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.

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 North Campus building and is part of the Department of Materials Science and Engineering. It is one on a number of analysis and characterisation facilities withing the Department, and is predominantly used by members of the Centre for Biomaterials and Tissue Engineering.

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.

Confocal laser scanning microscope

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.

Widefield Fluorescence Microscopy

There are now two inverted widefield fluorescent microscopes located within the Kroto Imaging Facility. These microscope are particularly good for imaging thin samples even with low levels of fluorescence. if you wish to use these microscopes there is no charge for users within the Kroto Tissue Engineering Group, and a £5 per hour charge for all other users. All users however must still book the facility using the Equipment Booking website.

There is a recently acquired IX73 Olympus microscope, with wLS LED illumination, Cairn MonoLED for brightfield imaging and a Retiga 6000 CCD camera. This micrscope currently 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

There is also a Leica microscope with a heated environmental chamber and the capability for controlling CO2 levels for longer term imaging of live cells. The Leica microscope can be used as a standard light microscope, for example for time lapse imaging of cells. It can generate both brightfield and phase contrast images. It also has a mercury lamp and the appropriate filters for imaging green, blue and red fluorophores.
How to use the Leica widefield microscope

In an epifluorescent microscope the excitation light, in this case from the mercury lamp, passes through the objective lens on the way to illuminating the sample, rather than passing first through the sample. A filter between the objective and the camera separates out the excitation light from emitted light. Most of the excitation light is actually transmitted through the specimen and therefore it is only any reflected excitatory light which reaches the objective together with the emitted light. This method therefore gives an improved signal to noise ratio.

Epifluorescence microscope schematic

It is actually very important to note that these are both inverted microscopes, with the objectives located below the stage and the images obtained by looking upwards into the sample and through any surface that the sample is sitting on.

Standard tissue culture plastic can only produce good images at lower magnifications,  since it is too thick when maging at higher magnifications where the objectives have much shorter working distances. Consequently, if you wish to obtain a good image at anything above 10x magnification, it is very important to have a the cells upon an appropriate surface with good optical properties.

This is particularly important when producing fluorescence images where both the excitation light and any emitted fluorescence passes through and is scattered and absorbed by whatever substrate the cells are sitting on. It is generally not possible to obtain a fluorescence image of any kind with the higher magnification objectives.
A number of products are available which have been developed for cell culture use and are optimised for imaging with inverted microscopes. Suitable products can be obtained from Ibidi, Thermoscientific and PAA

For training in use of the machine or any advice in this area please contact Dr Nicola Green

Confocal Imaging

What is 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.

Confocal Theory

In confocal imaging a point light source, generated by passing a laser light through an illumination pinhole, is used to illuminate a specimen. This ensures that only one point within a specimen is lit at any one time and therefore eliminates lateral light interference. Light from the sample is collected from a single focal plane. In the standard confocal set up this is achieved by using a pinhole in front of the detector to prevent light reflected or emitted from regions above and below the focal plane from entering the detector. This is called “optical sectioning”.

The size of the pinhole determines the thickness of the optical slice. Decreasing the pinhole reduces the thickness, but also decreases the strength of the signal reaching the detector.
A confocal system has:

- point light source for illumination
- pinhole at light source
- pinhole at image detector

These 3 points are optically conjugated together and hence described as confocal.

Schematic of confocal microscope

Fluorescent Imaging

When fluorophores are exposed to light of a specific wavelength some of their electrons are raised to a higher energy level. After a very short period of time they lose some of the energy before dropping drop back to their original level and emitting the remaining energy as light of a longer wavelength. This emitted light can then be detected as fluorescence.

When a single photon laser is focused upon a sample, the sample is exposed to the light at the focal point and also in the regions above and below this focal point. This exposure causes the fluorophores in all these regions to be excited and to fluoresce. The confocal microscope is then capable of preventing out of focus light from being detected using the pinhole as described above.

Jablonski diagram of single photon excitation and emissionSingle photon laser excitation and emission regions

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 micrometers will appear indistinct and out of focus because parts of the sample are outside the focal plane of the objective. 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
  • By changing the focal plane the user can obtain optical slices from increasing depths from within sample which 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
Two Photon Imaging

In two-photon imaging, samples are exposed to light at twice the normal excitation wavelength. In this situation two photons, hitting simultaneously from a femtosecond pulsed laser, are required. The two photons together raise the electron to a higher energy level after which it behaves in the same manner as that following single photon excitation.

The laser focal point is the only location along the optical path where the photons are crowded enough to generate significant occurrence of two-photon excitation. Consequently, two-photon imaging only activates fluorophores on the focal plane and a pinhole is not required as there is no out of focus fluorescence being generated.

It is worth noting that the two-photon excitation spectra for a given fluorophore can bear little resemblance to the corresponding single photon spectra. Although it does seem that most fluorophores function fairly well at twice the standard one photon excitation wavelength, there may be a number of other useful excitation wavelengths. This can extend the range of possible imaging parameters.

Jablonski diagram of two photon excitation and emissionTwo photon laser excitation and emission regions

As well as imaging molecules tagged with exogenous fluorescent labels, the two-photon laser can be used to excite indigenous fluorophores such as elastin, NAD(P)H, flavins and collagen which may be found within biological samples.

