# Objective Lenses for Confocal Imaging

Confocal imaging has the highest demands on objective lenses. To fully benefit from the resolution of confocal imaging, the focal spot created by the objective needs to be diffraction limited for the full application wavelength range and field of view. Important objective parameters for confocal imaging are the numerical aperture (NA), chromatic correction and flatness of field.

## Numerical aperture

The numerical aperture (NA) determines the resolving power and the brightness of the image. It is a measure for the width and depth of the  focal spot produced by the objective. It is defined by the sine of the half aperture angle a of the lens and the refractive index n of the immersion medium (Figure 1). According to this definition, the larger the numerical aperture, the more narrow the focal spot and hence the higher the resolving power.

Since the NA scales linearly with the refractive index n, for NA greater than 1 an immersion medium with a refractive index greater than 1, i.e. other than air, is required. Higher NA typically demands a more complex design involving a larger number of lens elements and tighter tolerances. Figure 2 provides a comparison of the foci produced by 40x objectives with different NA by means of their point spread functions (PSF).

Figure 1 Definition of numerical aperture (NA). The NA is a measure for the width of the focal spot. It depends on the sine of the half aperture angle alpha and the refractive index n of the immersion medium. NA = n • sin alpha (with n=1 for air).

Figure 2 Comparison of focal spot sizes as a function of numerical aperture. Calculated point spread functions plot normalized intensities against two spatial coordinates. Larger NAs result in a higher resolving power indicated by a smaller spot size.

## Chromatic correction

An ideal lens would focus a point light source into an ideal PSF which dimensions are determined by the NA of the objective and the wavelength of the light. However, the shape and position of a real PSF deviate from an ideal PSF due to imperfections of a real lens. The results of such imperfections are called aberrations. Aberrations causing a non-ideal shape of the PSF have a negative impact on resolution and contrast (spherical aberration, coma, astigmatism). A shifted position of the PSF results in a suboptimal image geometry (curvature, distortion) or image brightness and chromatic overlay (chromatic aberration in lateral and axial directions).

In confocal imaging, there are high demands on z-sectioning and multi-spectral imaging which is why chromatic aberrations and curvature deserve special attention. Chromatic aberrations are reduced by special lens designs to make sure different wavelengths are focused into the same spot in xy and z dimensions. Any deviation from the ideal reduces the quality of colocalization experiments in functional characterization, because one could draw false conclusions from the corresponding correlation  coefficient. Moreover, with a better chromatic correction, light is collected more efficiently because of a larger overlap between excitation and emission PSF (see Figure 3).

Figure 3 Illustration of how the confocal signal depends on chromatic aberrations (computed.) Assumptions are an ideal 63x / 1.40 NA lens with Airy radius ~ 190 nm, infinitely small pinhole and excitation at 405 nm

## A Class of Objectives for Confocal Scanning: HC PL APO CS2*

Better  corrections for chromatic, spherical or other aberrations go along with the introduction of more lens elements  and require higher precision during manufacturing. The design therefore becomes more complicated and production more expensive.

Objectives are categorized in three classes in increasing order of quality: Achromats, semi-apochromats and apochromats. Apochromats are color corrected for the widest spectral range. Within the class of apochromats, Leica offers lenses especially designed to match the highest specifications for confocal scanning (CS). The latest CS2 series has further improved over the previous CS series and the newly developed lateral color correction goes hand in hand with the innovative UV optics of the Leica TCS SP8. The excellent color correction of the CS2 objectives delivers an improved color overlay especially in the image periphery.

*Abbreviations:

PL           plan, field flatness correction

APO       apochromatic, color corrected for red, blue and green wavelength range

A more general overview of the objective classes offered by Leica Microsystems can be found here.

## Dedicated for Excitation with 355 nm: HC PL APO UVIS CS2

With the HC PL APO 63x/1.20 W CORR UVIS CS2 we offer an ultra-broadband objective dedicated for excitation with 355 nm. It provides excellent color correction from 345-730 nm. This makes it ideal for photoactivation, uncaging and physiological experiments using Ca2+-single line and ratio imaging, monitoring gene expression or autofluorescence.

## Objectives for Multiphoton Imaging and CARS: HC PL IRAPO

Figure 4 Selection of IRAPO objectives Infrared-apochromats are corrected for the near-infrared range of the wavelength spectrum. Therefore, they are ideal for MP imaging including excitation with OPO as well as for CARS.

At the opposite end of the color spectrum a new set of specialized objectives is now available for improved multiphoton imaging (MP). The new IR apochromats (Figure 4) are color corrected up to 1300 nm and highly transmissive in the visible and infrared wavelength ranges with > 85 % transmission from 470 – 1200 nm making them ideal for non-linear imaging like multicolor multiphoton imaging including excitation with OPO (optical parametric oscillator), and CARS (Coherent Anti-Stokes Raman Scattering). See also Figure 5.

Figure 5 Overview of different color corrections offered for confocal microscopy.by different objective types, CS2, UVIS CS2 and IR APO, respectively. The respective color corrections of IRAPO, PL APO CS2 and PL APO UVIS CS2 have been designed to support specific applications optimally by making sure colocalization and detection efficiency are high throughout the scan field. The graph shows how the optimal ranges (boxes with solid lines) and usable ranges (boxes with dashed lines) overlap to provide full coverage of the wavelength range from 355 to 1300 nm. For the dashed green area between 405 and 450 nm color correction is available with some CS2 objectives. Combined with the optimal scan optics one gains a lot of applicative flexibility by having the right objective for each experiment.