This guide for long-working distance (LWD) objectives explains how to choose an optimal objective for microscopy considering dynamic imaging parameters including glass quality (Achro vs FL vs Apo), phase contrast, LWD and CC, and optimal vessel thickness.

This guide will help researchers who have questions regarding objective markings, image quality, sample types and applications, for example:

  1. Choosing the objectives most suitable for different samples, applications, and microscopes (with specific focus on vessel matching)
  2. Clarifying meanings of glass quality, optimal vessel thickness and implications on image quality
  3. Defining the markings on the barrel of a microscope objective
  4. Selecting the glass type (Achro, Fluorite, or Apochromat) best suited for different applications
  5. Understanding differences between coverslip corrected (CC) and long-working distance (LWD) objectives
  6. Choosing between achromat (Plan), achromat phase (Plan Ph), fluorite (FL), fluorite phase (FL Ph) and apochromat (Apo) objectives
  7. Deciding which 40x and 60x LWD objective to use for my sample vessel

Learn to interpret commonly used objective barrel markings

Using the proper microscope objectives is essential to generating high-quality images. However, this can be complicated by the choices among objectives with different types, classes and price ranges. Much of this information can be found on the markings inscribed on the objective barrel, and understanding them can therefore make selections easier. Most modern objective barrels contain a variety of markings, some of which are widely understood [magnification, numerical aperture (NA)] while others, such as the optimal vessel thickness (typically 0.17 mm for objectives intended for imaging through a coverslip or 1.0 mm for objectives designed for imaging of samples in plastic vessels) are less clear to many users. The purpose of this article is to help you navigate the many objective options by easily interpreting the commonly used objective barrel markings, and to highlight they key determinants of image quality and relevant trade-offs.

Figure 1. Illustration of working distance (WD) and optimal vessel thickness for coverslip-corrected (CC) and long–working distance (LWD) objectives.

Comparing long–working distance and coverslip-corrected objectives

While it may be the least well understood, optimal vessel thickness can have a profound effect on image quality. Microscope objectives are divided into two major classes, long-working distance (LWD) and coverslip corrected (CC). Of the two classes, CC objectives generate superior image quality with samples mounted under a coverslip or cultured on a glass-bottom dish, but are limited in their ability to image through thicker substrates. Therefore, LWD objectives are required for samples in plastic dishes, flasks or multi-well plates.

LWD objectives are designed for use with thicker-walled vessels

Whereas CC objectives are designed to image through a microscope cover slip with a commonly accepted thickness of 0.17 mm, also commonly regarded to as a ‘#1.5 coverslip’, LWD objectives are designed for use with thicker sample vessels such as cell culture dishes with a variable thickness of 0.8 mm or above (Figure 1). This design specification is indicated by the ∞/--, ∞/0.17 mm, ∞/1.0 mm or ∞/1.2 mm inscriptions on the objective barrel. In the given examples, an objective with a ∞/-- designation can be used across a broad range of vessel thicknesses, whereas an objective with a ∞/0.17 mm or ∞/1.0 mm designation is best suited for use with vessels that are 0.17 mm or 1.0 mm in thickness, respectively. Deviations from this optimal design specification will result in degraded image quality and a ‘soft’ blurry image that can’t be brought into sharp focus.

Poor image quality results when LWD objectives are not properly matched to vessel thickness

Matching a ∞/0.17 CC objective to an optimal vessel is generally straightforward due to the common use of 0.17 mm thick (#1.5) coverslips in bioscience imaging applications. However, matching a ∞/1.0 or ∞/1.2 LWD objective to an optimal vessel is made challenging due to the variability in thickness between commonly used plastic vessels, such as flasks, dishes, and plates. The thickness between, and within, manufacturers of cell-compatible plastic vessels can range from about 0.8 mm to 1.6 mm in thickness. If vessel thickness and objective compatibility are not considered when choosing LWD objectives, the outcome will be significantly degraded image quality. This effect is particularly pronounced at high magnifications such as 40x and 60x, where it becomes imperative to match the objective design to the vessel thickness.

1.0 mm vs 1.2 mm LWD EVOS objectives

At magnifications from 2x to 20x the depth of field is sufficiently large that image quality will not vary significantly between vessels with different thicknesses in LWD applications. This is indicated by the label ∞/--. However, at 40x and 60x the depth of field is shallow and matching the objective to the vessel thickness becomes imperative in ensuring optimal image quality. Therefore, these objectives have dedicated vessel thickness markings of ∞/1.0 mm or ∞/1.2 mm (Figure 2).

Empirically determine the best match between objective and vessel thickness

As indicated by the markings on the barrel of the objective, ∞/1.0 mm LWD objectives are designed for imaging through vessels with a thickness of approximately 1.0 mm, whereas ∞/1.2 mm LWD objectives are designed for imaging through vessels with a nominal thickness of 1.2 mm. For optimal results, use 1.0 mm corrected objectives when imaging through a standard microscope slide and plastic dishes with a thickness of <1.2 mm, and use 1.2 mm corrected objectives when imaging through plastic dishes with a thickness of ≥1.2 mm. However, as mentioned previously vessel thickness can vary, therefore it is important to empirically determine the thickness of the vessel(s) that will be commonly imaged and use this data to inform the decision on which objectives to purchase for the lab.

