Converting an air objective into a dipping objective

If you’ve ever used an air objective to image into a liquid sample, you may have encountered the problem that as you image deeper, your image quality degrades. This is due to the refractive index mismatch causing aberration of the objective focus in the sample.  An easy way to think about this is by thinking about the optical path length between the objective and the focal plane.  As you image deeper into the sample, you’re replacing air (with a refractive index of 1) with liquid (with a higher refractive index).  This causes the optical path length to increase, and this gets worse the deeper in the sample you image (as you’re replacing more air with liquid).

SphericalAberration

Spherical aberration caused by the refractive index mismatch between the sample and the medium the objective was designed for.

This primarily introduces spherical aberration, although other aberrations are induced too. This is a particular problem with low magnification light sheet microscopes of the ‘Ultramicroscope’ type [1], where you use a low magnification air lens to image many millimeters into a cleared tissue sample. What’s particularly problematic is that the spherical aberration gets worse the deeper you image, requiring some adjustable correction to eliminate it.

I recently came across a simple way to fix this, which is mentioned in a recent Ultramicroscopy review [2]. The idea is that you attach a cap to the objective, converting the air objective into a dipping objective. Now as you focus into the liquid sample, the thickness of the air and the liquid  layers remains constant, so you have a constant, unchanging amount of spherical aberration. This can be corrected with a fixed spherical aberration correction, which is much easier to implement than a variable correction.

ObjectiveCap

Diagram of objective cap used to convert an air objective into a dipping objective. Notice how the thickness of the air and liquid layers remains constant during focusing into the sample.

The method is described in more detail in this patent.

References

  1. H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C.P. Mauch, K. Deininger, J.M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain", Nature Methods, vol. 4, pp. 331-336, 2007. http://dx.doi.org/10.1038/nmeth1036
  2. H. Dodt, S. Saghafi, K. Becker, N. Jährling, C. Hahn, M. Pende, M. Wanis, and A. Niendorf, "Ultramicroscopy: development and outlook", Neurophotonics, vol. 2, pp. 041407, 2015. http://dx.doi.org/10.1117/1.NPh.2.4.041407