TCD

Name of the infrastructure: TCD-TA

Location (town, country): Dublin, Ireland

Web site address: www.tcd.ie; www.crann.tcd.ie; www.tcd.ie/IMM

Legal name of organisation operating the infrastructure: Trinity College, Dublin

Location of organisation (town, country): Dublin 2, Ireland

 

Description of the Infrastructure

The CRANN facilities are housed in a 6000m2 building; comprised of a basement area, designed to meet the stringent environmental demands of precision nanoscale measurements, and a number of specialised labsdedicated to nanobiology and material synthesis research. The CRANN Advanced Microscopy Laboratory, aseparate 1000 m2 facility, provides e-beam lithography down to less than 10 nm, and access to state a of the artfocused ion beam and transmission electron microscope. This laboratory also houses the CRANN Helium IonMicroscope, which is one of just ten installations worldwide. The BioNano Laboratory is a recent developmentat CRANN dedicated to research at the interface between the physical and life sciences. The group possessesa breadth of research expertise for the investigation of molecular, cellular and physiological interactions usingnovel biophysical tools such as cell actuators, magnetic and ultrasound fields. The IMM in Trinity College Dublinoffers a number of state of the art imaging facilities, which include the national facility for High Content Analysis,which opened officially in 2008. The High Content Analysis suite is comprised of a number of high contentimaging platforms, automated liquid handlers and a comprehensive image and data analysis platform.The IMM includes core cell, tissue and bacteria culture facilities, core Microscopy and Cell Imaging/Cell analysisfacilities (including a real time laser confocal microscope linked to image reconstruction and analysis hardwareand software, two fluorescent inverted microscopes coupled to digital still and video cameras and computerizedequipment for image processing, microinjecting equipment, a microfluidics facility to mimic in vivo continuouscell flow and two flow cytometers. For high content screening (HCS), a Cellomics KineticScan HCS and two GEHealthcare InCell-1000 systems are available. This equipment allows the multi-parametric analysis of cells in96-384 well format in a controlled environment. Individual cells can be tracked throughout,. permitting temporaland high-resolution spatial analysis of physiological processes, dynamic distribution and activity of cellularconstituents, and morphologies in living cells. Finally, the IMM also offers a wide arrange of other facilities,including a dedicated containment laboratory for nucleic acid isolation, PCR machines, TaqMan technologyfor analysis of real-time PCR-gene expression, DNA microarray and gene sequencing facilities. The IMM alsoboasts confocal microscopy facilities suitable for live cell imaging, and both Raman and AFM capabilities. Thesetechnologies are currently being employed for the study of bio-nanoparticle interactions.

 

SUPPORTING FACILITIES AND SERVICES:

TCD will be offering access to components C and D as listed inthe project, as follows:

 

Particle Characterisation in situ & ex situ

 

Advanced Microscopy Laboratory

The CRANN Advanced Microscopy Laboratory is a specially built state-of-the-art 1000 m2 facility whichprovides e-beam lithography down to less than 10 nm, and access to state a of the art focused ion beam andtransmission electron microscope (TEM). This laboratory also houses the CRANN Helium Ion Microscope,which is one of just ten installations worldwide. It has novel, first of a kind engineering controls. Several of theinstruments are unique:

The Titan 80-300 TEM is the first and only TEM in Ireland with EELS capability. It offers sub-Ångstrom resolution(<0.1 nm), enabling imaging with atomic resolution in both normal imaging mode and scanning TEM (STEM)mode.

 

The Auriga focused ion beam microscope [FIB], is a state of the art one stop shop for nanofabrication. The coretechnology is an Orsay Physics Cobra ion column - the sharpest nano-engineering FIB instrument, which uses30 KeV or lower gallium ions to selectively mill and modify surfaces and samples on the nanoscale.

 

The He-Ion at CRANN is one of the world's first installations of this exciting new technology. The He-Ionmicroscope affords the highest resolution surface imaging of bulk materials from a scanning microscope, andbridges the gap in imaging resolution traditionally left between the scanning electron microscope [SEM] and theTEM. This is the only such tool installed in Ireland, and one of a handful in Europe.

 

Confocal Microscopy

Zeiss Meta LSM inverted confocal system has the capability of imaging standard or 3D confocal throughout theentire visible spectrum with the following lasers, diode 405nm, Argon-Ion 488nm, DPSS 561 nm, and a HeNe633 nm. The following objectives are available – 63x oil, 10x, 20x and 40x dry. The 40x is ideal for imaging 96well plates or similar. Live cell imaging is possible with the use of an external heated stage. Lambda scans canbe taken using the Meta detector which is of particular benefit for characterising the excitation and emissionwavelengths of new nano-materials. Imaging of these nanomaterials at the optimal detection wavelength canthen be done using the Meta detector. Full training in the use of the system, with on-site assistance, is availableat all times.

