The Microscopy Core Facility

The core Microscopy facilities in the Medical School provide a comprehensive biological imaging service to all departments.

We are in the process of developing the microscopy core facility for the School of Medicine and this page will be updated as new features are added. The microscopes are available 24/7 to trained users from within the university. Access for external users can be arranged during normal working hours. 

At present there are three confocal microscopes a spinning disk confocal and two Zeiss LSM 510 confocal fluorescence microscopes both equipped with Coherent Chameleon lasers that are suitable for both confocal and multiphoton imaging. The inverted microscope on K floor is suitable for conventional cell and tissue specimens mounted under glass cover slips or in glass bottomed dishes. The upright microscope situated in the Biological Services Labs on F floor and the spinning disk confocal are available for intravital microscopy or "open dish" cell/tissue specimens since they are fitted with "dipping" lenses which need to be immersed in water/culture medium. For more detailed specifications please see the confocal pages (top left on this page).

There are three inverted widefield fluorescence microscopes, two in room DU12 and one in the Jessop laboratories. These are equipped with long working distance lenses and are ideally suited to working with tissue culture plates and dishes. They can be used for phase contrast or fluorescence imaging of either fixed or live specimens stained or expressing a range of markers. For more detailed specifications please see the widefield microscopes pages (top left on this page). 

There is a computer workstation with a full version of AIM, Zeiss confocal software, Leica LAS “browser” version, IVIS living Image and the 64bit version of ImageJ installed. This is available for use free of charge to microscopy booking system account holders.

How to gain access to the microscopes and request training

Please request an account and/or training using the microscope booking system by following this link https://ppms.eu/sheffield/login/?LM_core. If you need any other help or to discuss any future requirements please contact me:

Colin Gray
Room LU104
colin.gray@sheffield.ac.uk
+44 (0)114 215 9580

Confocal/multiphoton training comes in two parts, a theory session of approximately 2 hours and a practical of approximately 3 hours that usually takes place a day or two later. Widefield microscopy training usually takes 2 hours and covers the basic operation of the microscope. Further training on more advanced features is done as required. There is a charge for training (see below for details) but further help or training (within reasonable limits) is included.  

Data storage

Users should back up their data via the LAN or on portable devices (e.g. memory sticks, CD-ROM, DVD) at the end of imaging sessions. Data left on computer hard disks will be erased regularly for system maintenance purposes without warning. Every user is entitled to use storage space on the D drive (d:\all_users_data\) while they are acquiring images. If storage space is in short supply, you may delete data belonging to anyone without warning so remember to back up your data before you lose something important.

Charges

Please note, at the moment use of the microscopes will be charged at a heavily subsidised introductory rate. This is intended to allow time for users to gain grant funding and is a temporary measure which is not sustainable in the longer term. Charges are applied to meet microscope maintenance and external service costs and do not support other areas of the medical school. If you are thinking of applying for a grant please contact Colin Gray (details above or via link below) to discuss your requirements and obtain a quote.

A discount is available for continuous time-lapse imaging sessions >5 hours. Also discounts for heavy users (>250 hours/year) can be arranged. Reduced rates apply to Sheffield University departments. Rates for users from other academic or commercial organisations are available on request. For details contact colin.gray@sheffield.ac.uk

Training is now charged at £20 per person for widefield microscopy plus the normal access charge. From 1/11/2018) the confocal / multiphoton microscopy training charge will be £110 per person plus the normal microscope access charge for the technique used. This charge includes £60 worth of microscope time which must be used within 2 weeks of completing training. At present this is a once only charge any subsequent help/support is free (within reasonable limits). 

In order to be fair and maximize system availability these charges will apply to bookings cancelled within 24 hours of the booked time if it is not used by someone else. People doing live cell/animal work who have a good reason for an occasional late cancellation (eg the cells died), and who report this by email at the time will be allowed to re-book without charge).