Second Harmonic Imaging

This is a relatively new imaging technique which can be performed using the laser scanning microscope in conjunction with the two-photon Ti-Sapphire laser. It is based on the optical effect of second harmonic generation (SHG), whereby laser light passing through molecules with a noncentrosymmetric structure (ie have no centre of symmetry) leads to the generation of light at exactly half the wavelength of the incident light. Some well-ordered biological materials such as collagen, muscle myosin and microtubules can be imaged in this way without the need for additional labelling.

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
Non-Descanned Detector (NDD)

The upright confocal microscope now has a non-descanned detector and two filter sets suitable for collecting light emitted by green and red fluorophores in conjunction with the two-photon laser. For details on how to use this detector download the instructions on the right hand side, or contact Dr. Nicola Green for more information.

The Benefits of Using an External or Non-Descanned Detector

The normal detectors on the confocal microscope are called de-scanned detectors. They collect the light emitted from the fluorophore using a system of dichroics, mirrors, filters and finally a pinhole in front of the detector to collect light of the required wavelength emitted from the focal plane and avoid out of focus light. The length of the light path that the emitted light has to travel is relatively long and the light therefore encounters more dust particles in the air which interfere with the signal. In addition the presence of the many optical elements and the pinhole cause a reduction in the amount of signal detected. This all reduces the signal to noise ratio and limits the depth within a sample from which a signal can be detected.

However, when using the two-photon laser to obtain images there is no requirement to use a pinhole in front of the detector to remove out of focus light, since only fluorophores within the focal plane are excited. Consequently it is possible to collect all the emitted light of the required wavelengths. This is why when using the de-scanned detectors in conjunction with the two-photon laser, the pinhole is set to fully open. This is one of the reasons why two-photon images can be obtained from greater depths within a sample. However, signal is still lost because of the presence of the optical elements in the emitted light path, together with the length of this path.

A non-descanned (external) detector has the shortest possible light path for the emitted light, avoiding the reducing signal loss. It also has no pinhole and fewer optical elements in the path. Consequently, the NDD system used in conjunction with two-photon excitation dramatically increases the amount of emitted light collected and provides a further increase in sample depth penetration.

The figure below shows the configuration for the NDD detector when imaging a green fluorophore.
The Zeiss confocal configuration for using the NDD on the upright microscope

A document describing the Bio-Rad NDD system, but which also provides a clear explanation of the NDD system in general and contains a good demonstration of the increased penetration possible using the NDD, is available to download: How to use NDD | The Benefits of Non-Descanned (External) Detectors in Multi-Photon Microscopy

Time-Correlated Single Photon Counting

There are three fundamental emission parameters that can be used in fluorescence microscopy:

1. Intensity
2. Wavelength
3. Lifetime

Most work utilises the first two of these - by either examining variations in fluorescence intensity across or between samples or by detecting specific fluorophores on the basis of their excitation and emission profiles.

However, fluorescence lifetime is much less used, in part due to the relative paucity of suitable stains, although this is changing. There are benefits to working with fluorescence lifetime. Unlike intensity measurements, it is generally independent of fluorophore concentration, allowing it to be calibrated absolutely.

It can also help to remove background autofluorescence. This can be an issue when, for example, imaging cells on collagen based and some synthetic scaffolds. It can be difficult to separate the scaffold fluorescence from that of the exogenous labels on the basis of wavelength. However, by using fluorophores with lifetimes in the microsecond range, differences in fluorescence lifetime can be exploited to separate the short lived (ns) autofluorescence from the fluorophores with longer lifetimes (µs).  

Graph showing short and long lived fluorescence over time

The upright microscope has the capability to perform time-correlated single photon counting (TCSPC) for phosphorescence and fluorescence lifetime imaging (PLIM and FLIM). This allows the user to generate an image based on fluorescence lifetime rather than wavelength. This can be seen below in an image of cells labelled with a platimum complex. The the image on the left is an intensity image, while that on the right shows the lifetime of the complex and it can be seen to differ between the nucleus and cytoplasm of the cells.

Fluorescent intensity and lifetime images of cells labelled with platinum complex

The method allows us to analyse the relaxation of a molecule from its excited state to a lower energy. After simultaneous excitation a fluorophore within a sample will return to the ground state with a certain probability, consequently the process has a decay rate, rather than a specific time scale. Our system utilises the two–photon pulsed laser as the light source and is capable of recording times when molecules emit photons following a laser pulse. This is repeated for additional excitation pulses until sufficient events have occurred to produce an intensity versus time graph that displays the exponential decay curve typical to these processes. From this, the fluorescence lifetime can be determined on a pixel by pixel basis.

It is possible to use the system at the Kroto Institute to perform both FLIM (nanosecond lifetimes) and PLIM (microsecond lifetimes) experiments. The choice will depend upon the lifetime of the fluorophores being used. Standard fluorescent molecules emit light with lifetimes around 0.1 to 10 ns, phosphorescent molecules have a  lifetime on the µs scale.

We have a modified confocal microscope which combines a Time-Correlated Single Photon Counting (TCSPC) detector with laser modulation of a 2 photon laser. The laser is pulsed on/off via a shutter and the time at which each photon arrives at the detector with respect to the laser pulse is determined. Fluorescence photons (obtained when the laser is on) are split from phosphorescent (laser off) and a phosphorescence decay curve is built up for each pixel through repeated scanning of the sample.

