Epifluorescence microscopy

In epifluorescence microscopy (also called widefield microscopy), a parallel beam of light is passed directly upwards through the sample, maximizing the amount of illumination. 

The excitation and emission light travel through the same objective lens in epifluorescence microscopy, where “epi” is borrowed from the Greek to mean “same”. Epifluorescence microscopy is particularly useful for imaging thick samples.

 

 

Life Technologies EVOS FL

Life Technologies EVOS FL is particularly good for total beginners to microscopy. It uses light cubes that cover a wide range of applications.  It provides quick, high quality images.

There are no eyepieces, instead an easy-to-use interface enables the user to take quick snapshots through to time-lapse imaging using the in-built monochrome camera. Fluorescence illumination is provided by LEDs within specific light cubes for imaging a variety of fluorescent proteins and dyes.

• Light cubes available for: DAPI, CFP, YFP, FRET, GFP, RFP, TexasRed, Brightfield
• Objectives: 1.25x, 4x, 10x, 20x, 40x and 100x (oil immersion)

 

 

Inverted Fluorescence Microscope (Leica DM IL LED)

This inverted fluorescence microscope is best used for screening.

• Filters:  DAPI, GFP, RFP and brightfield
• Objectives:  5x, 10x, 20x, 40x (all dry)
• Phase contrast (with 40x objective only)

Confocal microscopy

We have 6 point scanning confocals and a spinning disc confocal microscope.

 

Point scanning confocal microscopes

 

 

Zeiss LSM880

The LSM880 upright confocal is particularly particularly good for imaging problematic fluorophore combinations and achieving better resolution than conventional confocal microscopes. It is suitable for high quality 3D imaging using combinations of fluorescent proteins or dyes that would otherwise be difficult to distinguish. It can fit larger objects via the attached Axio-Examiner microscope stand, making it particularly useful for time-lapse imaging of whole plants. The Airyscan module permits super-resolution imaging, achieving lateral resolutions of up to 120nm.

Software

Zen confocal software including rapid 3D rendering

Lasers

405nm, 440nm, 458nm, 488nm, 514nm, 561nm, 633nm

Detectors

• 2xPMTs, 1x Spectral detector (32 Channel GaAsP QUASAR detector)
• 1x Airyscan detector
• T-PMT

Extras

Motorized stage

Objectives

• Dry: 10x, 20x
• Immersion: 40x 1.3NA oil, 63x 1.2NA water, 63x 1.4NA oil
• Water dipping: 10x, 20x, 40x, 63x 1.0NA

 

 

Leica TCS SP8

Upright confocal equipped with sensitive HyD detectors. This confocal has a vertical imaging mode, enabling roots grown vertically on petri dishes to be easily imaged as part of a time-lapse experiment. It is fully automated and particularly suitable for fast image acquisition. It contains both the tandem and resonant scanner for routine and high speed imaging, respectively. Three out of the four detectors are the high sensitivity HyD detectors suitable for low-light imaging.

The SP8 can be converted to a vertical imager in 30 minutes, enabling seedlings to be imaged directly on specific petri dishes as they grow and respond normally to gravity. This involves the microscope nosepiece illuminating to the side. A high performance piezo focusing kit enables rapid z-stacks to be taken and Live Data Software Mode enables multiple regions to be visited.

Software

Leica AF with Matrixscanner module and FRAP tools

Lasers

405nm, 442nm, 458nm, 476nm, 488nm, 496nm, 514nm, 561nm, 633nm

Detectors

3x HyD, 1x PMT, T-PMT

Objectives

• Dry: 5x, 10x, 20x
• Immersion: 63x 1.2NA water, 63x 1.4NA oil, 100x 1.44NA oil, 20x 0.75NA multi-immersion for long working distance/bleaching
• Water dipping: 25x 0.95NA, 40x 0.8NA, SP8-Vertical Imager mode

Extras

Piezo z-drive, fully enclosed incubator

 

 

Zeiss LSM700

The LSM700 upright confocal microscope is particularly good for combinations of GFP/YFP with the new generation of fluorescent proteins (e.g. mOrange/mCherry). It is fitted with custom filter sets that reduce the amount of undesirable autofluorescence from live plant samples. A new automatic stage, with the same controls and "feel" of a manual, facilitates easy location of samples/regions of interest while enabling multi-position visiting and tile-scanning.

Software

Zen confocal software including rapid 3D rendering.

