CENTRE

OF

BIOPHOTONICS

 Light-based innovation for the biosciences

We have multiple light microscopes and workstations dedicated to image processing. Some of our microscopes are commercial instruments but a significant number of them are innovative techniques developed by groups within the Centre, and therefore, are custom-built setups.  Our imaging facilities are additionally complemented by nano- and microfabrication techniques within a cleanroom environment and ultra-fast techniques including femtosecond spectroscopy, fluorescence up-conversion and transient-absorption spectroscopy. This provides a broad experimental platform to address important biological questions across temporal and spatial scales and drive innovation. Below, we list some of our instruments and facilities. If you are interested in having more information as a potential user please contact us. 

 

EQUIPMENT – Commercial light microscopes

DELTAVISION

The Deltavision microscope is a wide field fluorescence light microscope with deconvolution and a quantitative laser module with lasers of wavelengths 406nM, 488nM and 532nM to allow techniques like FRET, FLIP and FRAP.  The system has 100X and 60X oil immersion lenses and a 40X air lens. We have the following filters CFP, CY5, DAPI, FITC, M Cherry and YFP.

More details…

Location: ROOM B104, Biomedical Science Research Complex (BSRC), School of Biology.

LEICA TCS SP8

The system incorporates a motorised inverted DMI 6000 microscope, which has 4 objectives.  There is also a motorised x/y-stage allowing for multi-position imaging and tiling. For eyepiece visualization there are three fluorescent filters: Blue, Green and Red.  The SP8 has a Tandem scanner system, which provides for Conventional scanning, or Resonant scanning (8 000 Hz) – 28 fps at 512*512 for fast live cell imaging. It contains four lasers, allowing for excitation in the following wavelengths: 405 nm, 458 nm, 476 nm, 488 nm, 496 nm, 514 nm, 543nm, 561nm, 594nm and 633 nm.

More details…

Location: ROOM B122, Biomedical Science Research Complex (BSRC), School of Biology.

ZEISS AXIOPLAN 2

The Zeiss Axioplan 2 is an upright epi- fluorescent microscope that interphases with Zeiss’s Axiovision 4.8.1 software. This microscope system is fully automated in its ability to acquire, fluorescent, bright field, phase contrast and Differential Interference Contrast (DIC) images.  There is also a Z-stack module for thicker tissue specimens. Fluorescent filter blocks available are for the excitation and emission of blue, green and red fluorochromes.  Filters available: Band Pass DAPI filter block, Band Pass FITC filter, Band Pass GFP filter, Long Pass TRICT/Rhodamine and Texas Red filters.

More details…

Location: ROOM B122, Biomedical Science Research Complex (BSRC), School of Biology.

ZEISS PASCAL 510 CONFOCAL

The Zeiss Pascal Meta 510 Confocal Imaging system includes a Zeiss Axio-Imager M1 upright microscope that is equipped with X10, X40 and X63 objectives. The microscope is also fitted with epi-fluorescent filter blocks to visualise blue (e.g. DAPI,) green (e.g. FITC) and red (e.g. TRICT) fluorochromes.  The Zeiss Meta 510 confocal system, has three lasers (488 nm, 543 nm and 633 nm). The Exciter Software allows for the acquisition of simultaneous single-track images. There are pre-set Zeiss configurations of Main Dichroic Beam Splitters and Secondary Dichroic Beam Splitters to filter excitation wavelengths coupled with Long pass and Band Pass filters to detect emission wavelengths. These configurations can be used in two channel combinations such as the excitation of both 488 and 543 laser lines permitting for double and triple staining.

More details…

Location: ROOM B106, Biomedical Science Research Complex (BSRC), School of Biology.

ZEISS APOTOME

The Apotome is a widefield fluorescence microscope which allows you to create optical sections of your samples free of scattered light. With optical sectioning structured illumination (OS-SIM) only the focal plane appears in your final image. The Apotome projects a grid structure onto the focal plane of your specimen, then moves it into three positions using a scanning mechanism acquiring an image at each position. The system processes the three images into one optical section with improved contrast and increased resolution. The process occurs automatically making this as easy to use as an standard widefield fluorescence microscope.