This apparatus uses a Becker and Hickl detector system (ref Proc. SPIE 7903, Multiphoton Microscopy in the Biomedical Sciences XI, 790320 (2011); doi:10.1117/12.875204).
The two photon laser is run at its normal rate and in the ns time period between pulses we are able to collect FLIM (fluorescence lifetime) data. The longer time scale PLIM (phosporescence lifetime) data is obtained by combining the same two photon laser with the electronic shutter which blocks off the laser for microsecond time periods (and this is variable depending on the settings used). The length of this window is adjusted by altering the laser modulation period. The use of a pulsed laser is not necessary for FLIM/PLIM, however, by using such a pulsed laser we can build up a greater triplet population than would be achieved with a single laser pulse at the same power, allowing us to run the experiment at lower powers.

The software controls the system via the pixel clock, which starts when the scan head moves to the next pixel. By ensuring that the pixel dwell time is aligned to the decay time window in the software we have been able to use up to 100 us pixel dwell times where the laser on time is 10 us and the decay window where PLIM data could be collected is 90 us.

If you would like to try either FLIM or PLIM imaging of appropriate samples please contact Nicola Green to discuss this in more detail

Meta Detector and Lambda Imaging

Both microscopes are connected to LSM 510 Meta Detectors. The light emitted from any multi labelled image will contain a mix of all the contributing dye components. However it is possible to use the Meta detector to apply the computational approach of spectral imaging to separate overlapping fluorescent spectra, or remove background autofluorescence.

The Meta detector is capable of collecting all emitted light from the sample rather than only from a specific bandwidth. The light from each pixel is spread into its component wavelengths and detected by separate photomultiplier tubes (PMT) within the Meta detector. The minimum possible bandwidth of each channel is 10.7 nm. The Meta detector can collect a maximum of 32 channels with up to 8 channels acquired at the same time. Through this process a “lambda stack” is acquired where each subsequent image in the stack represents the next band of the emission spectrum.

Schematic showing the light that would be collected from two fluorophores at different wavelengths

These lambda stacks can also be combined with Z-stacks and/or time series.

This diagram shows the response from a sample labelled with two overlapping fluorophores. The space between each red line represents the light detected by one PMT. Each separate PMT response would provide the information in one image of the lambda stack. For the first four lambda stack images, the response would arise from both flurorophores. The final image in the stack would contain the light emitted from only one of the probes.

Linear Unmixing

This is the most accurate method for spectral imaging. The Meta detector is used to produce an emission spectrum for each of the fluorophores used within a multi-stained image. It can also be used to obtain a similar spectrum for the autofluorescence. This is best done where control samples with only a single label, or only the background fluorescence are available. Once the individual emission spectra have been collected they can be used to identify and separate the emission spectra obtained from a multi-stained sample, by determining how much each of the probes contributes to the total signal at each pixel.

This process is also explained in some detail in the two documents available for downloading

ACE and Multichannel Unmixing

It is also possible to employ the Zeiss ACE software in conjunction with “lambda stack” images from the Meta detector to capture spectra from each fluorophore with multi-stained samples. This method is less accurate than the one described above but can still work well if the stains are well segregated spatially within the image. This can also be used with autofluorescent samples.

Alternatively multichannel unmixing can be performed on a lambda stack obtained from the Meta Detector or even a standard single image. Here the software attempts to automatically separate the emission responses obtained into a number of distinct channels. However this is the least accurate method of the three and cannot deal with autofluorescence.

Spectral Separation ofMultifluorescence Labels with theLSM 510 META

Emission Fingerprinting

Resolution

The resolution of an image is the minimum distance between two points where the points can still be detected as separate entities.

The resolution that can be achieved is determined by a number of factors

  1. The size of the pinhole used to prevent out of focus light entering the detector. With a smaller pinhole better resolution is achieved. This is true until the optimal diameter of 1 Airy unit is reached. Below this there is no improvement in resolution but the signal can still be reduced.
  2. The wavelength of light being used also affects resolution, with shorter wavelengths providing a greater resolution. For example for a fixed objective (with a numerical aperture of 0.95) light at a wavelength of 360 nm gives 0.19 µm resolution while 700 nm only gives 0.37 µm resolution. Consequently the resolution achievable through multi-photon excitation is less than that from single photon excitation.
  3. The numerical aperture (NA) of the objective used to obtain an image will also have a big effect upon the resolution that can be obtained. The NA of an objective is a measure of its ability to gather light from the sample and relates to the angle at which light enters the objective. The greater the angle, the higher the NA and the better the resolution.

NA = n x sin ϑ
where ϑ is one-half the angular aperture and n is the refractive index of the media between the objective and the coverslip (see diagram below).

Air has a refractive index of 1, water is 1.33 and immersion oil is generally around 1.51. Thus to obtain an NA greater than 1 and thus improve resolution it is necessary to use immersion objectives.

The brightness of an image at a fixed magnification is also increases with an increase in NA However, as the diagram also shows, an increase in NA results in a decrease in the focal length of the objective.

Good explanations of NA and the impact upon resolution can be found at
http://www.gonda.ucla.edu/bri_core/na.htm
http://micro.magnet.fsu.edu/primer/anatomy/numaperture.html

A demonstration of how the numerical aperture varies with the size of the illumination cone

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 fluorphore was used to generate an image, it is necessary to give the details for each fluorophore separately.

Experimental Officer Inclusion in the List of Authors

If the published work has required significant input from the Experimental Officer in terms of image acquisition, experimental design or data analysis, please remember to include Dr Nicola Green within the list of authors. Please contact Dr Green if you would like to discuss this further.

