Objectives for Research Microscopy

EC Plan Neofluar® and LD Plan Neofluar® objectives

The Enhanced Contrast Plan Neofluar universal objectives with consequently minimized straylight: with a visible increase in contrast, important for all contrast methods: Brightfield, Darkfield, Phasecontrast, DIC, Fluorescence, Pol.

EC Plan Neofluar® objectives feature full chromatic correction for the focal plane, high resolving power and brilliant, rich-contrast, completely flat images for observation and documentation. 

Their high transmission of wavelengths from the near UV range (>40% starting at 340nm) and the use of glass types of low autofluorescence makes these objectives ideal for epi-fluorescence microscopy. 

Further special-purpose members of the family are EC Epiplan Neofluar® objectives for reflected-light work, the EC Plan Neofluar® Antiflex objective for reflection contrast, and EC Plan Neofluar® Pol objectives for the requirements of polarizing microscopy. 

All these objectives are suitable for all microscopes designed to accommodate eyepieces with visual field numbers up to 25 mm.

 

LCI Plan Neofluar® Objectives for best Live Cell Imaging

The LCI Plan Neofluar objectives 25x/0,8 and 63x/1,3 Imm. Korr. are especially designed for Live Cell Imaging and can be adjusted for special temperatures (23°C and 37°C).

The 25x/0.8 can be used with the following immersion mediums: water, glycerol and oil, while the 63x/1,3 can be used with water and glycerol.

With only one Correction collar it is easy to correct for spherical aberrations, caused by different coverglass thicknesses, temperature differences or immersion mediums.

Water is an immersion medium particularly suited to the examination of living cells. Also if the objective has to dip in directly into aqueous medium of an uncovered specimen. As an added advantage, water causes no cleaning problems.

Cleaned glycerol suggests itself for critical fluorescence examinations, as it has nearly no autofluorescence.

Multi-immersion objectives are convenient for use on inverted microscopes where they permit, within certain limits, the compensation of non-standard glass vessel bottom thicknesses (< 0.17 mm). They accommodate the popular practice of using water instead of oil at the cost of some of the numerical aperture.

 

EC Plan Neofluar® Antiflex

The EC Plan Neofluar® 63x/1.25 Oil Ph3 Antiflex is an objective for reflected-light reflection contrast. This method reveals structures invisible with normal brightfield microscopy, such as cells adhering to the bottom of a Petri dish. Moreover, this objective is excellent for the immunogold staining method (IGS or IGSS).

The method requires the use of an A-Pol filter set (polarizer, neutral beam splitter, analyzer). The contrast can be modulated by a quarter-wave plate provided in front of the front lens.

The built-in phase annulus allows this objective to be used also for phase microscopy.

 

EC Epiplan Neofluar® objectives

are a series of objectives for the examination of materials. They differ from Epiplan® objectives by their higher NA and, accordingly, higher resolving power and of course like all EC objectives for highest contrast.

Unlike the Epiplan® series, the EC Epiplan Neofluar® series includes 1.25x and 2.5x objectives. These low-power objectives provide maximum object field diameters of 20 mm.

EC Epiplan Neofluar® objectives are also suitable for transmitted-light work. Because of spherical aberration, specimens with cover glass should not be observed with objective powers above 20x. There are no restrictions for uncovered specimens.
 

EC Epiplan Apochromat® objectives

These high-power, apochromatically corrected objectives for reflected light satisfy the most exacting demands in material microscopy.

They are suitable for visual field diameters up to 25 mm and feature sufficiently high transmission in the 400-to-700nm spectral range.

The 150x dry objective provides an empty magnification required in some engineering applications.
 

Plan Apochromat® objectives

This is a first-class series of objectives for observation and photomicrography. Their performance is limited only by the laws of physics. The excellent correction and extremely high apertures of the Plan Apochromat® objectives provide a maximum of resolving power, color purity, contrast and image flatness for observation and photomicrography.

The extraordinarily high NA ratings, e.g. of Plan Apochromat® 10x/0.45, 20x/0.75 or 63x/1.4, makes these objectives superior in fluorescence microscopy. The enormous resolving power reveals structures that otherwise cannot be discerned.

As image brightness in fluorescence microscopy increases with the square of numerical aperture, Plan Apochromat® objectives are excellently suited to fluorescence applications.

The objectives can be used for visual field numbers up to 25 mm.
 

