Fluorescence

The NBL has an extensive suite of standard and advanced spectroscopic systems in order to sustain our wide and varied research objectives.

We have a mixture of standard steady-state spectrometers for routine analysis, several modified for polarized EEM measurements, and multiple fluorescence lifetime based systems for time-resolved studies.
We also have the capability to build our own systems as the need arises.

Steady state fluorescence.

Fluorescence Lifetime cuvette 1 & 2.

Fluorescence Lifetime cuvette 3 & 4.

UV-Visible spectrometers.

Sampling systems.

Historical systems.

Most of the general fluorescence equipment is located in the middle laboratory (Lab 7 in the Concourse), while the more advanced lifetime based systems are in the dedicated microscopy laboratory downstairs.

Fluorescence (steady state):

Aqualog (Horiba)

We are currently (Nov. 2024) using two Aqualogs from Horiba for polymer, protein, and liposome analysis. This includes specific studies on non-visible aggregation of polymers and protein-liposome interactions. This system with a multi-channel CCD detector provides a huge speed boost in terms of reducing data acquisition times while also enabling us to simultaneously measure the absorbance spectrum. One system is on loan from Horiba and we've been using it to investigate the feasibility of using polarized Excitation Emission Matrix (pEEM) for the quantitative analysis of large protein complexes, the kinetics of protein conjugation reactions, bioprocess monitoring, and the analysis of protein-polymer interactions. The pEEM approach will complement and extend the A-TEEM capability of the Aqualog, as part of our ongoing collaboration. This has also been upgraded with a Rapid Mixing accessory (SFA-20 from TgK Scientific) for kinetic studies. ‌

The second system with a sipper accessory to support our expanding bioanalytical projects was installed in Nov. 2024. This system is being configured for both high sample number experiments and flow based measurements.

Eclipse (Agilent).

Our other steady-state fluorescence spectrometers are the Cary Eclipses (left) the first of which was installed in July 2004.

It has a front surface accessory and standard polarizers for additional functionality. In 2008 we also acquired a thermostatted multi-cell holder for high throughput studies. This system is primarily used for cell culture media studies. This system has been in continual operation for more than 14 years now and we still have not yet had to change the bulb...not bad, considering that it has collected hundred's of thousands of emission spectra.

Two more Agilent Eclipse systems (2012) were fitted with wire grid polarizers polarizers for anisotropy research. We also have a multi-well accessory for these systems. A fourth system (2015) was installed for the pEEM/anisotropy based research projects.

Three Ocean Insight fibre coupled spectrometers are also available and used for routine in-situ fluorescence measurements.

We also have a Perkin-Elmer LS-50B fluorescence spectrometer which can perform a range of techniques from time resolved fluorescence, phosphorescence and chemiluminescence measurements. This is typically used for undergraduate physics and chemistry research projects.

A front surface sample holder is available for fluorescence measurements of optically dense materials such as crude oils and polymers.

This picture above (2017) shows the middle of the PAT lab with a Shimadzu UV-1601 UV-Visible and Perkin-Elmer LS-50B spectrometers.
This workstation is generally used for the characterization of novel fluorophores and undergraduate student projects.

Fluorescence Lifetime, cuvette systems 1 & 2:

The primary fluorescence lifetime system for routine work is a FluoTime 200 system from PicoQuant. The system was delivered in 2003 (funded by SFI) and replaces a system we had built in house (see below).

This system is based around a TimeHarp 200 TCSPC card located in a PC above the system (pictured on the right).

This system allows for the automated collection of lifetimes over the 300-850 nm range, the collection of Time-Resolved Emission Spectra (TRES) and Anisotropy data.

The following excitation sources are available:

LED's : 295, 340, 380, 460, 510, and 610 nm centre wavelengths.
UV and blue laser diodes: 375, 404, 408, 440, and 470 nm (~40-70 ps FWHM).
Red Laser Diodes: 635 and 750 nm centre wavelengths.

All sources have been obtained from PicoQuant, and have maximum pulse rates of 40 MHz.

A Sepia dual channel laser driver is also available.

The sampling chamber (right) is large enough to accommodate a range of sampling accessories including liquid nitrogen cooled cryostats.

A Quantum Northwest TLC-50F temperature controlled sample chamber can also be retrofitted to the system for temperature dependent studies.

This system was funded via a Science Foundation Ireland Principal Investigator award.

The second primary lifetime system is the Excitation-Emission Fluorescence Lifetime Spectrometer (EEFLS) which was developed and built in 2017-18.

We have now also installed (2017) a Becker & Hickl PML-SPEC16 TCSPC multi-channel system for fluorescence lifetime spectroscopy.