Figure 2. Correlation between image quality and vessel thickness with low-to-medium (2x to 20x) and high magnification (40x to 60x) LWD objectives. In group 1 there is no difference between images acquired through a relatively thin (1.0 mm), compared to a thicker (1.5 mm), plastic vessel. However, at 40x and above choosing an objective best matched to the thickness of the vessel has significant impact on image quality. Note that at 40x properly matching a ∞/1.0 objective to a 1.0 mm thick plate yields the best image quality, whereas sub-optimally matching a ∞/1.2 objective to a 1.5 mm thick plate yields better image quality compared to the ∞/1.0 objective but not as good as an optimal match.

∞/-- LWD EVOS objectives

It is important to note that the ∞/-- designation specifically applies to LWD applications, meaning imaging through a range of thick vessels. If imaging through a coverslip, a dedicated ∞/0.17 mm CC objective is recommended (Figure 3). If a dedicated CC objective is not available, we do not advise you to image through a coverslip using LWD objectives. Instead, we recommend that you flip the slide over and image through the slide for best results. A common misconception in microscopy is that imaging through the coverslip will always produce the best image, and while that is true for CC objectives, the example shown below demonstrates that is not the best practice with LWD objectives (Figure 3) due to the mismatching in substrate thickness and objective design.

Figure 3. Comparative images of a sample mounted on a microscope slide under a coverslip and captured by using a 20x LWD objective through the 1.0 mm slide (A) or 0.17 mm coverslip (B). Not that when using a LWD objective, imaging through the coverslip produces inferior image quality compared to imaging through the slide.

Achromat vs fluorite vs phase compatible LWD EVOS objectives

In addition to avoiding spherical aberration (blurry image) caused by vessel thickness mismatch, another parameter to consider is the objective classification regarding chromatic aberration, or the ability to bring different colors (wavelengths) into focus within a single focal plane. This correction factor is designated by the objective design ‘classification’. Commonly used classifications for LWD objectives are plan achromat (Achro), plan fluorite (FL) and plan apochromat (Apo), each of which promises a certain level of performance regarding aberration correction and image quality. Achromat, generally simply labeled 'Plan' or 'Plan Ph' objectives are designed to bring 2 wavelengths (blue, red) into a single focal plane whereas FL objectives are designed to bring 2–4 wavelengths (blue, green, red) into a single focal plane, while Apo objectives provide chromatic aberration correction for 4–5 colors. As image quality is also influenced by the medium through which light passes, Apo objectives are generally recommended only for coverslips and glass bottom vessels.

FL and Apo objectives are suitable for fluorescence microscopy

Though all three objective classes offer planar (Plan) correction for field curvature, only FL and Apo objectives are suitable for fluorescence microscopy. Achro objectives are therefore recommended for transmitted light microscopy only, either brightfield or phase-contrast imaging using transmitted white light. Fluorite objectives and Apo, on the other hand, can be used for multicolor fluorescence as well as brightfield/phase contrast imaging. Due to their superior correction they generate images with significantly improved image quality compared to achromat objectives.

Achro and FL objectives are available with an internal phase ring for phase-contrast imaging

For EVOS microscopes both Achro and FL objectives are also available with an internal phase ring (Plan Ph and FL Ph objectives) for phase contrast imaging using transmitted light. Combined with an annulus in the microscope condenser (Figure 4), phase contrast enhances contrast compared to brightfield illumination and is typically used to visualize unstained cells in a cell culture environment to assess confluence, morphology, proliferation, viability, etc.

Figure 4. Live CHO K1 cells imaged using brightfield/transmitted light compared to phase contrast. While both methods enable visualization of cells without a label in the same field of view, phase imaging offers superior contrast and cellular boundary detection.

Phase rings reduce light throughput, so weighing the benefits of phase contrast imaging is important

However, the phase ring inside the objective also results in a significant drop in light throughput, thus significantly compromising the brightness and contrast of fluorescently labeled targets. Therefore, it is important to consider these trade-offs and weigh the potential benefits gained by phase contrast versus the diminished performance in fluorescence (Figure 5); while a single 40x fluorite phase objective is versatile and cheaper than purchasing separate 40x Achro Ph and 40x fluorite objectives, the trade-off in image quality can be significant. FL Ph objectives are therefore recommended only in applications where phase contrast and fluorescence are required in the same experiment, otherwise a mixed set of Achro Ph and FL objectives may be more desirable.

Figure 5. Impact of image quality comparing three types of LWD objectives with the same sample and exposure settings. In fluorescence mode the results with fluorite is superior to fluorite phase and achromat phase. However, in transmitted light phase contrast imaging the lower price of the Achro Ph make them a good choice for this application. Fluorite phase objectives are convenient and economical when both fluorescence and phase imaging is required.

The objective selection flow chart (Figure 6) can help you determine which EVOS LWD objectives are most appropriate for your application. If you have questions, please consider calling our Tech Support experts or your local sales rep.

In summary, a range of microscope objectives are available and the optimal choice for a given sample and application will depend on several technical factors. Prices can vary from a few hundred dollars to several thousand and in many cases is a real consideration as well. By an understanding of the factors that influence image quality (contrast, signal-to-noise, resolution, etc.), as well as the terms that describe objective classes and types, well-informed decisions can be made and data appropriate for the purpose of the experiment more easily generated.

Figure 6. EVOS objective decision tree. Use the following guidelines to determine the optimal objective(s) for your imaging application. Note that each objective type is intended for a specific use, therefore choosing multiple objectives of the same magnification but with design parameters may be advantageous in order to achieve maximum flexibility in the lab environment without compromising image quality.