Microscope depth of field explorer

See how objective NA and aperture affect the thickness that appears in focus.

Choose your objective

Colour bands are a general guide; use the NA marked on your own objective.

Match the two markings on your eyepiece, for example 10×/22. Eyepiece magnification affects the visual-focus estimate; FN changes the visible specimen width, not the calculated depth. Camera viewing needs a different criterion.

For fluorescence, use the detected emission wavelength. White light uses 550 nm as an example.

Objective aperture100% open

Approximate diffraction depth of field
1.30 µm
Physical depth of focus · specimen side0.65 µmFull-aperture diffraction component
Visual depth of focus · through eyepieces1.20 µmIncludes an eyepiece viewing term; 10× eyepieces selected.
Approximate visible field diameter · specimen side0.55 mmFN 22 ÷ 40× objective. Assumes 1× tube factor and sufficient objective field.
Depth of field at full objective NA1.30 µm
Effective objective NA0.65

Side view: objective, aperture and specimen

40× / 0.65 NA · Biological dry

40× / 0.65ObjectiveObjective iris FOCUS DEPTH1.30 µmSpecimen
Specimen layersDepth appearing in focus

The blue cone shows collected light angle. The highlighted specimen thickness grows as the simulated objective iris closes. Schematic, not to scale.

General guide only. Values are theoretical approximations, not measured specifications or guaranteed performance. Illumination-aperture slider values are illustrative. Actual focus range varies with objective design, the glass or plastic thickness beneath or above the specimen, sample depth and refractive index, immersion medium, wavelength, contrast method, illumination and microscope adjustment. Check the objective documentation and verify on your own system for critical work.

Compare two objectives at full aperture

The same selected wavelength is used for both. Each lens shows its full-aperture depth of field and physical focus-depth convention in microns.

Your selected objective40× / 0.65 NA1.30 µmPhysical focus depth: 0.65 µm
Through eyepieces: 1.20 µm
8.80 µmPhysical focus depth: 4.40 µm
Through eyepieces: 10.1 µm

Bar lengths compare the diffraction term on a logarithmic scale, so very low and high NA objectives remain visible. Micron values are the useful comparison.

What changes when you adjust an aperture?

Depth of field · specimen side

Thickness of the specimen that looks acceptably sharp. The full-aperture estimate above is in microns. Closing an adjustable objective iris increases the calculated estimate.

Illumination diaphragms

A transmitted condenser aperture or reflected-light incident aperture can change apparent depth and contrast. The slider illustrates the change in apparent focus, while the listed micron values remain full-objective-NA baselines. Its percentage is not a calibrated NA or micron scale.

Physical and visual focus depth

Your table’s physical value is λn/(2NA²): 50× / 0.80 NA at 550 nm gives 0.43 µm. The visual eyepiece estimate adds the glossary’s magnification term. Both describe specimen-side focus ranges; sensor-side tolerance is different.

About these results: This educational tool gives approximate, specimen-side values under simplified assumptions. Manufacturer specifications, practical imaging and different depth-of-field definitions may give different figures. The preset objectives represent example magnification/NA combinations, not an exhaustive catalogue or a promise that every combination fits every microscope. The illumination-aperture slider uses an illustrative scaling, not a calibrated measurement. For decisions requiring a specified tolerance, use the exact objective data and measure the complete microscope system under the intended conditions.

Sample holder and thickness: A slide and coverslip, flask, Petri dish or multiwell plate may put different thicknesses and materials in the light path. Objectives are designed for particular coverslips, vessel bottoms and working distances. Imaging deeper into a thick specimen can add refractive-index mismatch and aberration, especially at high NA. These factors can change image sharpness and the practically usable focus range, but the vessel name or sample thickness alone cannot supply a reliable correction to the theoretical micron values above. Use a compatible objective and its specified cover-glass or vessel-bottom correction, then check the result on the actual specimen.

Eyepiece field number: FN is the diameter of the intermediate-image field in millimetres. The displayed specimen field diameter is FN ÷ objective magnification, assuming a 1× tube factor and an objective/tube able to deliver that field. Tube or zoom factors and optical vignetting can reduce the actual view. FN does not enter the depth-of-field or visual-focus formula; eyepiece magnification does enter the latter. Use the markings on your eyepiece rather than treating the options as universal manufacturer pairings.

How this estimate works: The full-NA micron estimate and objective-iris numeric estimate use the diffraction term d ≈ λn/NA² (λ in µm; n = 1.00 for air, 1.33 for water, approximately 1.45 for glycerol, 1.515 for oil). The second number is physical focus depth λn/(2NA²), matching the convention in the supplied objective table. The eyepiece result uses the supplied glossary formula λn/(2NA²) + (n × 1000)/(7NA × M), with M = objective magnification × eyepiece magnification and λ in µm. Its coefficient and viewing criterion come from that document; it is an estimate for visual observation. These are different definitions of specimen-side focus thickness and should not be added together. Both omit the detector/eye circle-of-confusion term, which depends on magnification, sensor sampling and the chosen sharpness criterion. The objective iris slider assumes effective NA changes in proportion to its percentage setting; real iris markings and pupil geometry may not follow that scale. Most ordinary objectives do not have an adjustable iris. Condenser and incident-light apertures change illumination angle and contrast without changing the objective’s marked collection NA. Their displayed micron changes are illustrative apparent-depth estimates, scaled by 2/(1 + opening fraction²), anchored at the full-aperture baseline. This simple visualisation is not a calibrated optical law: an iris opening percentage does not directly specify illumination NA or a measured depth of field. Do not use those estimated micron changes as physical depth-of-focus specifications. A reflected-light field diaphragm controls the illuminated area and should not be confused with the aperture diaphragm. Technique, specimen, aberrations and setup alter actual results. Both displayed micron measures refer to the object/specimen side; image-side depth of focus is a different quantity. The physical value excludes the eyepiece viewing term. Camera image-side tolerance and visual accommodation vary with the imaging system and observer.

Educational guide from Auckland Scientific. References: Nikon MicroscopyU: depth of field · ZEISS: reflected-light path · Objective iris explanation.

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