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.

Available Equipment

Lasers

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

Dichroic and Filter Nomenclature

Both microscopes posses a range of dichroic reflectors and filters. A dichroic reflector will reflect light of a specific wavelength or wavelengths while allowing other light to pass through. These ensure that the sample is exposed to light of the required wavelength and that light reaching the detectors is at the wavelength of interest. The software uses the following nomenclature to describe these components.

HFT - Main Dichroic beam splitter
HFT x/x/x - Reflects these x nm wavelengths and transmits all other
KP x - Short pass i.e. wavelengths less than x nm transmitted, greater than reflected
LP x - Long pass i.e. wavelengths greater than x nm transmitted, less than reflected
BP x-y - Band pass i.e. wavelengths between x and y transmitted, others reflected
NFT - Secondary Dichroic beam splitter (these are long pass)
NFT x - Wavelengths greater than x nm transmitted, less than reflected
NT 80/20 - 80% of all light at all wavelengths transmitted and remaining 20% reflected

When using the software to set up an imaging configuration, this information can be helpful for checking the beam path and the wavelengths being detected.
Here is a configuration used for the imaging of FITC labelled molecules. FITC has a peak excitation wavelength of 490 nm and peak emission at 525 nm. In this case the Argon laser at 488 nm can be used to excite the sample. By following the beam path it can be seen that laser light at 488 nm (and if there was any at 700 nm) is reflected by the primary dichroic HFT KP 700/488 onto the sample. The emitted light with a maxima at 525 nm passes up through the primary dichroic, but any stray 488 nm laser light reflected from the sample will not pass this filter. The mirror then reflects all the light towards a second mirror which then sends the light through a band pass filter, which permits light between 500 and 550 nm through to the Channel 2 detector. This includes the FITC emission peak at 525 nm.

Objectives

A number of different objectives are available for use. Different classes of objectives are suitable for different specimens. It is important when imaging that the correct objectives are used to ensure optimal image quality. This table below gives the recommended objective classes for particular types of biological specimens.

Information on suitable objectives to use

Below is a table of the objectives that are currently available to all users within the facility, with those fixed on the inverted scope marked #. For further information about specific objectives and objective classes see the Zeiss Objective Assistant. If you would like more help in selecting the appropriate objective for your needs please contact Dr Green.

Details of Objectives Currently Available within the Kroto Institute

Details of avialable objectives

Inverted LSM510 Meta Confocal Microscope

This microscope is ideal for imaging fixed samples and live cells, especially where maintaining sterility is a priority. It also has an environmental chamber which allows the temperature of the sample to be controlled. It is capable of employing the motorised stage to obtain images from consecutive areas and combine them into a single image of a larger area.

System Specifications

Axiovert 200M microscope
Inverted stage

Available Dichroics

NT 80/20, HFT 488/543/633/KP 725, HFT KP 700/488, HFT KP 700/543, HFT 458/514, HFT 458/543/633, HFT 488/543, HFT KP 660

PMT and Emission Filters

Channel D
Transmission DIC imaging
Channel 2
KP 660 IR, BP 390-465 IR, BP 435-485 IR, BP 465-510 IR, BP 500-530 IR, BP 500-550 IR
Channel 3
LP 505, LP 560, BP 500-530 IR, BP 500-550 IR, BP 520-555 IR, BP 535-590 IR, BP 560-615 IR, BP 650-710 IR
Meta Detector
Further 8 user defined detection bandwidth channels

Fluorescence correlation spectroscopy (FCS)

This microscope is also connected to a ConfoCor2 module. This allows multi-point fluorescence correlation spectroscope (FCS), enabling users to study the dynamic properties of diffusion and chemical reaction rates.

Axiovert 200M microscope manual

LSM 510 Concocor Manual

Upright LSM510 Meta Confocal Microscope

This microscope, used in conjunction with the water dipping objectives, is ideal for imaging live cells and tissues. It is also good for imaging fixed samples and slides.

System Specifications

Axioskop 2 FS mot microscope
Upright stage
Available Dichroics

NT 80/20, HFT 488/543/633, HFT KP 700/488, HFT KP 700/543, HFT 458/514, HFT 458/543/633, HFT 488/543, HFT KP 650
PMT and Emission Filters

Channel D
Transmission DIC imaging
Channel 2
KP 685, BP 390-465 IR, BP 435-485 IR, BP 485-520 IR, BP 500-530 IR, BP 500-550 IR
Channel 3
LP 505, LP 560, BP 500-530 IR, BP 500-550 IR, BP 535-590 IR, BP 565-615 IR, BP 650-710 IR
Meta Detector
Further 8 user defined detection bandwidth channels.

Axioskop Manual

LSM510 Meta Manual

Axioskop 2 FS Microscope Brochure

Henry Royce Equipment

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

This equpment 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

Lightsheet Z1

Funding from the Henry Royce Institute has allowed us to purchase a Lightsheet Z1 microscope for imaging of relatively large, living samples that are fluorescently labelled in some way. The equipment has incubation capabilities and so can be used with live samples. Due to licence limitations, this facility cannot be used for imaging living organisms, but is suitable for viewing live cells or samples ex vivo or those grown in the laboratory. It can also be used to image fixed samples that do not require incubation.

