Optical trapping and fluorescence microscopy

While a large majority of biological optical trapping studies are performed using only brightfield illumination to image the microscope field-of-view, much more information about cellular and biomolecular mechanics can be obtained by the use of fluorescent tags. Using fluorescence microscopy, cell function studies monitoring the movement of proteins under mechanical perturbation and advanced fluorescent techniques such as Förster resonance energy transfer (used to determine the proximity and conforma- tion of certain biomolecules under optical tension in single molecule studies [67]) are possible. Epi-fluorescence is a standard microscopy technique that can easily be used

in combination with optical tweezers but, for some studies, higher spatial resolution is required, in the form of confocal, multiphoton or super-resolution microscopy tech- niques. The optical sectioning provided by these microscopy methods allow precise 3D localisation of regions of interest but require an increase in complexity in the combined optical system. Movement of the objective to capture az-scan of an object also moves the trapping plane of the optical tweezer so it is only possible to capture a single plane of a trapped object. Several methodologies have been used to alleviate this problem, ranging from simple to highly complex.

For specialised applications, once the necessary optical manipulations have been per- formed, the trap can be turned off and imaging performed in any requisite plane. This technique has been employed for imaging cell-pathogen interactions [81] and virological synapses [82], events where once the initial connection has been induced, will proceed autonomously. In other applications, only a single plane is required for imaging so no movement of the objective is necessary, although this does not allow for any possible movement out of the plane that might occur during trapping and imaging.

To decouple the movement of the trapping and imaging planes when using the same objective, additional optics are required in the trapping or imaging beam path. This was first explored in Hoffman at al [83], who coupled a trapping laser into a confocal microscope. The trapping laser was directed in by means of a laser-coupled fibre and lens combination, the fibre was seated on a translation stage so that axial movement of the fibre changed the axial position of the trapping plane. The movement of the fibre was synchronised to the movement of the objective so the absolute height of the trapping plane remained the same, enabling trapping and 3D imaging of highly motile plant chloroplasts and axial displacement of granules in mammalian cells. Goksör et al.[84] employed an opposite technique; by keeping the objective height the same but moving a lens in the trapping beam path to change the height of the optical trap, it was possible to scan an object through the imaging plane. The dual-path trapping system (with different paths for confocal or multiphoton imaging) was based on a dual-trap

tweezer designed by Fallman et al. [85]. This system was also coupled into a STED (stimulated emission by depletion) microscope for super-resolution imaging of proteins on optically trapped DNA [86]. Both systems described above are highly complex, requiring precise software control and complex optical set-up respectively.

SLM-based optical tweezers can be used to change the height of an optical trap at the microscope focus. An SLM placed into the optical trapping beam path can shape the light into traps of varying axial positions. These dynamic traps have been used to create arrays of trapped yeast cells at different heights so each nuclei is in the same plane [87], increasing acquisition speed because only a single plane needs to be imaged to collect all the required nuclear information. They have also been used to create multiple dynamic traps for controlled rotation of immune cells, which were seen to rotate passively in a single trap, adversely affecting 3D imaging [88].

A third option, negating the use of expensive but highly reconfigurable HOTs, is to decouple the imaging and trapping beam paths completely. By bringing a trapping laser in to the opposite side of the sample to the imaging objective, by means of an objective or optical fibre, the trapping and imaging planes are independent of each other. Yevnin et al. [89] mounted a second objective above the trapping objective for confocal imaging of arrays of silica particles and yeast cells. Decoupling in this way also allows lower magnification imaging, if required, than that provided by the trapping objective, which requires high NA and therefore suffers from a restricted field of view. Although low achievable NAs (< 1.0) limit their axial trapping efficiency, single beam optical fibre traps provide a simple-to-implement and easily configurable optical trap [90] that can be introduced to any inverted microscope and therefore combined with any number of imaging modalities. Surprisingly, the combination of an optical fibre trap with a fluorescence microscopy platform has yet to be reported, opening up an avenue for investigation in this thesis. The use of a fibre trap for epi-fluorescence imaging of immune cells is reported in Section 7.3, with the potential for combination with super-resolution imaging.