| Parameter |
Description |
| Magnification |
- Determines the apparent size of structures in the image.
- Must match the level of detail required
for the application.
|
| Numerical Aperture (NA) |
- Ability of an objective to collect light over a wider angle.
- Wider collection angle corresponds
to higher NA.
- Higher NA results in higher spatial resolution. Increasing NA reduces the working distance,
limiting compatibility with thick samples.
- High-NA objectives deliver superior optical performance but are
associated with higher costs.
|
| Working Distance |
- Defines the distance between the front lens of the objective and the closest surface of the
sample (or the coverslip) that is in sharp focus.
- Is inversely proportional to NA. Long working distance
objectives are required for imaging thicker samples (e.g. organoids) and when using additional equipment (e.g.
micro-injection needles, perfusion lines).
- Short working distances enable higher NA but restrict compatible
sample types.
|
| Depth of field (focal depth) |
- Indicates the axial thickness of a sample that appears in focus at one axial (Z) position.
- Primarily controlled by the objective's NA.
- High NA = smaller depth of field = better optical sectioning.
- Low NA = higher depth of field = beneficial for thicker and uneven samples.
|
| Aberration correction |
- Refers to objective design features that minimise optical imperfections, ensuring images are
sharp, colour-accurate, and well-focused across the field of view.
- Different aberrations require different
correction levels:
- Field curvature: Distortions due to field curvature impair the properstrong> Distortions due to field curvature impair the proper
focus of an object onto a flat plane. Plan objectives correct for field curvature, highly recommended if budget
allows.
- Spherical aberration: Lens curvature causes rays of light passing through the edges
of the objective to be out-of-focus compared to those passing through the centre, resulting in a blurred image.
Objectives can correct for spherical aberration to different degrees: Achromat (1 wavelength), Fluorite (2-3
wavelengths) and Plan Apochromat (3-4 wavelengths).
- Chromatic aberration: Failure to focus
all wavelengths on the same plane due to different diffraction of wavelengths by the objective. High-quality
objectives correct for chromatic aberration as follows: Achromat (2 wavelengths), Fluorite (2-3 wavelengths),
Plan Apochromat (4-5 wavelengths).
- Objective classes (increasing correction capabilities and costs): Achromat/Plan-Achromat: basic correction, spherical aberration for 1 wavelength, chromatic aberration
for 2 wavelengths
- Fluorite, Plan-Fluorite: improved correction, spherical aberration
correction for 2-3 wavelengths, chromatic aberration correction for 3 wavelengths
- Apochromat and
Plan-Apochromat: highest correction, spherical aberration correction for 3-4 wavelengths, chromatic
aberration correction for 4-5 wavelengths
|
| Immersion media |
- Objectives are optimised for specific immersion media (air, water, oil, silicone, glycerol).
- To
maximise the collection of light rays and avoid diffraction, the immersion medium of the objective should match
the refractive index of the sample environment.
- Air objectives are convenient and cost-effective but limited in
NA.
- Oil objectives support higher NAs and enable high-resolution imaging.
- Silicone and glycerol objectives are
better suited for imaging deeper into samples due to reduced spherical aberration with imaging depth.
- Water
objectives are particularly suitable for imaging deeper into live, aqueous samples.
|
| Transmission characteristics |
- Describe how efficiently an objective transmits light across relevant wavelength ranges.
- High
transmission efficiency is essential for sensitive detection of weak signals.
- Specialised objectives are
available for specific wavelengths (e.g., UV-compatible for DAPI, NIR for near-infrared imaging).
|
| Modality compatibility |
- Defines which imaging technique the objective can support such as fluorescence, confocal imaging,
brightfield, darkfield, phase contrast, DIC, and polarisation.
- Imaging modes including confocal imaging, DIC,
and phase contrast require specific objective design.
- Objective compatibility with the intended imaging modality
should always be verified.
|