Also note, you must book enough time to allow your data to be saved at the end of the session. If you inconvenience another user by over-running your allocated time you will be charged for the microscope time (minimum 1 hour) plus an extra £20 for any part of an hour which will be paid to the booked user's account as compensation.

The Histomorphometry / fluorescence Leica DMRB upright microscope, the Bioquant microscope and the Bright field and fluorescence Leica DMRB upright microscope belong to Skelet.AL and are not part of the microscopy core facility.

Other facilities at Sheffield


Confocal Microscopes and Widefield Microscopes

Inverted Zeiss LSM 510 NLO microscope - Room K139

This inverted microscope is equipped with; Ar (458, 477, 488, 514 nm) 30 mW laser suitable for imaging fluorescent markers such as CFP, GFP, Alexa 488, FITC, Syto13, CY2, YFP etc; He/Ne (543nm) 1.2nW laser suitable for imaging markers such as TRITC, CY3, Alexa 543, Alexa 555, Lysotracker, Texas Red etc; HeNe laser (633 nm) 5 mW suitable for imaging markers such as CY5, Alexa 633, Draq 5 etc. Using conventional detectors up to 3 fluorescence channels + transmitted light can be imaged simultaneously and even more channels sequentially. This microscope is also fitted with a spectral detector that with suitable specimens allows fluorescence colours that are too close to separate using conventional filters to be imaged eg green Alexa 488 and green autofluorescence or gfp and yfp can be separated. The LSM 510 is also fully automated with a motorised stage and focus control for acquiring z stack images for 3d reconstruction. It is also possible to “tile scan” a specimen, mark areas of interest and then scan these at higher resolution. The microscope also has laser bleaching capability and can be used for techniques such as Förster Resonance Energy Transfer (FRET) by acceptor bleaching, Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Localisation After Photobleaching (FLAP) and photoactivation.

In addition to the single photon fluorescence capabilities mentioned above this microscope is fitted with a Coherent Chameleon femto-second pulsed laser suitable for multiphoton microscopy. This is suitable for experiments where deeper penetration into the specimen than is possible using single photon fluorescence is required. It is also suitable for techniques such as FRAP in thick specimens because bleaching is restricted to a small volume at the point of focus.

The microscope is fitted with 10x/0.3 dry; 20x/0.8 dry; 40x/1.2 water immersion; 40x/1.3 oil immersion and 63x/1.4 oil immersion lenses. All these lenses are designed to be used with a 0.17mm thick (#1.5) coverslip. Please note only the 10x lens has enough working distance to be used with plastic culture dishes and plates. Free image browser software is available here Zeiss Image Browser (click link to install PC only). 

Upright Zeiss LSM 510 NLO microscope - Room FU319

This upright microscope is equipped with; Ar laser (458, 477, 488, 514 nm) 30 mW laser suitable for imaging fluorescent markers such as CFP, GFP, Alexa 488, FITC, Syto13, CY2, YFP etc; He/Ne (543nm) 1.2mW laser suitable for imaging markers such as TRITC, CY3, Alexa 543, Alexa 555, Lysotracker, Texas Red etc; HeNe laser (633 nm) 5 mW suitable for imaging markers such as CY5, Alexa 633, Draq 5 etc. Using conventional detectors up to 3 fluorescence channels + transmitted light can be imaged simultaneously and even more channels sequentially. This microscope has motorised focus control for acquiring Z stack images for 3d reconstruction. The microscope has laser bleaching capability and can be used for techniques such as FRET (acceptor bleaching), FRAP, FLAP and photoactivation.
In addition to the single photon fluorescence capabilities mentioned above this microscope is fitted with a Coherent Chameleon femto-second pulsed laser suitable for multiphoton microscopy. This is suitable for experiments where deeper penetration into the specimen than is possible using single photon fluorescence is required. Because this microscope is primarily designed for imaging tissue it is fitted with non de-scanned detectors (NDD’s) which collect light normally lost when using the conventional de-scanned optical path. The microscope also has the option to be used for Fluorescence Lifetime Imaging (FLIM) a technique which can be used for FRET by measuring the change in fluorescence lifetime.