Picture of lightsheet equipment

There are 5 laser lines available with this instrument (405 nm, 488 nm, 514 nm, 561 nm and 638 nm) and the equipment is suitable for imaging whole samples up to approx. 200 µm thick.
The Lightsheet comes with 3 detection objectives (5x, 10x and 20X) along with a zoom facility which allows between 0.3 x and 2.5 x magnification. As a result the equipment can achieve magnifications between 1.5x and 50x.

The light sheet can be booked through the confocal facility booking pages. If you require access to this, more information or training for use of the equipment please contact Dr Nicola Green

Cellvizio Dual Band In Vivo Confocal

Through the Henry Royce Institute funding we now have available a confocal laser system designed for visual inspection of tissues using a fibre optic probe. It is capable of generating two colour images using 488 nm and 660 nm laser excitation. Detection can be achieved at 502-633 nm and 673-800 nm. The device can be used with 488 nm excitation only, 660 nm excitation only or both.

Photo of Celvizio confocal microscope

The fibre optic probe is put into contact with the tissue being imaged and can be used on in vivo, ex vivo or laboratory engineered tissue. Users have 3 probes available to them for use with this system.

S1500
This is a general use probe used for surface imaging of the sample.
MiniZ
This probe obtains images from within the sample rather than the surface.
UltraMiniO
This probe is capable of generating higher resolution images than the other two. These images are also obtained from within the sample rather than at the sample surface.

Please note that the probes cannot be autoclaved, but can be cleaned carefully with ethanol. This means that they are not recommended for use on samples during the culture period as they are likely to cause bacterial contamination of your sample, but they can be used at the end of an experiment when you no longer intend to return your samples to an incubator.
The characteristics of the probes are described in more detail in the table below.

Table showing key characterisitcs of optical fibre probes available

Users can also access software for data analysis and image reconstruction. Software is available for image mosaicing, vessel detection and movie handling (IC viewer). Please contact Dr Nicola Green for more information

Recent Publications

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

Modulation of neuronal cell affinity of composite scaffolds based on polyhydroxyalkanoates and bioactive glasses. L.R. Lizarraga-Valderrama, R. Nigmatullin, B. Ladino, C.S. taylor, A.R. Boccaccini, J.C. Knowles, F. Claeyssens, J.W. Haycock, I. Roy. Biomed Mat (2020) 15:045024

A Dinuclear Ruthenium(II) Complex Excited by Near-Infrared Light through Two-Photon Absorption Induces Phototoxicity Deep within Hypoxic Regions of Melanoma Cancer Spheroids. A. Raza, S.A. Archer, S.D. Fairbanks, K.L. Smitten, S.W. Botchway, J.A. Thomas, S. MacNeil, J.W. Haycock. J Am Chem Soc (2020) 142:4639-4647

Biomimetic surface delivery of NGF and BDNF to enhance neurite outgrowth. A.M. Sandoval-Castellanos, F. Claeyssens, J.W. Haycock. Biotech Bioeng (2020) 1-12

Carbon dot-protoporphyrin IX conjugates for improved drug delivery and bioimaging. J.R. Aguilar Cosme, H.E. Bryant, F. Claeyssens. PLOS ONE (2019) 14(7): e0220210

A Novel Bilayer Polycaprolactone Membrane for Guided Bone Regeneration: Combining Electrospinning and Emulsion Templating. B. Aldemir Dikici, S. Dikici, G.C. Reilly, S. MacNeil, F. Claeyssens. Materials (2019) 12(16):2643

A dinuclear ruthenium(II) phototherapeutic that targets duplex and quadruplex DNA. S. Archer, A. Raza, F, Drohe, C. Robinson. A.J. Auty, D. Chekulaev, J.A. Weinstein, T. Keane, A.J.H.M. Meijer, J.W. Haycock, S. MacNeil, J.A. Thomas. Chem Sci (2019) 10:3502-3513

Slow polymer diffusion on brush-patterened surfaces in aqueous solution. C.G. Clarkson, A. Johnson. G.J. Leggett, M. Geoghegan. Nanoscale (2019) 11:6052-6061

Unidirectional neuronal cell growth and differentiation on aligned polyhydroxyalkanoate blend microfibres with varying diameters. L.R. Lizarraga-Valderrama, C.S. Taylor, F. Claeyssens, J.W. Haycock, J. Knowles, I. Roy. J Tissue Eng Regen Med (2019) 13:1581-1594

Two photon excitable graphene quantum dots for structured illumination microscopy and imaging applications: lysosome specificity and tissue-dependent imaging. H. Singh, S. Sreedharan, K. Tiwari, N.H. Green, C. Smythe, S.K. Pramanik, J.A. Thomas, A. Das. Chem Comm (2019) 55:521-524

Pre-clinical evaluation of advanced nerve guide conduits using a novel 3D in vitro testing model. M. Behbehani, A. Glen, C.S. Taylor, A. Schuhmacher, F. Claeyssens, J.W. Haycock, Int J Bioprint (2018) 4(1):UNSP 123

Composite porous scaffold of PEG/PLA support improved bone matrix deposition in vitro compared to PLA-only scaffolds. B. Bhaskar, R. Owen, H. Bahmaee, Z. Wally, P. Sreenivasa Rao, G.C. Reilly, J Biomed Mat Res (2018) 6(5):1334-1340

An Improved In Vivo Methodology to Visualise Tumour Induced Changes in Vasculature Using the Chick Chorionic Allantoic Membrane Assay. N. Mangir, A. Raza, J.W. Haycock, C. Chapple, S. MacNeil,  In Vivo (2018) 32(3):461-472

