Optical tweezers have revolutionized our understanding of the microscopic world. Axial optical tweezers, which apply force to a surface-tethered molecule by directly moving either the trap or the stage along the laser beam axis, offer several potential benefits when studying a range of novel biophysical phenomena. This geometry, although it is conceptually straightforward, suffers from aberrations that result in variation of the trap stiffness when the distance between the microscope coverslip and the trap focus is being changed. Many standard techniques, such as back-focal-plane interferometry, are difficult to employ in this geometry due to back-scattered light between the bead and the coverslip, whereas the noise inherent in a surface-tethered assay can severely limit the resolution of an experiment. Because of these complications, precision force spectroscopy measurements have adapted alternative geometries such as the highly successful dumbbell traps. In recent years, however, most of the difficulties inherent in constructing a precision axial optical tweezers have been solved. This review article aims to inform the reader about recent progress in axial optical trapping, as well as the potential for these devices to perform innovative biophysical measurements.

Conventional optical tweezers suffer from several complications when applying axial forces to surface-tethered molecules. Aberrations from the refractive-index mismatch between an oil-immersion objective's medium and the aqueous trapping environment both shift the trap centre and degrade the trapping strength with focal depth. Furthermore, interference effects from back-scattered light make it difficult to use back-focal-plane interferometry for high-bandwidth position detection. Holographic optical tweezers were employed to correct for aberrations to achieve a constant axial stiffness and modulate artifacts from backscattered light. Once the aberrations are corrected for, the trap height can be precisely determined from either the back-scattered light or Brenner's formula.

Advances in light shaping techniques are leading to new tools for optical trapping and micromanipulation. For example, optical tweezers made from Laguerre-Gaussian or donut beams display an increased axial trap strength and can impart angular momentum to rotate a specimen. However, the application of donut beam optical tweezers to precision, biophysical measurements remains limited due to a lack of methods for calibrating such devices sufficiently. For instance, one notable complication, not present when trapping with a Gaussian beam, is that the polarization of the trap light can significantly affect the tweezers’ strength as well as the location of the trap. In this article, we show how to precisely calibrate the axial trap strength as a function of height above the coverslip surface while accounting for focal shifts in the trap position arising from radiation pressure, mismatches in the index of refraction, and polarization induced intensity variations. This provides a foundation for implementing a donut beam optical tweezers capable of applying precise axial forces.

Advances in light shaping techniques are leading to new tools for optical trapping and micromanipulation. For example, optical tweezers made from Laguerre-Gaussian or donut beams display an increased axial trap strength and can impart angular momentum to rotate a specimen. However, their application to precision, biophysical measurements remains limited as their are a number of challenges to applying this tool to optical force spectroscopy.One notable complication, not present when trapping with a Gaussian beam, is that the polarization of the trap light can significantly affect the tweezers' strength as well as the precise location of the trap. In this article, we provide a practical implementation of a donut beam optical tweezers for applying axial forces. We show how to precisely calibrate the height of the optical trap above the coverslip surface while accounting for focal shifts in the trap position that arise due to radiation pressure, mismatches in the index of refraction, and polarization induced intensity variations.

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