This microscope is fitted with 2.5x/0.075 dry, 10x/0.3 dry, 20x/0.8 Ph2 dry, 63x/1.4 Ph3 oil immersion and 10x/0.3, 40x/0.8, 60x/1.0 water dipping lenses. Free image browser software (PC only) is available from the downloads link above.

Upright Nikon Spinning Disk Confocal - Room FU323

This upright Nikon microscope is fitted with a Yokogawa spinning disk confocal system and Photometrics camera which will support faster scanning than is possible with the Zeiss confocal microscopes. The microscope is located in the Biological services department and is suitable for intravital microscopy. It is equipped with a recently refurbished Melles Griot gas laser, 488nm  suitable for imaging fluorescent markers such as GFP, Alexa 488, FITC, Syto13, CY2 etc; 561nm suitable for imaging markers such as TRITC, CY3, Alexa 543, Alexa 555, Lysotracker, Texas Red etc. There is a Physic Instumente, piezo fast focus device for z-stacks.

The microscope is currently fitted with a  40x/0.8 water "dipping" lens although there are also 10x and 20x that can be used by prior arrangement. More lenses will be purchased as the need arises.

Leica AF6000LX inverted microscope - Room DU12

This fully automated microscope primarily designed for live cell imaging. It is fitted with an environmental chamber maintained at 37°C, it is also equipped with a gas mixer for the supply of CO2 and a range of stage top incubators designed to fit various plates and dishes. The microscope can be used to acquire via Transmitted Light BrightField (TL-BF) or Phase contrast (TL-Ph) images. The microscope is fitted with appropriate filters for imaging a range of fluorophores.

  • Filter A4 is used for DAPI, Hoesch and other blue markers
  • Filter L5 for GFP, Alexa 488, FITC and other green markers
  • Filter N3 for TRITC, Alexa 543, 555 or 568 and other red markers
  • Filter Y3 for CY5, Topro, Alexa 647, Draq5 etc
  • Filter CHE for mCherry, Alexa 594 etc.

The N3 filter is not ideally suited to longer wavelength red fluorophores eg Alexa 594, mCherry etc but, well stained specimens with these markers can still be imaged. There is now a new CHE filter for mCherry or Alexa 594 which will work well with the A4 and L5 blue and green filters. However, there will be significant "cross talk" between images taken using either the N3 and CHE or CHE and Y3 filters.

The microscope control software has been upgraded to (LAS-X) which can support so called 6 dimensional imaging, xyz, time, colour and multiple locations (eg imaging every 10 minutes for 12 hours of transmitted phase contrast and GFP with 4 focus levels in each well of a 24 well plate =13824 images). The motorised stage can also be used to acquire “tiled” images. This microscope can also collect "tiled" images which can cover quite substantial areas of the slide or dish.
The microscope is fitted with 5x/0.12, 10x/0.3 Ph1, 20x/0.35 Ph1, 40x/0.6 Ph2 and 63x/0.7 Ph2 dry objectives which are all designed to work with tissue culture flasks, dishes and plates.

A 20x/0.7 Ph2 dry lens and a 100x/1.4 Oil immersion lens are also available but these can only be used with thin glass bottomed plates/dishes or specimens mounted under thin glass coverslips. These lenses are only available on a "first come first served" basis and must be returned to the shelf above the AF6000 computer at the end of the session. Please ensure that the normal lens is replaced or a plug is fitted into the microscope turret after removing any lens. Take care to ensure that dry lenses do not become contaminated with oil when using the 100x/1.4 lens. Only use lens tissue (never ordinary tissue or paper towel) when removing oil from lenses. Only use the oil provided and do not mix with oil from other sources.