Synthesis, Characterization and 3D Micro-Structuring via 2-Photon Polymerization of Poly(glycerol sebacate)-Methacrylate-An Elastomeric Degradable Polymer. S. Pashneh-Tala, R. Owen, H. Bahmaee, S. Rekstyte, M. Malinauskas, F. Claeyssens Front Phys (2018) 6:41

Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis. T.E. Paterson, G. Gigliobianco, C. Sherborne, N.H. Green, J.M. Dugan, S. MacNeil, G.C Reilly, F. Claeyssens, APL Bioengineering (2018) 2:026103

Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits. D. Singh, A.J. harding, E. Albadawi, F.M. Boissonade, J.W. Haycock, F. Claeyssens. Acta Biomateriala (2018) 78:48-63

Measurement of friction-induced changes in pig aorta fibre organization by non-invasive imaging as a model for detecting the tissue response to endovascular catheters. L. Bostan, C. Noble, N. Smulders, R. Lewis, M.J. Carre, S.E. Franklin, N.H. Green, S. MacNeil, Biotribology (2017) 12:24

Poly(n-butyl Methacrylate) with Primary Amine End Groups for Supporting Cell Adhesion and Proliferation of Renal Epithelial Cells. K. Cox-Nowak, O. Al-Yamani, C.A. Grant, N.H. Green, S. Rimmer, Int J Polymeric Mat Polymetic Biomat (2017) 66(15):762-767

Development of a UV crosslinked biodegradable hydrogel containing adipose derived stem cells to promote vascularization for skin wounds and tissue engineering. G. Eke, N. Mangir, N. Hasirci, S. MacNeil, V. Hasirci, Biomaterials (2017) 129:188-198

A new mode of contrast in biological second harmonic generation microscopy. N.H. Green, R. Delaine-Smith, H.J. Askew, R. Byers, G.C. Reilly, S.J. Matcher, Sci Rep (2017) 7:13331

Selective laser-melting enabled electrospinning: Introducing complexity within electrospun membranes. T.E. Paterspn, S.N. Beal, M.E. Santocildes-Romero, A.T. Sidambe, P.V. Hatton, I.O. Ascencio. Proc Insti Mech Eng Part H - J Eng in Med (2017) 231:565-574

Imaging cellular trafficking processes in real time using lysosome targeted up-conversion nanoparticles. S.K. Pramanik, S. Sreedharan, H. Singh, N.H. Green, C. Smythe, J. Thomas, A. Das, Chem Com (2017) 53:12672

Oxygen Mapping of Melanoma Spheroids using Small Molecule Platinum Probe and Phosphorescence Lifetime Imaging Microscopy. A. Raza, H.E. Colley, E. Baggaley, I.V. Sazanovich, N.H. Green, J.A. Weinstein, S.W. Botchway, S. MacNeil, J.W. Haycock, Sci Rep (2017) 7:10743

Investigating Neovascularization in Rat Decellularized Intestine: An In Vitro Platform for Studying Angiogenesis. L. Dew, W.R. English, C.K. Chong, S. MacNeil, Tissue Eng Part A (2016) 22(23-24):1317-1326

Fabrication of Biodegradable Synthetic Vascular Networks and Their Use as a Model of Angiogenesis. L. Dew, W.R. English, I. Ortega, F. Claeyssens, S. MacNeil, Cells Tissues Organs (2016) 202(5-6):319-328

Antimicrobial Graft Copolymer Gels. A.C. Harvey, J. Madsen, C.W.I Douglas, S. MacNeil, S.P. Armes, Biomacromolecules (2016) 17(8):2710-2718

Developing Repair Materials for Stress Urinary Incontinence to Withstand Dynamic Distension. C.J. Hillary, S. Roman, A.J. Bullock, N.H. Green, C.R. Chapple, S. MacNeil, PLoS ONE (2016) 11(3):e0149971

Photochemically modified diamond-like carbon surfaces for neural interfaces A.P. Hopper, J.M. Dugan, A.A. Gill, E.M. Regan, J.W. Haycock, S. Kelly, P.W. May, F. Claeyssens, Mat Sci Eng C (2016) 58(1):1199–1206

Second Harmonic Generation microscopy reveals collagen fibres are more organised in the cervix of postmenopausal women. B.F. Narice, N.H. Green, S. MacNeil, D. Anumba, Reprod Biol Endocrinol (2016) 14(1):70

Controlled peel testing of a model tissue for diseased aorta. C. Noble, N. Smulders, R. Lewis, M.J. Carre, S.E Franklin, S. MacNeil, Z.A. Taylor, J Biomech (2016) 49(15):3667-3675

Creating a model of diseased artery damage and failure from healthy porcine aorta. C. Noble, N. Smulders, N.H. Green, R. Lewis, M.J. Carre, S.E. Franklin, S. MacNeil, Z.A. Taylor, J Mech Behav Biomed Mat (2016) 60:378-393

Production and characterization of a novel, electrospun, tri-layer polycaprolactone membrane for the segregated co-culture of bone and tissue. S. Puwanun, F.J. Bye, M.M. Ireland, S. MacNeil, G.C. Reilly, N.H. Green, Polymers (2016) 8(6):221-229

Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications. G. Stevenson, S. Rehman, E. Draper, E, Hernandex-Nava, J. Hunt, J.W. Haycock, Biotech Bioeng (2016) 113(7):1586-1599

