NANO-scale VIsualization to understand Bacterial virulence and
invasiveness - based on fluorescence NANOscopy and VIBrational
microscopy
Background and motivation
In the biomedical field, fluorescence is the by far most widely
used modality for cellular imaging, offering a unique combination
of spatial and temporal resolution, sensitivity and specificity.
There has been a remarkable development of fluorescence-based
super-resolution microscopy techniques in the last decade.
"Super-resolution" means that the resolution of the imaging is not
limited by the wavelength of the light used to image a sample, and
the main scientists behind this development were awarded the Nobel
Prize in 2014.
By the development of fluorescence-based super-resolution
microscopy it has become possible to image cellular proteins marked
with fluorescence emitters (fluorophores), with a ten-fold higher
resolution than with any other fluorescence microscopy technique.
In a previous EU FP7 project (Fluodiamon),
this was used to analyze how certain proteins were spatially
distributed in breast and prostate cancer cells, compared to in
corresponding non-cancer cells, and was demonstrated as a new basis
for cancer diagnosis. Two of the NanoVIB partners (KTH and
KI) have
also more recently applied super-resolution microscopy to study
bacterial surface proteins of Streptococcus Pneumoniae
(pneumococci). Pneumococcal infections represent a major cause of
morbidity and mortality world-wide. These super-resolution
microscopy studies could shed new light on how specific surface
proteins of these bacteria are spatially distributed on the cells,
and provided important evidence that the virulence (capacity to
generate disease) and invasiveness of these bacteria is strongly
coupled to such spatial protein distribution patterns (Figure 1).
Clearly, imaging of these patterns with yet higher spatial
resolution can lead to a significantly increased understanding of
the mechanisms underlying pneumococcal virulence and invasiveness.
Figure 1: Stimulated emission depletion (STED) super-resolution
image showing the distribution patterns of the proteins PSPC1 (red)
and PSPC2 (green) on bacteria (Streptococcus Pneumoniae).
(PSP simply stands for pneumococcal surface protein). These
bacterial proteins can bind a host protein, called factor H, which
in turn can reduce the immunological response against the bacteria.
Recent studies between two partners of the NanoVIB project (KI and
KTH) have shown that super-resolution STED imaging makes it
possible to resolve how PSPC1 and PSPC2 are distributed on the
bacteria under different stages of division, and that these
proteins are localized in such a way that they protect specifically
vulnerable surface regions of the bacteria upon cell division. The
spatial distribution patterns of these specific proteins thus play
a decisive role for the ability of the bacteria to withstand the
immunological defense of the host.
More details of this study can be found in Pathak et al, Nature
Comm. 2018.
Now,
MINFLUX, a next generation super-resolution concept, has
recently been demonstrated, offering another order of magnitude
higher spatial resolution than current super-resolution microscopy
techniques (Figure 2). This means, spatial distribution patterns of
proteins, which could be resolved within spatial scales of hundreds
of nanometers by regular state-of-the-art fluorescence
microscopes, and within tens of nanometers with super-resolution
microscopy techniques, can now be resolved within a few nanometers,
i.e. at a spatial scale corresponding to the size of the proteins
themselves. Two of the NanoVIB partners (Abberior Instruments
and IFNANO) have been closely
involved in this development, originating from the 2014 Nobel Prize
laureate Stefan W Hell and his research group in Göttingen.
MINFLUX imaging can be performed with much lower irradiation doses
and based on less bright fluorophores. This means that fluorophores
in the near-infrared (NIR) spectral range (in our case 700-900nm)
can now be used, which because of low brightness and photostability
have not been eligible for super-resolution microscopy before.
Imaging in the near infrared (NIR) can offer several distinct
advantages: i) strongly reduced background scattering and
autofluorescence from the sample, ii) lower phototoxicity, iii)
deeper penetration depths, and iv) an additional spectral window,
allowing for simultaneous imaging over multiple, spectrally
separated color channels. To take MINFLUX into the NIR range
however, requires detectors with high sensitivity in this spectral
range. Such detectors will be developed in the NanoVIB project by
the partner Pi Imaging,
building further on their world-leading single photon counting
detector technologies.
To take full benefit of the information contained in the highly
resolved protein localization patterns within cells, as obtainable
by MINFLUX, we also want to overlay these patterns onto
morphological images. To do this, without adding additional
fluorophore markers that would sterically or spectrally interfere
with the fluorophore reporters for MINFLUX, we will use in-elastic
scattering from specific molecules already present in the cells.
So-called stimulated Raman scattering (SRS) imaging requires
sophisticated laser systems, with multiple, narrow linewidth
emission lines, and precise tunability in the emission
wavelength(s). The NanoVIB partner APE is in the
absolute forefront of the development of such lasers. By further
development and implementation of these laser systems into the
MINFLUX prototype system, correlative imaging of nano-scale protein
localization patterns and cellular morphology/chemical environment
will be realized in the project.
Objectives
In this coherent and interdisciplinary project, we will prototype a
next-generation super-resolution microscope system based on the
MINFLUX concept, capable to reveal intricate, detailed molecular
mechanisms underlying inter- and intracellular processes and
disease. As a lead application, we will demonstrate its unique
capabilities to understand virulence and invasiveness of pathogenic
bacteria, more precisely pneumococci. By concerted development of
laser and detector technologies, microscopy devices and image
acquisition procedures, we will be able to retrieve information,
which is not within reach by any other microscopic or
photonics-based technique. We will demonstrate how cellular
nanoscale protein localization patterns can be resolved, which will
not only help us revealing mechanisms of bacterial disease, but
which also are likely to be of large relevance for many other
diseases. Thus, the microscope system prototype will prove its
broad applicability in biomedical research, as a tool to understand
intra- and intercellular processes and the cellular origin of
diseases.