Lasers*

405nm, 488nm, 555nm, 639nm

*all solid state

Detectors

2xPMTs, T-PMT

Objectives

• Dry: 10x, 20x
• Immersion: 40x 1.3NA oil, 63x 1.2NA water, 63x 1.4NA oil
• Water dipping: 10x, 20x, 40x, 63x 1.0NA 

 

 

Leica-Renishaw SP8-FLIMan

This system is a new type of confocal enabling label-free organelle imaging and Raman detection of molecules. It enables you to image, in living tissue, the ‘chemistry of cells’. A partnership between the Sainsbury Laboratory and three companies; Leica Microsystems, Leica Industrial and Renishaw, the FLIMan allows a diverse range of specialist techniques in addition to high-end confocal microscopy:

• Real time imaging of protein-protein interactions and biosensors using FLIM
• Label-free organelle imaging using Raman, in addition to fluorescently labelled reporters
• Direct imaging of molecular constituents of cells and tissues with applications to phytohormones

Leica SP8 confocal with dual channel FLIM, 2x HyD SMD detectors, 2x PMTs, 1x t-PMT, automatic stage for use in both confocal and Raman modes. Renishaw InVia Raman system with Rencam CCD and Andor EMCCD detectors. Conversion kit for liquid samples.

Software

Leica LAS X with FLIM module, Picoquant software for photon counting. WiRE software for Raman spectral acquisition and
mapping. 

Lasers

(Confocal) Pulsed 405nm, 440nm, 470nm. Continuous 440nm, 488nm, 514nm and 552nm
(Raman) 532nm, 785nm.

Objectives

(SP8+Raman) 20x dry, 20x 0.75NA multi-immersion, 25x 0.95NA water dipping, 63x 1.2NA water, 63x 1.4NA oil.
(Raman only) 5x dry, 20x dry, 50x dry, 20x 0.75NA multi-immersion.

 

 

Leica SP8-iPhox

The Leica SP8-iPhox inverted confocal is equipped with cooled ultra-sensitive detectors and all-solid-state lasers. This confocal has been designed for high-end applications, including automated time-lapse imaging over long periods, single photon detection, rapid scanning and detailed quantification.

The Leica SP8-iPhox uses solid state lasers and ultrasensitive detectors for rapid low-signal imaging. Hardware autofocus prevents unwanted drift during time-courses and a new Stage Overview mode allows quick and easy scanning over large areas.

Equipment

Leica SP8 on DMI8 inverted microscope. 2x HyD SMD detectors, 2x PMTs, 1x t-PMT, fully automated stage, hardware autofocus, Resonant scanner option for high-speed scanning.

Software

LAS X with Live Data mode

Lasers

Solid-state 405nm, 448nm, 488nm, 514nm, 552nm.

Objectives

• Dry: 5x, 10x, 20x
• Immersion: 63x 1.2NA water, 63x 1.4NA oil
• Upon request: 100x 1.44NA oil and 20x 0.75NA multi-immersion

 

 

Leica StretchP8

The Leica StretchP8 upright confocal contains solid state lasers for imaging and time-lapse of whole plants, organs and tissues, together with mechanical perturbations using external devices mounted on the stage. The confocal supports single cell ablations and has the Lightning module that allows on-the-fly image processing to extract more information during scanning to give finer detail.

Equipment

Leica SP8 on upright microscope. 2x HyD detectors, 1x PMT, 1x t-PMT, automated xy stage

Software

LAS X with Lightning module

Lasers

Solid-state 405nm, 488nm, 552nm

Objectives

5x, 20x dry, 20x 0.5NA, 25x 0.95NA and 63x 0.9NA water dipping

 

 

Nikon Ti Eclipse Spinning Disk Microscope

This microscope is particularly good for high sensitivity timelapse imaging.

Assembled by Cairn Research in the UK, the inverted spinning disk system is centred around a Nikon Eclipse Ti microscope and a Yokogawa disk CSU-X1. It is most suitable for ultra-fast imaging or for specimens where unwanted photobleaching may be a problem. In the Sainsbury Laboratory, it is commonly used for long time-lapse imaging ranging from overnight to several days.

Offers multidimensional acquisition from two cameras (EMCCD from the Yokogawa, High resolution CCD from the Nikon Ti), sample incubation, FRAP and photoactivation. The Nikon Perfect Focus system prevents specimen z-drift.

Software

Metamorph

Lasers

405nm, 445nm, 488nm, 561nm, 514nm

Cameras

Photometrics Evolve (512x512 EMCCD) and Coolsnap HQ.

Objectives

Nikon objectives include standard and long working distance lenses for dry, immersion and water dipping. Please contact the facility manager for further details.

Extras

FRAP module, fully enclosed incubation, sensitive xyz stage movements, Nikon Perfect Focus

 

 

Scanning electron microscopy

We have two scanning electron microscopes - Zeiss EVO HD15 and Hitachi TM4000PLUS Desktop SEM.