Inkjet printing Schwann cells and neuronal analogue NG108-15 cells. C. Tse, R. Whitely, T. Yu, J. Stringer, S. MacNeil, J.W. Haycock, P.J. Smith, Biofabrication (2016) 8(1):015017

Utilising Inkjet Printed Paraffin Wax for Cell Patterning Applications C.C.W. Tse, S.S. Ng, J. Stringer, S. MacNeil, J.W. Haycock, P.J. Smith, Int J Bioprint (2016) 2(1) doi:10.18063/ijb.2016.01.001

Decellularisation and histological characterisation of porcine peripheral nerves. L. Zilic, S.P. Wilshaw, J.W. Haycock, Biotech Bioeng (2016) 113(9):2041-2053

Characterization of diblock copolymer order-order transitions in semi-dilute aqueous solution using fluorescence correlation spectroscopy C.G. Clarkson, J.R. Lovett, J. Madsen, S.P. Armes, M. Geoghegan, Macromol Rapid Commun (2015) 36(17):1572-1577

Rocking media over ex vivo corneas improves this model and allows the study of the effect of proinflammatory cytokines on wound healing. P. Deshpande, Í. Ortega, F. Sefat, V.S. Sangwan, N. Green, F. Claeyssens, S. MacNeil, Invest Ophthalmol Vis Sci (2015) 56(3):1553-1561

Thermoresponsive, stretchable, biodegradable and biocompatible poly(glycerol sebacate)-based polyurethane hydrogels. M. Frydrych, S. Román, N.H. Green, S. MacNeil, B. Chen Polym Chem (2015) 6:7974-7987

Simple surface coating of electrospun poly-L-lactic acid scaffolds to induce angiogenesis. G. Gigliobianco, C.K. Chong, S. MacNeil, J Biomater Appl (2015) 30:50-60

Towards the fabrication of artificial 3D microdevices for neural cell networks. A.A. Gill, Í Ortega, S. Kelly, F. Claeyssens, (2015) 17(2):27

Nerve tissue engineering using blends of poly(3-hydroxyalkanoates) for peripheral nerve regeneration. L.R. Lizarraga-Valderrama, R. Nigmatulin, C. Taylor, J.W. Haycock, F. Claeyssens, J.C. Knowles, I Roy Eng Life Sci (2015) 15(6):612-621

Fabrication of biodegradable synthetic perfusable vascular networks via a combination of electrospinning and robocasting. Í. Ortega, L. Dew, A.G. Kelly, C.K. Chong, S. MacNeil, F. Claeyssens, Biomater Sci. (2015) 3(4):592-596

Emulsion templated scaffolds with tunable mechanical properties for bone tissue engineering. R. Owen, C. Sherborne, T. Paterson, N.H. Green, G.C. Reilly, F. Claeyssens, J Mech Behav Biomed Mater (2015) 54:159-172

Nerve guides manufactured from photocurable polymers to aid peripheral nerve repair. C.J. Pateman, A.J. Harding, A. Glen, C.S. Taylor, C.R. Christmas, P.P. Robinson, S. Rimmer, F.M. Boissonade, F. Claeyssens, J.W. Haycock, Biomaterials (2015) 49:77-89

Arginine–glycine–aspartic acid functional branched semi-interpenetrating hydrogels. R.A. Plenderleith, C.J. Pateman, C. Rodenburg, J.W. Haycock, F. Claeyssens, C. Sammon, S. Rimmer, Soft Matter (2015) 11(38):7567-78

An anatomical study of porcine peripheral nerve and its potential use in nerve tissue engineering. L. Zilic, P.E. Garner, T. Yu, S. Román, J.W. Haycock, S.-P. Wilshaw, J Anat (2015) 227(3):302-314

Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission Microscopy Probes for Cellular DNA. E. Baggaley, M.R. Gill, N.H. Green, D. Turton, I.V. Sazanovich, S.W. Botchway, C. Smythe, J.W. Haycock, J.A. Weinstein, J.A. Thomas, Angew Chem Int Ed Engl (2014) 53(13):3367-3371

Two-photon phosphorescence lifetime imaging of cells and tissues using a long-lived cyclometallated Npyridyl^Cphenyl^Npyridyl Pt(II) complex. E. Baggaley, I.V. Sazanovich, J.A.G. Williams, J.W. Haycock, S.W. Botchway, J. Weinstein, RSC Advances (2014) 4:35003-35008

Monitoring Fibrous Scaffold Guidance of Three-Dimensional Collagen Organisation Using Minimally-Invasive Second Harmonic Generation. R. Delaine-Smith, N.H. Green, S.J. Matcher, S. MacNeil, G.C. Reilly, PLOS ONE (2014) 9(2):0089761

Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts. R.M. Delaine-Smith, A. Sittichokechaiwut, G.C. Reilly, FASEB J (2014) 28(1):430-439

Amine functional hydrogels as selective substrates for corneal epithelialization. E. Hassan, P. Deshpande, F. Claeyssens, S. Rimmer, S. MacNeil, Acta Biomater (2014) 10(7):3029-3037

Amine functionalized nanodiamond promotes cellular adhesion, proliferation and neurite outgrowth. A.P. Hopper, J.M. Dugan, A.A. Gill, O.J.L. Fox, P.W. May, J.W. Haycock, F. Claeyssens, Biomed Mater (2014) 9(4):045009

