The working distance in microscopy is a critical parameter that determines the distance between the objective front lens and the top of the cover glass when the specimen is in focus. This comprehensive guide delves into the technical specifications, numerical aperture relationships, objective design considerations, and real-world examples to provide a thorough understanding of working distance in microscopy.
Specifications and Identification of Objectives
Working Distance (WD)
The working distance (WD) is the distance between the objective front lens and the top of the cover glass when the specimen is in focus. This parameter is crucial in determining the accessibility and clearance required for the specimen and any additional equipment or accessories.
Free Working Distance (FWD)
The free working distance (FWD) is the distance between the objective front end and the coverslip. This application-related feature of the objective is particularly important for imaging through thick glass walls or for chemical and metallurgical microscopy applications.
Numerical Aperture and Working Distance
Relationship
The working distance and numerical aperture (NA) of an objective lens are inversely related. As the magnification and numerical aperture increase, the working distance decreases. This relationship can be expressed mathematically as:
WD = k / NA
Where:
– WD is the working distance
– k is a constant that depends on the refractive index of the medium between the objective and the specimen
– NA is the numerical aperture of the objective
Example
For a working distance of 1 cm (10 mm), an objective lens with an NA of approximately 0.2 is required, assuming an air lens (n = 1).
Objective Designs and Working Distances
Long Working Distance Objectives
Long working distance (LWD) objectives are designed for applications that require a long free working distance, such as imaging through thick glass walls and for chemical and metallurgical microscopy. These objectives typically have lower numerical apertures compared to standard objectives, resulting in a larger working distance.
Super Long Working Distance Objectives
Super long working distance (SLWD) objectives exhibit significantly longer working distances but correspondingly lower numerical apertures compared to extra-long working distance objectives. These objectives are particularly useful for applications that require a large working distance, such as imaging thick samples or working with bulky equipment.
Examples of Working Distances for Different Objectives
Nikon Objectives
Nikon offers a range of objectives with varying working distances:
ELWD (Extra Long Working Distance):
– 20x: 11.0 mm
– 50x: 8.7 mm
– 100x: 2.0 mm
SLWD (Super Long Working Distance):
– 10x: 20.3 mm
– 20x: 20.5 mm
– 50x: 13.8 mm
– 100x: 4.7 mm
Olympus Objectives
Olympus also provides a variety of objectives with different working distances:
ACH (Achromat):
– 10x: 6.10 mm
– 20x: 3.00 mm
– 40x: 0.45 mm
– 60x: 0.23 mm
– 100x (Oil): 0.13 mm
PL (Plan Fluorite):
– 4x: 22.0 mm
– 10x: 10.5 mm
– 20x: 1.20 mm
– 40x: 0.56 mm
– 100x (Oil): 0.15 mm
PL APO (Plan Apochromat):
– 1.25x: 5.1 mm
– 2x: 6.20 mm
– 4x: 13.00 mm
– 10x: 3.10 mm
– 20x: 0.65 mm
– 40x: 0.20 mm
– 60x (Oil): 1.10 mm
– 100x (Oil): 0.10 mm
Advanced Considerations
Parfocal Length
The parfocal length is the distance between the objective’s front focal plane and the mounting surface of the objective. This parameter is important for maintaining focus when switching between objectives with different magnifications.
Immersion Media
The choice of immersion media, such as air, water, or oil, can also affect the working distance. The refractive index of the immersion medium influences the numerical aperture and, consequently, the working distance.
Aberration Correction
Objectives with advanced aberration correction, such as apochromatic (APO) objectives, can provide improved image quality and a larger working distance compared to standard objectives.
Depth of Field
The depth of field, which is the range of distances in the specimen that appear in focus, is inversely related to the numerical aperture. Objectives with a lower numerical aperture will have a larger depth of field, which can be advantageous for certain applications.
Practical Applications and Considerations
Working distance is a crucial parameter in various microscopy applications, including:
- Thick Samples: Long working distance objectives are essential for imaging through thick glass walls or containers, allowing for greater accessibility and clearance.
- Chemical and Metallurgical Microscopy: Long working distance objectives are preferred in these fields to accommodate bulky equipment and provide sufficient clearance for sample manipulation.
- Micromanipulation: Objectives with a large working distance enable the use of micromanipulators and other accessories within the microscope’s field of view.
- Live Cell Imaging: Objectives with a long working distance can accommodate specialized cell culture chambers or incubation systems, allowing for continuous monitoring of living cells.
- Correlative Microscopy: The working distance must be considered when integrating multiple imaging modalities, such as light microscopy and electron microscopy, to ensure compatibility and optimal sample positioning.
Conclusion
The working distance in microscopy is a critical parameter that directly impacts the accessibility, clearance, and overall performance of the microscope system. By understanding the relationship between working distance, numerical aperture, and objective design, researchers and microscopists can select the appropriate objectives for their specific applications, ensuring optimal imaging conditions and successful experimental outcomes.
References
- Olympus LS. (n.d.). Objectives: Specifications and Identification. Retrieved from https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/specifications/
- von Diezmann, A., et al. (2017). Super-resolution microscopy with very large working distance by using a large number of sources. Scientific Reports, 7(1), 1–10. doi: 10.1038/s41598-017-03743-4
- Leica Microsystems. (n.d.). Labeling of Objectives. Retrieved from https://www.leica-microsystems.com/products/microscope-accessories/microscope-objective-lens/labeling-of-objectives/
- Dutton Institute. (n.d.). Working Distance. Retrieved from https://www.e-education.psu.edu/mcl-optpro/theory/node801
- Nikon’s MicroscopyU. (n.d.). Working Distance and Parfocal Length. Retrieved from https://www.microscopyu.com/microscopy-basics/working-distance-and-parfocal-length
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