Location: School of Psychology and Neuroscience

CREST X-LIGHT CONFOCAL

The Crest X-Light unit is mounted on a custom upright body. The Crest X-Light allows high-speed, large field of view spinning disk confocal imaging. It specially suitable for capturing fast dynamics in cell motility, signalling and tansport applications. It consist of a flexible illumination source (laser or LED) and enables the capture of large FOV samples with minimal scanning and stitching, often in  a single image capture.

Location: School of Psychology and Neuroscience

  YOKOGAWA CSU-W1 SPINNING DISK CONFOCAL

The CSU-W1 is a high-end real time confocal imaging system with 2 sets of spinning disks for low and high resolution imaging. The system features a  Perfect Focus System (PFS) (active focus control), facilitating long time investigations (hours to days) of cells and tissue, showing very low phototoxicity/minimal photo-damage, which is also well suited for samples with low fluorescence emission. CSU-W1 offers single camera split-view mode with wide FOV.

Location: Wolfson laboratories. School of Medicine.

NIKON N-SIM

3D Structured Illumination equipped 405nm, 488nm, 561 nm exciation wavelengths. The N-SIM improves lateral resolution by a factor of 2 compared to conventional light microscopes and axial resolution also improved by a factor of 2 with 3D SIM. Other specifications include: 1 sec/frame temporal resolution suitable for live cell imaging, temperature and CO2control and large field of view (66um x 66um with 100x objective)

Location:  School of Physics and Astronomy

ZEISS AXIOSCAN Z1

The Axio Scan Z1 is a fast and flexible slide scannmer for fluorescence and brightfield. The CoB has two Z1 microscopes. The setup comprises a modular tray for specimen slids of 26×77, 52×77 and 106×77 mm an can digitize up to five fluorescence channels. It uses a LED light source Colibri 7 can switch excitation wavelength (400-700 nm) within milliseconds for transmitted light and LED (385, 423, 511, 555, 590, 631 and 735 nm) for fluorescence.
ld of view (66um x 66um with 100x objective)

Location:  School of Medicine

NIKON N-SIM

3D Structured Illumination equipped 405nm, 488nm, 561 nm exciation wavelengths. The N-SIM improves lateral resolution by a factor of 2 compared to conventional light microscopes and axial resolution also improved by a factor of 2 with 3D SIM. Other specifications include: 1 sec/frame temporal resolution suitable for live cell imaging, temperature and CO2control and large field of view (66um x 66um with 100x objective)

Location:  School of Physics and Astronomy

ZEISS AXIOVERT – MICROINJECTION

The Zeiss Axiovert has been adapted for micro-injection of adherent cells and is also capable of phase-constrast time-lapse imaging,

Location:  School of Physics and Astronomy

LEICA LASER MICRO-DISSECTION LMD6500

The LMD6500 performs sample preparation for molecular biology analysis from the tissue section using a UV laser. It can be used for precise targeting of cells (i.e., extruding tumour cells from biopsies for downstream analyses) but also potentially for ablation studies in living tissues. Downstream applications of LCM-dissected samples include various NGS, transcriptomics and mass spectrometry. The LMD6500 is based on a high-end upright microscope coupled to a 355 nm laser operating at 80Hz (4 ns pulse) with a maximum pulse energy of 70 microjoules. The LMD6500 is fully integrated with fluorescence imaging and enables live cutting within brightfield and fluorescence. The LMD6500 allows specimen cutting from above and collection by gravity, with no limitation of size and shape of dissectate. The microscope software offers complete control of laser settings, drawing tools and cutting modes.

