Assembly and optimization of a super-resolution STORM microscope for nanoscopic imaging of biological structures

Fluorescence microscopy is a widely used technique for imaging of
biological structures due to its noninvasiveness although
resolution of conventional fluorescence microscopes is limited to
about 200-300 nm due to the diffraction limit of light.
Super-resolution fluorescence microscopy offers an extension of the
original method that allows optical imaging below the diffraction
limit. In this thesis, a microscope for localization-based
super-resolution fluorescence microscopy techniques such as
Stochastic Optical Reconstruction Microscopy (STORM) or
Photoactivated Localization Microscopy (PALM) was established. An
epifluorescence microscope was built for this purpose that provides
both widefield and Total Internal Reflection Fluorescence (TIRF)
excitation modalities and focus was put on the special requirements
of localization-based super-resolution methods. This included a
high mechanical and optical stability realized by a compact design
and implementation of a home-built perfect focus system. The setup
was further designed to allow both two- and three-dimensional
imaging. The work also included both the development of a setup
control software and a software for the analysis of the required
data. Different analysis methods and parameters were tested on
simulated data before the performance of the microscope was
demonstrated in two and three dimensions at appropriate test
samples such as the cellular microtubule network. These experiments
showed the capability of super-resolution microscopy to reveal
underlying structures that cannot be resolved by conventional
fluorescence microscopy. Resolutions could be achieved down to
approximately 30 nm in the lateral and 115 nm in the axial
dimension. Subsequently, the established method was applied to two
biological systems. The first is a study of the budding of the
human immunodeficiency virus type 1 (HIV-1) from the host cell. In
this step of the viral reproduction cycle, the virus hijacks the
cellular endosomal sorting complex required for transport (ESCRT)
machinery to achieve membrane fission. ESCRT consists of the
subcomplexes ESCRT-0, -I, -II and -III and additional related
proteins, from which HIV-1 recruits certain components. The fission
process is initiated by the HIV-1 Gag protein, which recruits the
ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX to the
virus assembly site. Subsequently, ESCRT-III proteins CHMP4 and
CHMP2 form transient lattices at the membrane, which are actively
involved in membrane fission. However, the actual geometry of the
ESCRT machinery assembling at the HIV-1 budding site that is
driving the fission process is still not fully understood.
Different models proposed either constriction of the budding neck
by lattices surrounding the neck, by ESCRT structures within the
neck or within the bud itself. A problematic aspect in previous
studies was the usage of modified, tagged versions of the involved
proteins for visualization. In this study, super-resolution
microscopy was therefore applied to endogenous Tsg101, ALIX and
CHMP2 isoform CHMP2A and to a version of CHMP4 isoform CHMP4B with
a small HA-tag to elucidate the size and the distribution of the
structure relative to the HIV-1 assembly sites. ESCRT structures
colocalizing with HIV-1 exhibited closed, circular structures with
an average size restricted to 45 and 60 nm in diameter. This size
was significantly smaller than found for HIV-1 assembly sites and
the constriction of the size, which was not observed for
non-colocalizing ESCRT structures at the cell membrane, ruled out
an external restriction model. Super-resolution imaging of ALIX
often revealed an additional cloud-like structure of individual
molecules surrounding the central clusters. This was attributed to
ALIX molecules incorporated into the nascent HIV-1 Gag shell.
Together with experiments that confirmed the non-physiological
behavior of tagged Tsg101 and a relative orientation of ESCRT
clusters towards the edge of the assembly site, the results
strongly point toward a within-neck model. A second project focused
on the influence of external constriction on cell migration. The
latter plays an important role in various processes in the human
body ranging from wound healing to metastasis formation by cancer
cells. Migration is driven by the lamellipodium, which is a
meshwork of fine actin filaments that drive membrane protrusion.
Endothelial cells were grown on micropatterns that confined the
freedom of movement of the cells. Three-dimensional
super-resolution imaging revealed that the lamellipodia of these
cells showed a much broader axial extension than was the case for
control cells that grew without confinement of migration. The
different organization of the actin filament network showed a clear
effect of environmental conditions on cellular migration. Overall,
it was possible to build a super-resolution fluorescence microscope
over the course of this study and establish the required analysis
methods to allow STORM and PALM imaging below the diffraction limit
of light. Two applications further showed that these tools are
capable of answering currently discussed questions in the
biological sciences.