C-Apochromat® objectives

10x/0,45 W, 40x/1,2 W Korr und 63x/1,2 W Korr
Water immersion objectives for supreme demands:

If you want to examine a biological specimen having a refractive index close to that of water (n = 1.33) with a high-aperture objective, an oil objective can only supply a useful result unless you focus at too great a depth below the specimen surface.

If you want to look deeper into the specimen, high spherical aberration will annihilate contrast and resolution and drastically reduce image brightness. The latter will be noticed especially in confocal microscopy by a substantial deterioration of the signal-to-noise ratio.

Therefore, an oil objective, however good it may be, is not the optimum for a water-like specimen. In the designing of objectives, the refractive index of the intended specimens (and the immersion medium) plays an essential part.

Oil objectives are designed and corrected on the assumption that the refractive indices of the immersion and embedding media are equal (n = 1.52). For water objectives, this index is assumed to be n = 1.33 for both media.

Special objectives:

C-Apochromat 40x and 63x corrected from UV to IR !

Other water immersion objectives:

For use without coverglass:

- Achroplan® W objectives, corrected for specimens without cover glass and for long working distances, especially for use in microinjection and patch-clamp techniques on upright microscopes.

- Objektiv Plan Apochromat® W 63x/1.0 corrected from VIS to IR.

For use with coverglass:

- LCI Plan Neofluar® multi-immersion objectives, corrected for immersion media with refractive indices between those of water and oil, and for a single cover glass thickness.

The examination of an aqueous specimen to exacting standards, e.g. with a laser scanning microscope, involves a number of special requirements that have to be satisfied:

High aperture for high resolution in optical thin sections, and high brightness of fluorescence
Long working distance for 3D examinations
Low axial chromatic aberration so that the focal planes of different wavelengths coincide
Low lateral chromatic aberration so that different fluorescence images register.
Flat field to enable true-to-life 3D reconstruction

To satisfy these requirements, the C-Apochromat® 40x/1.2W Corr was designed to have the following properties and features:

Water immersion for aqueous specimens (vital sections, cell cultures, specimens with aqueous embedding medium)
Correction collar for compensating differences in cover glass thickness (0.14...0.18 mm) and temperatures (scales for 24ºC and 37ºC)
The correction collar can also be used for compensating slight deviations of the specimen's index of refraction from that of water
Magnification 40x
Numerical aperture: 1.2
Free working distance: 220 µm
Image flattened over 25 mm (conventional) or 12 mm (confocal)
Transmittance (absolute): 50% at 350 nm, 85% at 400 nm, 90% at 500...700 nm
High chromatic correction (as for Plan Apochromat® objectives)
No spherical aberration in aqueous specimens.

Background

If the embedding and immersion media differ in refractive index, the optical paths will differ in length. Depending on the depth of the focal point below the specimen surface, the difference will range between negligible and quite distinct. As path difference increases, so does spherical aberration, with considerable losses in definition, contrast, brightness and depth resolution. Moreover, measurements in axial directions will be deteriorated by scale contractions and expansions along the Z axis. To exclude deformation along Z, the refractive indices need to be equal. Subsequent correction by the computer is unsatisfactory, because it cannot compensate spherical aberrations. It is therefore imperative that the immersion medium be matched to the specimen.

Objectives with extra-high apertures (1.2 being the very limit for water immersion) are highly sensitive to differences in cover slip thickness. Therefore, the C-Apochromat® 40x/1.2W Corr has a correction collar to compensate for cover slip thicknesses between 0.14 and 0.18 mm. For maximum optical performance, however, it is recommendable to use cover slips with closely toleranced thicknesses (e.g. 0.16...0.17 mm). If the objective is used on the laser scanning microscope, the cover glass thickness can easily be measured and the correction be set to a high precision.

The effect of adaptation to the refractive index can be seen in the pictures below. These are projections of optical sections made of fluorescence-marked culture cells (preparations by Dr. Kartenbeck, Heidelberg, micrographs by Carl Zeiss). Cytokeratin was stained with fluorescein. In the LSM, fluorescence was excited with 488 nm and the emission filtered out with an LP 515 long pass filter (which is equivalent in effect to filter set No. 09). Cover slip and specimen were separated by a water sheet of 40 µm thickness.

Fluar® objectives

Fluar® objectives have been designed especially for qualitative and quantitative analyses of ion movements and for particularly critical fluorescence methods (e.g. chromosome studies in human and cytogenetics).

These objectives are distinguished by extremely high numerical apertures and high transmission for wavelengths from 340 nm. Field flattening is sufficient to allow the use of CCD cameras.

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