MVLscope CONCEPT 02 · 10–15 MINUTES
Find your plane of focus.
Focus and Depth of Field
Inside the specimen
3D · drag to orbitPreparing the 3D specimen…
The cyan sheet is the focus plane. Its translucent band shows the depth reference. Beads and vertical distances are enlarged for visibility.
Through the microscope
Simulated widefieldKEEP THE EVIDENCE
More depth, or finer detail?
Focus stays at z = 0 µm. Capture one image at NA 0.25 and one at NA 0.80. Compare the central 0.60 µm bead pair and the two outer layers.
A VOLUME IS MORE THAN ONE IMAGE
Build a z-stack.
Scan from −3 to +3 µm. First try a 2 µm step and inspect what it misses. Then use 0.25 µm steps. Every frame is calculated at its own focal plane.
Projection keeps the brightest value at each pixel across the stack. It collapses depth; it is not a single focused image or a 3D reconstruction.
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Research notes · model, assumptions & sources
What this experiment models
A synthetic fluorescent-bead phantom with three layers at −2, 0 and +2 µm. All beads emit at the selected wavelength; colors in the 3D diagram identify depths, not different dyes. The two inner beads in each layer are 0.60 µm apart. The 3D view shows known specimen geometry, not a reconstruction from camera images.
The diffraction depth reference is D = nλ / NA², with n = 1 for air. Here D is the full band thickness centered on the focal plane. This is a teaching reference, not a sharp boundary of visibility or a measured axial resolution. Real acceptable focus also depends on the detector, contrast, illumination and the criterion used. Depth of field is in specimen space; depth of focus is in image space.
The detector sums energy-normalized Gaussian point-spread functions. The in-focus width is σ₀ = 0.21λ / NA; defocus broadens it as σ(z) = σ₀√[1 + (2Δz / D)²]. This is a smooth approximation to the central diffraction spot, not a full wave-optical PSF. It does not reproduce Airy rings or predict exact defocused intensities. A fixed display mapping is used for comparisons and stacks.
Magnification, field width and medium are fixed. The aperture control represents an ideal pupil stop, not an immersion-medium switch or a condenser diaphragm. Collected photon counts are held constant to isolate blur; exposure, camera noise, scattering, bleaching, aberrations and mechanical collision are not simulated. The 3D objective and bead sizes are schematic, with exaggerated depth.
The suggested z step ≤ D/2 is a teaching sampling rule for this model, not a universal Nyquist criterion. A fine widefield stack still contains out-of-focus light. Maximum-intensity projection does not optically reject that light or recover hidden axial detail.
Nikon MicroscopyU · Depth of Field and Depth of Focus ↗
Leica Microsystems · Depth of Field in Microscope Images ↗