Leica DMI4000B inverted microscope - Room DU12

This microscope is very similar to the AF6000 and has the same software control (LASAF) but, it is not automated. It is ideally suited to imaging fixed cells/tissue in culture plates or on slides. The microscope is fitted with a dual mode camera, black and white for fluorescence and colour for histochemical stained specimens (eg Haemotoxilin & Eosin, DAB etc), either mode can be used for brightfield or phase contrast imaging with transmitted light.  When using Phase Contrast imaging the "phase ring" in the condenser should be positioned as follows:

  • For the standard 10x/0.30 lens  use position number 7
  • For the standard 20x/0.35 lens use position number 7
  • For the special 20x/0.70 lens use position number 1
  • For the standard 40x/0.60 lens use position number 1
  • For the standard 63x/0.70 lens use position number 1
  • For bright field (no contrast enhancement) number 6 or 2

Fluorescence excitation is provided by a CoolLed LED light source (so no warm up/cool down time). Filters appropriate for imaging a range of fluorophores are fitted. 

  • Filter A is used for "live/dead" assays but can be used for images when the camera is working in colour mode.
  • Filter A4 is used for DAPI, Hoesch and other blue markers
  • Filter L5 for GFP, Alexa 488, FITC and other green markers
  • Filter N3 for TRITC, Alexa 543, 555 or 568 and other red markers
  • Filter BGR is not really suitable for taking images but can be used when the camera is working in colour mode.

The filters are not ideally suited to longer wavelength red fluorophores eg Alexa 594, mCherry etc but, well stained specimens with these markers can still be imaged. Far red markers such as CY5, Alexa 647 etc will not work on this microscope as the camera is not sensitive to these wavelengths. 

The microscope is fitted with 2.5x/0.07, 5x/0.15, 10x/0.3Ph1, 20x/0.35Ph1, 40x/0.6/Ph2 dry objectives as standard, higher magnifications may be available.

A 20x/0.7 Ph2 dry lens and a 100x/1.4 Oil immersion lens are also available but these can only be used with thin glass bottomed plates/dishes or specimens mounted under thin glass coverslips. These lenses are only available on a "first come first served" basis and must be returned to the shelf above the AF6000 computer at the end of the session. Please ensure that the normal lens is replaced or a plug is fitted into the microscope turret after removing any lens. Take care to ensure that dry lenses do not become contaminated with oil when using the 100x/1.4 lens. Only use lens tissue (never ordinary tissue or paper towel) when removing oil from lenses. Only use the oil provided and do not mix with oil from other sources.

Olympus IX73 inverted microscope - Room Jessop JB4, 65

This microscope is primarily designed for tissue culture and general lab microscopy can be used to image cells and tissue using transmitted light with either phase contrast or epifluorescence. It can also be used for time-lapse imaging but at present only with specimens requiring room temperature. Fluorescence excitation is provided by a CoolLed LED light source suitable for imaging Blue (eg DAPI), Green (eg ALexa488, GFP, FITC etc) and Red (eg Alexa 543, 555 or 568, TRITC, Texas Red etc). The microscope has fairly simple to use control software (Micromanager). 

The microscope is fitted with 10x/0.3 UPlanFLNPh1, 20x/0.5 UPlanFLN Ph1, 40x/0.75 UPlanFLN Ph2 and 63x/0.7 Ph2 dry objectives which are all designed to work with glass slides, tissue culture flasks, plates etc and a 60x/1.35 UPlan S apo-chromatic oil immersion lens which can only be used with thin glass, bottomed plates/dishes or specimens mounted under thin glass coverslips. 

Take care to ensure that dry lenses do not become contaminated with oil when using the 63x/1.35 lens. Only use lens tissue (never ordinary tissue or paper towel) when removing oil from lenses. Only use the oil provided on the shelf above and do not mix with oil from other sources.

Downloads for the microscopes

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