Location:  School of Biology, BSRC Annex

EQUIPMENT – Custom-built microscopes

TOTAL-INTERNAL REFLECTION (TIR) MICROSCOPY

The CoB has three custom-built total internal reflection microscopes for cellular and molecular imaging down to the single-molecule level. One of the TIR microscopes uses an objective-type configuration and it is mounted on an inverted Nikon Eclipse Ti2 and the other two are prism-type setups mounted on IX81/IX71 Olympus inverted microscopes (picture above). These TIR microscopes are equipped with a range of fiber-coupled CW lasers (488, 532 and 647 nm) and EMCCD cameras (Andor, Ixon plus).  The time resolution achievable ranges from 33 ms/frame at full chip. Slightly faster resolution can be obtained using binning or by acquiring smaller regions of interest within the camera chip. Image analysis can be carried out using Image J and custom-built routines made in Matlab.

Location: Prism-type TIR Level 1 , Biomedical Science Research Complex (BSRC), School of Biology. Objective-type TIR: School of Medicine, Wolfson labs.

SINGLE-MOLECULE FRET

Two custom-built TIR and one confocal microscope are available at the CoB for single-molecule FRET measurements. The TIR microscopes are equipped with EMMCD cameras (Andor IXon Plus). Excitation can be performed a three wavelengths (488, 532 and 638 nm) using 50 mW CW lasers operating individually or sequentially switched. The TIR microscopes can operate in two or three colour emission modes using an OptoSplit III module (Cairn Research). Commonly used FRET dyes include Cy3, Cy5, Cy7 and Alexa and Atto families. In TIR-FRET configuration, usually donor and acceptor intensities of ~200-300 molecules are recorded simultaneously at speeds up to 16 ms/frame (half-chip mode). In confocal-FRET mode, only the intensity trajectory of a single-molecule is recorded but at faster integration times (~100-200 microseconds).

Location: Level 1, Biomedical Science Research Complex (BSRC), School of Biology.

SINGLE-MOLECULE SPECTROGRAPH

The single molecule spectrographic microscope enables collection of fluorescence spectra for single dyes instead of the conventional intensity recording. The microscope is mounted on an IX71 Olympus inverted microscope and can operate in both conventional epi-fluorescence or objective type TIR. It comprises a Omni-300 spectrograph 3-turret system for different gratings. The microscope is coupled to a Newton 971 EMCCD camera (Andor). As excitation sources, the microscope is coupled to a 532 nm and 638 nm diode-pumped solid-state CW lasers and to a NKT Supercontinuum laser (SuperK-FIANIUM, spectral coverage: 410-2400 nm) at 78 MHz repetition rate and 2.2W total output power. When using the NKT source, the microscope can simultaneously image the excitation and emission fluorescence profiles of individual fluorophores.

Location: ROOM 268, School of Physics and Astronomy

SINGLE-MOLECULE FCS-MFD

The single-molecule fluorescence correlation microscope (FCS) is mounted on an inverted IX81 microscope and consists of four avalance photodiode detectors (Excelitas) for simultaneous collection of four emission signals (two colors and two polarizations (vertical/horizontal) for each color, in addition to intensity and fluorescence lifetime. In this multi-parameter fluorescence detection (MFD) configuration, it is normally used to determine structural dynamics, polarization, lifetimes and diffusion coefficients. It can operate in FCS, FCCS, FCS-FRET and FCCS-FRET modes. It is equipped with 488 and 532 pulsed lasers with ~50 ps pulse width.  For time-tagging correlation of pulses it is equipped with two SPC-130EM time-correlated-single-photon counting modules (Becker & Hickl). 

Location: Biomedical Science Research Complex (BSRC), School of Biology.