 

Nanosight NS500 with latest NTA

The Nanomedicine and Molecular Imaging Group based in the Institute of Molecular Medicine, Trinity College Dublin are equipped with Nanosight NS500 running NTA version 2.3 and among the 12 laboratories involved in the QNANO NTA RR. This device is equipped with a 405 nm laser, coupled with a 430 nm long pass filter, an EMCCD camera and is thermostatically controlled. The device has the ability to characterise nano objects in the size range 10-1000 nm, depending on particle material. Nano objects are characterised using the Nanoparticle Tracking and Analysis (NTA) software, which utilises the properties of both light scattering and Brownian motion in order to obtain particle size distributions of samples in liquid suspension.

 

Raman and AFM

The Raman/AFM platform consists of a Renishaw inVia Raman spectrometer with both a cobalt solid state laserand a diode laser operating at 473 nm and 785 nm respectively. The AFM is from NT-MDT and is mounted onan inverted Olympus microscope which enables bright field imaging (100x oil, 60x oil, 10x and 20x dry). BothRaman and AFM imaging can be carried out in either liquid or dry phase, a heated stage is also available forlive cell imaging. This hybrid system allows for the generation of high resolution AFM images (in various modes)of nano-materials and cells (and tissue) and characteristic Raman spectra of these samples. Using this uniquesetup, characterisation of nanomaterials/cells interactions can be performed in live cells in a non-invasive way.Full training on the system use with on-site assistance is available at all times.

 

Ultrafast Femtosecond Laser Systems

A stand-alone 80 MHz Ti:Sapphire laser system operating with ~130fs pulse width. It is a highly-tuneable,high power laser system which is able in conjunction with a wide range of experiments that use a UV or visibleexcitation wavelength. An optical parametric oscillator (OPO) is used to extend the tunable range of the systeminto the near-IR which covered telecommunications wavelength. An amplified femtosecond-pulsed laser systemoperating at 100KHz with ~130fs pulse width. Optical parametric amplifiers (OPAs) are used to generate thetunable la ser in the region of visible and near-IR. Difference frequency generators (DFG) extend tunable light inthe mid-IR spectral region. A regenerative amplified laser system operating at 1kHz with ~130fs pulse width. Thesystem enables >1mJ per pulse for Laser ablation.

 

Particle Exposure Assessment

 

High Content Analysis

High Content AnalysisThe national centre for High Content analysis incorporates several state of the art imaging platforms, liquidhandling devices and image processing software that allow for high throughput, high content analysis of cellularinteractions on both a population and individual cell level. There are a number of liquid handling platforms inthe facility, which allow for rapid precision dispensing of liquids in nanolitre volumes. Cellular responses andinteractions can be investigated in both live and fixed cell populations in adherent cells allowing for accuratedetailed information. The high content imaging platforms, contains both live cell and 3D pseudo-confocalcapabilities. The image and data analysis suite comprises image processing packages are available for analysisof data.

 

Research supported by the infrastructure

CRANN’s strengths in fundamental nanoscience research provide a successful platform for engaging withindustry Participants. With research activities covering most of nanoscience, CRANN researchers are pioneering developments in several key and emerging areas such as organic and molecular spintronics, flexible electronics,nanoscale polymer imprint, ultra-sensitive bio-sensors and nanoscale metrology.

At the IMM, Prof Volkov has developed internationally-recognized expertise in cell adhesion and migrationin inflammation and cancer, intracellular signalling, cytoskeletal dynamics and biomedical applications ofnanotechnologies, live cell and molecular imaging.

 

Services currently offered by the infrastructure

The 22,000 square foot CRANN Central Equipment Facility [CEF], is an integrated infrastructure, equipment,and human capital platform offering a core of key facilities and techniques to the CRANN client base. Facilitiessuch as microscopy centre, nanobio facility, femto-second laser laboratory, and scanning probe microscopes aremanaged by technical specialists and accessed by researchers from a wide variety of research backgrounds.Routine access to these state of the art facilities underpins and supports much of the research undertaken here,and enables CRANN to adopt an integrated, systematic, and holistic approach to tackling large scale, long termscientific and industrially relevant research problems.

 

The High Content Imaging Facility at the IMM currently services approximately 30 research projects, whichrange from large scale gene silencing (siRNA) screens, where the functional characteristics of each individualgene in the whole human genome is assessed by disrupting its function. As well as gene silencing it isstrongly committed in the area of nano-biology, where the facility is being currently utilised for study of thebiocompatibility of a range of advanced nano-materials which have potential therapeutic and diagnosticapplications.