 

Zeiss EVO HD15 Scanning Electron Microscope

Both conventional and cryo Scanning Electron Microscopy is carried on a Zeiss EVO HD15 that is equipped with various detectors. Preserved or dried material can be first coated with Gold using the Safematic CCU-010 and imaged in high vacuum. Fresh (hydrated) material can be rapidly frozen in nitrogen slush, cryo fractured and coated with either Gold/Palladium, Platinum or Iridium and imaged on the cryo-stage in the SEM chamber.

 

The EVO HD cryoSEM, built by Zeiss and Quorum technologies, allows high magnification imaging of cryo-preserved tissue in full vacuum conditions. Magnifications routinely achieved are between 90x and 50,000x with maximum magnification of approximately 300,000x. Excellent contrast is maintained through the use of a built-in gold/palladium, platinum, iridium sputter coater enabling user-defined coating on the nanometre scale. Fracture tools enable fractures through organs and cells revealing delicate structures in the cell walls and membranes. Sublimation protocols enable removal of surface ice or reduction of cytoplasm to reveal organelles. The time between plunge-freezing and imaging takes as little as 15 minutes.

 

 

 

Specifications

Filament: LaB6

Detectors: SE, VPSE G3, EPSE, BSD (5 segment), STEM (for single TEM grids)

Stage: (standard SEM mode) 9 place stage or single specimen Debien Coolstage. For cryoSEM a cryo stage maintains samples at –145C with an anti contaminator maintained at –175C.

Cryo prep-deck: maintained at –145C with two fracture knives, platinum sputter coater with thickness monitor. Associated workstation for plunge freezing of fresh tissue with cryo-transfer under vacuum.

 

Software

SmartSEM including SmartStitch module for tile imaging of large areas at high mag.

 

Access and technical support

cryoSEM drop-in sessions, where users bring samples for cryo preparation and imaging by the facility manager, run from Wednesday 1pm to Friday evening. Please contact the facility manager to reserve a place.

Associated equipment consists of a Safematic Gold sputter coater that can apply nanometre-precision coatings.

 

 

 

 

Hitachi TM4000PLUS Desktop SEM

The Hitachi TM4000PLUS Desktop SEM is equipped with a Deben CoolStage and has a low vacuum mode for quick SEM imaging of fresh plant material without coating.

It has Backscatter (for routine images of cell outlines) and SE detectors (for high resolution) and can switch between low and high vacuum modes. The chamber takes approximately 2 mins to pump to vacuum allowing for quick imaging of multiple samples. The CoolStage operates between -50C - +50C. The 4 segment backscatter detector combined with Mountains Map software enables 3D reconstructions of images to be generated.

 

Photolithography Facility

The photolithography facility contains various equipment for making microfluidic devices based on custom designs. These devices can then be used for the study and manipulation of bacteria, single isolated plant cells, whole tissues and seedlings. They are often used for long term imaging experiments on the various SLCU microscopes.

 

 

 

Kloe Dilase 650 LaserWriter

  The Dilase 650 is a high-resolution direct laser lithography system. It has a 375nm laser which can be focused on either a 1mm or 10mm spot sizes. The system allows the writing on any type of substrate (photomask, semiconductors, glass, polymers or crystals) over a surface area of up to 4 in².

The Dilase 650 is designed for etching out both simple and complex patterns of channels and cavities for custom building of microfluidic devices. Its 375nm laser accurately constructs channel arrays with nanometre precision on silicon wafers that are processed in a cleanroom environment and then used as a template, or mould, for accommodating bacteria and single plant cells during live cell imaging.

 

UV Lamp

365nm UV lamp. This can be used for exposing larger features with a photomask.

 

 

 

 

 

 

 

 

 

 

 

 

 

SUSS LabSpin 6 Spin Coater

Suss spin coater for the coating of thin layers (less than 10mm) of photoresist onto wafers. A range of chucks are available to accommodate different wafer sizes and shapes.

 

 

 

 

Polis Spin Coater

Polis spin coater for the coating of thicker layers (greater than 10mm) of photoresist.

 

 

 

 

 

 

Stuart Hotplates

3x Stuart Hotplates with a temperature range up to 350°C for curing the photoresist before and after exposure.

 

 

 

 

Grant-bio Orbital Shaker

Grant-bio orbital shaker for the agitation of wafer cleaning solutions prior to spin coating and the development of wafers post exposure.

 

 

 

 

Leica DM2700 M Microscope and Camera

Leica DM2700 M microscope and HD camera for checking microfluidic features after development. Objectives include 5x, 20x, 50x, 100x and 150x.

For access to the facility please contact Gareth Evans or Christian Schwall.

Expertise and training

Our team's expertise covers a range of imaging and analytical tools developed for the plant sciences. The Microscopy Facility team has many years of experience light, fluorescence and electron microscopy and actively support researchers with setting up a new study, troubleshooting or helping to sort out an unexpected result. They provide inductions and training on all microscopy equipment and specialist techniques.