensional imaging up to 1 mm depth.

Additionally, when capturing the maximum field of view at 8192 × 8192 pixels, the pixel pitch reaches 69 nm. Combined with TruSight deconvolution processing, structures that previously required super-resolution microscopy for visualization can now be clearly resolved.

Furthermore, by continuously acquiring adjacent fields and stitching them together, wide-area, high-resolution imaging becomes possible. These combined capabilities deliver an imaging system that enables integrated analysis of both macro-scale tissue architecture and micro-scale cellular details within a single dataset, meeting the demands of fields such as developmental biology and neuroscience.

Authors

Naofumi Kobayashi, Optical Engineering Imaging Optics, Micro-Imaging Solutions R&D,  Evident
Hiromi Utsunomiya, Life Science High-End Imaging, Product Management, Evident

References

1. Israel, K. 2020. “Barrier‑Breaking and Now Edison Award‑Winning Objectives.” Insights, Evident, June 12, 2020.

2. Utsunomiya, H. 2026. “TruResolution Automated Spherical Aberration Correction for High-Resolution Deep Tissue Imaging .” Evident. Accessed on January 28, 2026.