We develop advanced optical imaging techniques for in vivo
imaging of biological tissues. Currently, one of our primary projects is
constructing and validating custom 2-photon microscopes capable of
performing simultaneous 2-photon imaging and 2-photon optogenetics of
neurons in small animals. The microscopes are highly optimized and
customizable to meet the demands of neuroscience research at NTU LKC
Medicine. Upcoming plan includes incorporating adaptive optics to
further enhance the capability of the 2-photon microscopes to preserve
spatial resolution when imaging through highly-scattering biological
tissues.
Team: Josiah CHONG, Peter
TÖRÖK, Aalim KHAN
We are developing optical coherence tomography at novel
wavelengths, i.e. 1700 nm wavelength for deep mouse brain imaging. The
infrared photons at 1700 nm wavelength experience much less scattering
in mouse brain tissues, which will allow us to improve the achievable
spatial resolutions and penetration depth.
Team: Josiah CHONG, Peter
TÖRÖK, Aalim KHAN
Light sheet microscopy revolutionised biological 3D imaging by
significantly increasing the imaging speed and reducing light doses in
comparison to traditional confocal scanning. Recent developments in the
field of lightsheet microscopy aim at further increasing the imaging
speed and the accessible sample volumes. We are working in this
direction and probing new concepts for lightsheet microscopy based on
the use of adaptive optics to manipulate the coherence and spatial
distribution of light. In this way for example Bessel beams (already
well-established in lightsheet microscopy) can be produce; their
"self-healing" properties allow for forming thin lightsheets over
extended areas and make them more robust against scattering artefacts.
Team: SHANG Wanqi, Peter
TÖRÖK
Remarkable progress in the manufacturing of opto-electronics lead to devices such as deformable mirrors or spatial light modulators, which offer a large degree of freedom in controlling the beam characteristics without any mechanically moving components.Specific arrangements of modulated beams enable coherent/incoherent superposition,parallelization or time-averaging. The liquid crystal on silicon (LCoS) devices, we are using, allow us to shape the beam by manipulating the amplitude and the phase field separately.
The initial setup tests generating an arbitrary axial intensity distribution based on off-axis holography and Fourier optics. A fibre-coupled diode laser is collimated by an off-axis parabolic mirror and a polariszer generates a high polarisation ratio light beam. The laser beam then passes through a half wave plate which rotates its polarisation direction to fit well the ferroelectric liquid crystal SLM. We can get different intensity distributions around the fourier plane responding to different computer-generated patterns displayed on the SLM. Incoherent light sheet array will appear in coming updated experimental configuration.
We are developing a custom microscope for confocal Brillouin and
Raman micro-spectroscopy. Brillouin and Raman scattering are both
non-elastic light scattering processes that can provide label-free image
contrast in a variety of biological as well as material sciences
samples. In Raman scattering, the photons exchange energy with molecular
vibrational modes and the spectra contain qualitative as well as
quantitative information on the chemical composition of the sample.
Raman spectral fingerprints have proven a powerful tool for non-invasive
microbial strain identification. In Brillouin scattering, the photons
exchange energy with density waves (acoustic phonons) in the material
under study. For homogeneous media, this information can be readily
interpreted in terms of elastic moduli of the material. In this way,
Brillouin spectroscopy is commonly employed in material science for
non-invasive mechanical testing. However, in inhomogeneous systems such
as any biological material, the interpretation of Brillouin spectra is
much more complicated and a simple theoretical model is lacking.
Therefore, Brillouin microscopy has not yet become widespread in
biological and biomedical research, despite its high application
potential in this field.
Team: Peter TÖRÖK, Aalim
KHAN, Josep RELAT GOBERNA, Radek MACHÁŇ
It has been shown that the Brillouin shift in biological samples correlates with water content [Wu P. et al. (2018) Nat Methods 15, 561–562]. We aim to circumvent the ambiguities of Brillouin spectra of biological systems by utilising the chemical information provided by Raman spectra and to map simultaneously chemical composition and mechanical properties of the systems under study.
The microscope is built around a commercial inverted frame
(Olympus, IX-71); it contains two lasers, 561 nm for Brillouin and
Raman scattering and 488 nm for confocal fluorescence imaging. Raman
spectra will be recorded by a custom-designed spectrograph with a CCD
camera. The technical challenge in recording Brillouin spectra lies in
the closeness of the Brillouin peaks to the peak of elastically
scattered photons (Rayleigh peak), which is many orders of magnitude
more intensive than the Brillouin peaks. This means that high spectral
resolution together with efficient suppression of the Rayleigh peak
are necessary. To meet this challenge, we use custom-designed
common-path interferometric filters (two in a series) and a virtually
imaged phase array (VIPA) spectrograph equipped with a scientific CMOS
camera. The design is an improved version of a previously described
setup [Karampatzakis
A. et al. (2017) npj Biofilms Microbiomes 3, 20] and its detail
can be found at our GitHub
repository. Alongside the hardware, a user software has been
developed for intuitive an efficient operation of the setup.