COMBINED MAGNETIC TWEEZERS AND FLUORESCENCE IMAGING

The magnetic trap is mounted on an inverted IX81 Olympus microscope equipped with an oil immersion objective (PLANAPO 1.4 NA 100X). The applied magnetic field is generated by a pair of magnets (NdFeB) located above the sample stage and separated by 1 mm. Variation of the magnetic field is achieved by translating the magnets using a Physik instrument XYZ nanopositioner (M-126 PD1). A red LED is used to create a diffraction pattern of the magnetic bead from which to calibrate and track its position using BFP interferometry. The microscope is also couple two 532 and 638 nm lasers for fluorescence excitation and a Ixon Plus EMCCD camera for imaging.

Location: ROOM 268, School of Physics and Astronomy

TRAFIX MULTIPHOTON MICROSCOPE

State-of-the-art inverted multiphoton microscope, including temporal focusing for deep sample imaging.
Conventional scanning two- and three-photon microscopy, enabled by Galvo-galvo scanning mirrors for 0.1-1 fps imaging and high-sensitivity photomultiplier tube. The system comprises two lasers: Coherent Chameleon Ultra II pulsed laser (two-photon), tunable for 680 – 1080 nm excitation and Coherent Monaco + Opera F pulsed laser (two- and three-photon), tunable between 650 – 920 nm and 1120 – 2500 nm high pulse energy excitation.
Widefield multiphoton detection for rapid imaging, enabled by Andor iXon EMCCD (30 frames/s) and temporal focussing widefield illumination for axial sectioning/
TRAFIX: temporal focussing with single pixel detection for imaging at depth, including low photobleaching/photodamage and dynamic light shaping for precision multiphoton excitation

Location:  School of Physics and Astronomy

CUSTOM LIGHT SHEET MICROSCOPES

Open top LSFM equipped with a Hamamatsu ORCA-Flash4.0 V3 sCMOS camera for high sensitivity and speed (up to 100 fps) and multiphoton capability. The following laser sources are available : 488 nm continuous wave laser (iBeam smart, TopticaPhotonics) for single photon excitation, adjustable 690-1064 nm Coherent, Chameleon Ultra II (80MHz Repetition Rate) laser for two photon excitation and two-stage optical parametric amplifier (Opera-F, Coherent) pumped by a 40 W diode-pumped femtosecond laser (Monaco, Coherent) (1MHz Repetition Rate), providing a broad tuning range (650-900 nm and 1200-2500 nm) for two and three photon excitations. 3D acquisition capabilities include: thin optical sectioning (light sheet thickness ranges from 1.5-14 µm) with high SNR, high-speed imaging with very low phototoxicity and photobleaching, easy mounting sample holder for different types of samples, long working distance, water-dipping objective tailored for zebrafish and drosophila embryos, histology/pathology samples, and transparent specimens. Additional features include attenuation compensation capability for optimised imaging of turbid samples and advanced image analysis with deep learning algorithms to achieve super resolution

Location:  School of Physics and Astronomy

COMBINED RAMAN/DIGITAL HOLOGRAPHIC MICROSCOPY

Raman Spectroscopy:
Setup equipped with Ti:Sapphire Laser (MSquared, UK, Solstis) with incident laser wavelength of 785 nm (can be used for WMRS). The light is collected in reflection through 60X objective and focussed into a 200 µm fibre. Raman photons are collected by a monochromator (Shamrock SR-303i, Andor Technology) with a 400 lines / mm grating, blazed at 850 nm, and deep depletion, back illuminated and themoelectrocally cooled CCD camera (Newton, Andor Technology).
DHM: Off-axis transmission DHM system has the source light from a continuous wave diode-pumped solid-state laser (Spectra-Physics Millennia Vs, wavelength of 532 nm). This light is coupled into a single mode optical fibre splitter. The split beam is fed into reference arm and signal arm (with OB1 and OB2). The two beams are combined using a beam splitter on a CMOS detector (Imaging Source, DFK 42AUC03, 1024×960, 8bit, 25fps). For the latest changes, the camera is replaced with the one you gave me (details of which I shall pass to you after I get back to the lab maybe in the next week) and a 2X afocal telescopic system of lenses is placed in the reference arm.

