Confocal imaging

Laser scanning confocal microscopy is a significant advance in the field of optical microscopy, primarily because it permits sample visualization deeper within living and fixed cells, tissues and other samples.

Image take of scaffolds used in the human body to aid tissue regrowth

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The confocal imaging facility is located in the Kroto building, North Campus, and is one of a number of analysis and characterisation facilities within the school.

The microscopes within this facility were supported by two BBSRC grants: BB/D524983/1 - Imaging of 3D Engineered Tissues and BB/E012981/1 - Two Photon Imaging: From Polymeric Materials to Engineered Tissues.

The facility contains two Zeiss LSM510 Meta confocal microscopes which can be booked and operated independently of each other. Both microscopes have their own set of lasers and both can also be connected to a tuneable two-photon Ti-Sapphire laser.

There is an offline PC, running the Zeiss image analysis software, which can be booked for data analysis.

There is also a Leica inverted epifluorescent microscope with an environmental chamber and camera, and an Olympus inverted epifluorescent microscope with camera.

This open access facility is available for use by researchers based in the Department of Materials Science and Engineering, other users from within Sheffield University and external researchers from both academia and industry.

The facility also supports users of the Zeiss Lightsheet z.1, and Cellvizio Dual Band Invivo confocal. These two pieces of equipment are within Royce@Sheffield, as part of the Henry Royce Institute

For further information or to discuss your requirements in more detail, please contact Dr Nicola Green.

Dr Nicola Green

Senior Lecturer in Bioengineering
School of Chemical, Materials and Biological Engineering

Biohybrid constructs | In vitro 3D models | Mechanical stimulation

Email: n.h.green@sheffield.ac.uk
Telephone: +44 114 222 3932

Widefield Fluorescence Microscopy

There is an inverted widefield fluorescent microscope in the Kroto Imaging area which is particularly good for imaging thin samples, even with low levels of fluorescence.

Available equipment

It is an IX73 Olympus microscope, with wLS LED illumination, Cairn MonoLED for brightfield imaging and a Retiga 6000 CCD camera. This microscope has 4x, 10x and 20x objectives and is capable of obtaining images from blue, green and red emitting fluorophores as well as standard brightfield images. The details of the excitation and emission filter set used can be found here, with the same filter cube being used at all times. The LEDs used to illuminate the sample are varied to generate images from the different fluorophores.
The 4x and 10x objectives can obtain images of cells grown on standard tissue culture well plates. The 20x objective requires a glass coverslip, or chamber optimised for optical imaging (e.g. Ibidi, Eppendorf, or Thermofisher chambered slides).
How to use the Olympus widefield microscope

For advice in this area please contact Dr Nicola Green

Confocal Imaging

Laser scanning confocal microscopy is a significant advance in the field of optical microscopy, primarily because it permits sample visualization deep within living and fixed cells, tissues and other samples. It provides the ability to collect sharply defined optical sections from which three-dimensional renderings can be created.

Please remember however, that the quality of the image that you will get from this process is greatly affected by the quality of your sample. Or to put it more simply - RUBBISH IN, RUBBISH OUT!!! Consequently, great care and planning is required for all stages of sample preparation to ensure a good image is obtained.

Why Use the Confocal Microscope

An epifluorescent microscope is capable of imaging some fluorescent samples very effectively. It uses an objective lens to focus light upon the specimen and to collect the light being emitted. However, any sample thicker than about 5 µm will appear indistinct and out of focus, because parts of the sample are outside the objective focal plane. In addition, epifluorescent microscopes generally use the line-spectrum produced by a high pressure mercury bulb for sample excitation, whereas a confocal microscope uses monochromatic lasers to excite the sample. This allows the user to choose very specific fluorescence excitation wavelengths and expands the range of fluorophores that can be used together on a single sample.