We are developing a new generation of high content
screening platform for simultaneous 3D live imaging of more than 1,000
organoids. The data obtained on gastruloids and liver organoids will be
analysed using unsupervised machine learning to provide new quantitative
statistical analysis tools characterizing the diversity of morphogenetic
movements within each organoid. Using correlative clustering, we will
then demonstrate how such quantification can lead to individual organoid
outcome prediction in a non-destructive way. Our group is responsible
for designing and building the optical and optomechanical parts of the
two prototypes and the final instrument.
Team: Peter TÖRÖK, Aalim
KHAN
Collaborators: Virgile Viasnoff (MBI), J-B. Sibarita (Univ. of Bordeaux)
We have designed an optical system for fluorescence imaging of a
large field of view of a highly curved corneal surface at chief ray
normal to surface. Cornea positioning and fluorescence excitation beam
homogenized over a large area will improve image signal-to-noise ratio.
The system promises to overcome the limitations of current methods for
diagnosis of corneal diseases, the third major cause of blindness
worldwide.
Team: Aalim KHAN, Peter
TÖRÖK
Collaborators: Leopold SCHMETTERER (SERI)
The project aims to develop an ultra-wide band optical
coherence tomograph to be used to assess retinal function of the human
eye that could lead to the detection of neuronal dysfunction in both
Alzheimer’s disease and glaucoma.
Team: Peter TÖRÖK , Aalim
KHAN, Josiah CHONG
Collaborators: Leopold SCHMETTERER (SERI), Jonathan Crowston (Duke-NUS Medical School)
We aim to time-resolve how multi-species bacteria communities
colonize wet surfaces with single-cell resolution. During early-stage
biofilm formation, we use continuous live-cell imaging to capture every
cell event as they land, spin-walk-swim, divide, leave or explore around
the surface as they meet other same-species or different-species cells,
all the while leaving extracellular materials and signals along their
trails. With full spatial-and-temporal causal understanding behind how
biofilms develop, there is a transformative potential to untangle the
myraid complex correlations observed in microbiology and genomics
studies. We hope to tap this to study how bacteria infections
fundamentally develop. We collaborate with Professor Gerard Wong at UCLA
Bioengineering, whose team pioneered the field of quantitative bacteria
tracking for single species. Our main challenge is to apply this rigor
to multi-species communities where bacteria species may look identical
but behave differently together as they coexist or fight against each
other, giving rise to unexpected emergent patterns at very different
timescales. The bacteria species have to be genetically engineered to
express different fluorescent reporters. Continuous flowcell
environments are inoculated with bacteria and continuously imaged under
bright-field with high-temporal frequency and intermittent fluorescent
imaging, and we develop deep learning techniques to help us tackle the
huge terabyte datasets of detailed bacteria cell activity on the
surface.
Team: LAI Ghee Hwee
Collaborators: Gerard
Wong (UCLA)
S. epidermidis and C. acnes are two of the most
common bacteria found on the human skin. C. acnes, being an
aerotolerant anaerobe, cannot grow as a monoculture biofilm in an
aerobic environment. However, when co-cultured with S. epidermidis,
C. acnes is able to grow. This lead to a hypothesis that S.
epidermidis is receiving some metabolites from C. acnes
thus increasing the respiration rate of S. epidermidis. This
in turn creates an environment with lower oxygen concentration to
support the growth of C. acnes.
Team: FOO Yong Hwee
Collaborators: Scott RICE (SCELSE)
In collaboration with Dr Viduthalai Rasheedkhan Regina and Prof. Scott Rice (SCELSE, NTU), O2 NS was synthesized for the detection of oxygen levels in S. epidermidis and C. acnes biofilm. The goal is to use the O2 NS to track oxygen consumption rates of S. epidermidis over time when co-cultured with C. acnes in a biofilm. Plate reader assay will provide high throughput, while confocal imaging will determine if there are zones with differences in oxygen gradient in the biofilm.
Investigating structures of a small bacterial cell is challenging
especially structures that are smaller than the diffraction limit (~200
nm) of a light microscope. Super resolution imaging techniques, which
won the Nobel Prize for Chemistry in 2014, can distinguish structures
below 200 nm. SMLM is capable of resolving structures as small as ~20
nm, was applied to image bacterial membrane and nucleoid. Optimising
SMLM of bacteria forms a part of the study. Point Accumulation for
Imaging in Nanoscale Topography (PAINT), a type of SMLM, image of Nile
red dye in the membrane of live Pseudomonas aeruginosa is
shown as an example. We collaborate with Dr Anuj Pathak from Prof.
Birgitta Henriques-Normark’s group in LKCMedicine on this project.
Team: FOO Yong Hwee
Collaborators: Birgitta HENRIQUES-NORMARK (LKCMedicine)