This is configured for UV-visible lifetime measurements.

This system was funded via a Science Foundation Ireland Principal Investigator award.

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This system uses a supercontinuum laser source from Leukos.

This is a SMHP-4W system equipped with a frequency doubler for UV generation.

The system was installed in 2017 and is shown in operation here with visible light output.

Tech Specs:

>4W total optical output (vis-NIR)
>600 mW in the visible.
260-350 nm tuneable output.
Variable pulse rates (60. 30, 20, 15, & 10 MHz)

This system was funded via a Science Foundation Ireland Principal Investigator award.
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Fluorescence Lifetime, other systems 3, & 4:

These systems can be built up as required using either a PicoQuant TtimeHarp 100 TCSPC card or a PicoQuant PicoHarp 300 . Both of these have been recycled from earlier primary systems.
This can be coupled either to the confocal microscopes or to the various cuvette and sample holders depending on requirements.
The PicoHarp allows us to undertake Time-Tagged Time-Resolved (TTTR) measurements, as well as standard FCS, and lifetime measurements.
At present (Nov. 2024) the system is attached to a confocal microscope for FCS measurements.

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UV-visible spectrometers

We currently have four systems in place which are mainly used for solution phase measurements.

A Shimadzu UV-1800 double beam spectrometer was installed in 2017 to facilitate routine solution measurements of media and proteins.
A Cary 60 single beam instrument (Installed Sept. 2015) is available for routine solution measurements using both immersion probe and cuvettes.
We also have a dedicated high specification UV-VIS-NIR system, a Perkin Elmer Lambda 950 (Installed March 2008).
A Shimadzu UV-1601 is used for routine solution phase UV-Visible measurements (~installed 2000).

The Lambda 950 system comes with a Universal Reflectance Accessory for the analysis of thin films and other solid substrates.

We have also available a Harrick Praying Mantis diffuse reflectance accessory for the analysis of powders and solid materials.

The system is configured with a range of predefined analysis routines for complex and difficult adsorption measurements.

This facility was provided under the HEA funded (PRTLI-IV) National Biophotonics Imaging Platform.

This is the Shimadzu UV-1800 double beam spectrometer (installed in 2017) with the Osmometer on the right hand side.

Both of these systems are mostly used for media analysis.

This picture above (Feb. 2017) shows one of the spectroscopy stations in the middle lab with a Cary Eclipse fluorescence spectrometer and a Cary 60, single beam, UV-visible spectrometer.
Both are equipped with temperature control and used for solution state studies.

Additional Extras / Sampling Systems:

Two Ocean Optics portable fluorescence spectrometers (a USB2000-FLG and a USB4000), and fibre accessories are also available for developing a range of new instrumentation/precision sampling systems for our fluorescence based research.
We have currently (Nov. 2024) coupled one of the spectrometers and the EEFLS with a TLC-50F temperature controlled cuvette holder. This will enable us to collect fluorescence steady-state and lifetime data at the same time, while running various temperature profiles.

A VGI 2000M custom built humidity chamber for microscopy and spectroscopy is also available and we use it for studying hygroscopic polymers. The system is usable over a 10-40 C temperature and 0-100% RH range and can be coupled to Raman, FT-IR, or Fluorescence spectrometers.

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The image on the right shows the heated sample chamber with gas and water lines attached. The left hand image shows the gas /humidity controller and heating control unit. We mostly used this hooked up to an Ocean Optics spectrometer to measure spectral changes in thin polymer films as a function of humidity (see publications for details).

As we acquire more equipment and accessories I will update the webpages with details and photos.

Historical systems:

Our first ever fluorescence lifetime system was based around a PicoQuant TimeHarp 100 TCSPC module (35 ps resolution) with LED and laser diode excitation sources. The system is contained used to be housed a light proof metal box (on left of picture) and was built up over the spring and summer of 2000. The system has been dismantled and was re-engineered using fibre coupling to assemble a system suitable for the time-resolved analysis of micro scale systems or extreme environmental conditions (low temperature studies of crude oils, protein behavior on surfaces, drug eluting polymers, etc.). This new system was coupled with the TLC-50 cell and the ocean optics spectrometer to provide multi-dimensional fluorescence data.

The picture on the right shows the inside of the first fluorescence lifetime system we built and used until 2003. The detector (on the right) is a Single Photon Counting Avalanche Photodiode from Perkin-Elmer and has a dark count of <100 cps.

Contact

Prof. Alan G. Ryder
Room 213, Nanoscale Biophotonics Laboratory,
University of Galway, University Road, Galway, H91TK33, Ireland.
Tel: +353 (0)91 492943 or ext. 2943 (internal)

E-mail: alan.ryder