Advantages of confocal imaging

Collects light from a single focal plane
User can change the focal plane to obtain optical slices at increasing depths within sample. These can then be combined to produce 3D images
No stray lateral interference, improving image contrast
Specific wavelengths used to excite sample

Disadvantages of confocal imaging

Signal strength reduced by requirement for detector pinhole
Reduced signal to noise ratio so increased sensitivity to noise
Technique is more labour intensive and requires more training and experience to be successful

Both confocal microscopes are connected to their own switchable Class 3b laser module which contains a number of lasers emitting light in the UV and visible spectral ranges. This provides each microscope with the following excitation capabilities

    Argon/2 - 458, 477, 488, 514 nm
    HeNe1 - 543 nm
    HeNe2 - 633 nm

Both are also connected to a class 4 tuneable Ti-Sapphire two-photon laser capable of excitation wavelengths between 690 and 1040 nm.

Comparison of Single and Two-Photon Imaging

Advantages of Two-Photon Imaging

Optical sections can be obtained from deeper within the sample

No attenuation of excitation source from outside focal plane
Longer excitation wavelengths used which scatter less
No pinhole required so all emitted light can be captured for imaging

Reduced damage to the sample

No fluorophore excitation in bulk of specimen, production of phototoxins is therefore limited only to focal plane
Excitation uses lower energy photons which have less impact on sample

Disadvantages of Two-Photon Imaging

Slightly lower resolution
Potential for thermal damage to specimen if it contains chromophores that absorb the excitation wavelengths
Two-photon excitation spectra is not fully characterised for all fluorophores

In addition to standard imaging protocols the microscopes are able to perform non-descanned detector imaging (NDD), time-correlated single photon counting and second harmonic imaging.

Please contact Dr Nicola Green for more information.

Including Confocal Images in Publications

Materials and Methods Information

The precise details of the imaging process are so variable between users and samples that it is difficult to provide a generic piece of text for inclusion in the Materials and Methods section of publications. However, below is a suggested template which you could use as a starting point, together with the addition of details specific to your own image acquisition process. Areas where you will need to replace the text with the specific details from your experiment are highlighted in bold to indicate this.

Images (pixel size x pixel size) were obtained using a Zeiss LSM 510Meta upright or inverted confocal microscope and objective details, with a pixel dwell time of dwell time.
Fluorophore 1 was excited using a laser wavelength nm laser (% transmission) and emission detected between [or "above" - if using a long pass filter] give wavelengths of light detected as a result of filter used.
All image analysis was performed using software for image analysis.

N.B. If more than one fluorophore was used to generate an image, it is necessary to give the details for each fluorophore separately.

Acknowledging the Imaging Facility

If you include any images obtained from the Kroto Imaging Facility within your published work please ensure that you acknowledge the Kroto Imaging Facility.

The following is an example of the wording that you could use:-

Imaging work was performed at the Kroto Research Institute Confocal Imaging Facility, using the LSM510 Meta inverted/upright (delete as appropriate) confocal microscope.

And finally - if you also send Dr Nicola Green the details and, if possible, a PDF copy of any papers published as this documentation will help to justify the continued existence of the Facility, demonstrate its value to the research community and significantly enhance future funding applications.

Henry Royce Equipment

The Kroto Confocal Facility is also home to the Lightsheet Z1 and Cellvizio confocal; two pieces of equipment from the Henry Royce Institute.

This equipment is available for use by industrial collaborators and academics within the Henry Royce Institute, as well as researchers here in Sheffield.

Please contact Dr Nicola Green or Royce@Sheffield.ac.uk if you require more information

Recent Publications

The following are some of the publications which include images or data obtained using the Kroto Imaging Facility

Publication year

2023, 2022, 2021, 2020, 2019, 2018, 2017, 2016, 2015, 2014, 2013, 2012, 2011, 2010, 2009

2023

Selectively inhibiting malignant melanoma migration and invasion in an engineered skin model using actin-targeting dinuclear Ru(II)-complexes. A. Raza, S.A. Archer, J.A. Thomas, S. MacNeil, J.W. Haycock. RSC Med Chem. 2023 14(1):65-73

Aligned Polyhydroxyalkanoate Blend Electrospun Fibers as Intraluminal Guidance Scaffolds for Peripheral Nerve Repair. C.S. Taylor, M. Behbehani, A Glen, P. Basnett, D.A. Gregory, B.B. Lukasiewicz, R. Nigmatullin, F. Claeyssens, I. Roy, J.W. Haycock ACS Biomater Sci Eng. 2023 epub ahead of print