Figure: The optical instruments highlighted in red and orange depict Raman system and the instruments in Green and orange depict DHM system. Here OB1 is long working distance objective (10X, 0.23NA), OB2 is 60X objective lens (Nikon, 0.85 NA), FC are the fiber coupling, L are the Lenses, NFi are the notch filters, M are the mirrors, BSi are the beam splitters, ND is the neutral density filter, DETi are the detectors and WLS is the white light source for microscope.

Location:  School of Physics and Astronomy

ACOUSTIC TRAPPING/RAMAN SPECTROSCOPY PLATFORM

Based on a Nikon inverted microscope, the setup comprises a microfluidic chamber equipped with a syringe pump and a 785 nm tunable laser (M2 Solstis). Acoustic trapping features include the ability to trap up to 20 micrometer-size beads or cells (semi-monolayer in the middle of the chamber) and adjust the trapping force and height. With regards to Raman capabilities, the setup acquires wavelength modulated Raman spectra in the range (500-1800 cm-1) and removes any autofluorescence cellular background. The plaftorm scans a 5 micrometer confocal volume and includes a motorized X-Y stage for scaning, a Z-stage for focusin along beam direction and live-cell spectroscopy with temperature control. The user-graphic interface is designed in Matlab.

Location:  School of Physics and Astronomy

OPTICAL COHERENT TOMOGRAPHY (OCT)

Based on a Mach-Zehnder interferometer in a home-made cage system. The setup includes a super-luminescent diode (SLD) laser (Superlum, Cork, Ireland, S850, central wavelength 850 nm, Δλ = 30 nm), motorized XYZ Galvo scanning, offset-OCT control, motorized sample and reference arm shutters, manual stage for sample holder and Matlab based GUIO control software. For OCT imaging capabilites:
    • A-scan acquirement

    • B-scan 2D imaging

    • C-scan 3D volume imaging

    • 17 um lateral resolution

    • 25 um axial resolution

    • Scan range: 5x5x2mm

Location:  School of Physics and Astronomy

Nanofabrication facilities

Ultra-fast fluorescence spectroscopy

We have facilities to manufacture micro- and nano-patterned photonics structures via electron-beam lithography and to characterize them via AFM, DEKTAK profiler and EM. If necessary, the fabrication process can be carried out in a clean room facility and entirely in a nitrogen atmosphere glovebox. These facilities are hosted in the School of Physics and Astronomy and managed by Prof Samuel, Prof Turnbull, Prof. Malte Gather and Prof di Falco. In addition, instrument developers at the CoB have access to excellent electronic and mechanical workshops to assist the researchers in the design, drawing and manufacturing process of specialized components. (Image courtesy Andrea di Falco).

We have access to a number of ultrafast spectroscopy techniques including:

Femtosecond lasers (SpectraPhysics Mai Tai Ti:Sapphire oscillator – 80 MHz output, 750-850 nm, 100 fs FWHM pulse width, SpectraPhysics Merlin laser at 527 nm, 50 KHz and 200 ns pulse width)

Light amplifiers (SpectraPhysics Hurricane, SpectraPhysics OPA, Light-Conversion TOPAS-White and SpectraPhysics Spitfire.

Hamamatsu Streak Camera – Detection of luminescence from thin films or solutions, with an instrument response function of ~ 2 ps (FWHM).

CDP FOG 100 – Optical gating allows luminescence to be detected on the femtosecond timescale. By sum-frequency mixing of the gating pulse and sample luminescence, upconverted light is produced that is detected. This technique allows temporal luminescence kintetics with time constants as low as 50 fs to be resolved.

Homebuilt Transient Absorption – Utilising two beams, a pump and a probe, with one of them delayable, allows transient kinetics in the absorption of samples to be recorded on the femtosecond timescale.

These facilities are hosted at the School of Physics and Astronomy and coordinated by the labs of Prof . Samuel and Prof. Turnbull. (Image courtesy of Ifor Samuel/Graham Turnbull.)