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: to enhance the efficiency of recombinant DNA utilization. O.L. Mozely, B.C. Thompson, A. Fernandez-Martell, D.C. James, Biotechnol Prog (2014) 30(5):1161-1170

Combination of microsterolithography and electrospinning to produce membranes equipped with niches for conreal regeneration. I. Ortega, F. Sefat, P. Deshpande, T. Paterson, C. Ramachandran, A.J. Ryan, S MacNeil, F. Claeyssens, J Vis Exp (2014) 12(91):51826

Development of a one-step approach for the reconstruction of full thickness skin defects using minced split thickness skin grafts and biodegradable synthetic scaffolds as a dermal substitute. K. Sharma, A. Bullock, D. Ralston, S. MacNeil, Burns (2014) 40(5):957-965

The development of a 3D immunocompetent model of human skin. D.Y. Chau, C. Johnson, S. MacNeil, J.W. Haycock, A.M. Ghaemmaqhami, Biofabrication (2013) 5(3):035011

Simplifying corneal surface regeneration using a biodegradable synthetic membrane and limbal tissue explants. P. Deshpande, C. Ramachandran, F. Sefat, I. Mariappan, C. Johnson, R. McKean, M. Hannah, V.S. Sangwan, F. Claeyssens, A.J. Ryan, S. MacNeil, Biomaterials (2013) 34(21):5088-5106

Generic methods for micrometer- and nanometer-scale surface derivatization based on photochemical coupling of primary amines to monolayers of aryl azides on gold and aluminum oxide surfaces O. El Zubir, I. Barlow, E. Ul-Haq, H.A. Tajuddin, N.H. Williams, G.J. Legget, Langmuir (2013) 29(4):1083-1092

Development of a microfabricated artifical limbus with micropockets for cell delivery to the cornea. I. Ortega, P. Deshpande, A.A. Gill, S. MacNeil, F. Claeyssens, Biofabrication (2013) 5(2):025008

Combined microfabrication and electrospinning to produce 3-D architectures for corneal repair. I. Ortega, A.J. Tyan, P. Deshpande, S. MacNeil, F. Claeyssens, Acta Biomaterialia (2013) 9(3):5511-5520

Laser exposure of gold nanorods can induce intracellular calcium transients. C. Paviolo, J.W. Haycock, P.J. Cadusch, S.L. McArthur, P.R. Stoddart, J Biophotonics (2013) 7(10):761-765

Laser exposure of gold nanorods can increase neuronal cell outgrowth. C. Paviolo, J.W. Haycock, J. Yong, A. Yu, P.R. Stoddart, S.L. McArthur, Biotech Bioeng (2013) 110(8):2277-2291

The diffusion of dextran within poly(methacrylic acid) hydrogels. A.M. AL-Baradi, M. Mears, R.A.L. Jones, M. Geoghegan, J Polym Sci B: Polym Phys (2012) 50:1286-1292

An aligned 3D neuronal-glial co-culture model for peripheral nerve studies. M.F.B. Daud, K.C. Pawar, F. Claeyssenss, A.J. Ryan, J.W. Haycock, Biomaterials (2012) 33(25):5901-5913

Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus. R.M. Delaine-Smith S. MacNeil, G.C. Reilly, Eur Cell Mater (2012) 24:162-174

Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue R. Kaewkhaw, A.M. Scutt, J.W. Haycock, Nature Protocols (2012) 7:1996-2004

Two-photon polymerization-generated and micromolding-replicated 3D scaffolds for peripheral neural tissue engineering applications A. Koroleva, A.A. Gill, I. Ortega, J.W. Haycock, S. Schlie, S.D. Gittard, B.N. Chichkov, F. Claeyssens, Biofabrication (2012) 4:025005

Micrometer and Nanometer Scale Photopatterning of Proteins on Glass Surfaces by Photo-degradation of Films Formed from Oligo(Ethylene Glycol) Terminated Silanes G. Tizazu, O. El Zubir, S. Patole, A. McLaren, C. Vasilev, D.J. Mothersole, A. Adawi, D.G. Lidzey, G.P. Lopez, G.J. Legget, Biointerphases (2012) 7(1-4):54-63

Protein Micro- and Nanopatterning Using Aminosilanes with Protein-Resistant Photolabile Protecting Groups. S.A. Alang Ahmad, L.S. Wong, E. ul-Haq, J.K. Hobbs, G.J. Leggett, J. Micklefield, J Am. Chem. Soc. (2011) 133(8):2749-2759

Magnetic field dependence of the diffusion of single dextran molecules within a hydrogel containing magnetite nanoparticles. A.M. Al-Baradi, O.O. Mykhaylyk, H.J. Blythe, M. Geoghegan, J Chem Phys (2011) 134:094901

Biocompatible hydrogels based on hyaluronic acid cross-linked with a polyaspartamide derivative as delivery systems for epithelial limbal cells. C. Fiorica, R.A. Senior, G. Pitarresi, F.S. Palumbo, G. Giammona, P. Deshpande, S. MacNeil, Int J Pharm (2011) 414(1-2):104-111

Ruthenium(II) metallo-intercalators: DNA imaging and cytotoxicity. M.R. Gill, H. Derrat, C.G. Smythe, G. Battaglia, J.A. Thomas, Chembiochem (2011) 12(6):887-880

NF-kB is activated in oesophageal fibroblasts in response to a paracrine signal generated by acid-exposed primary oesophageal squamous cells. N.H. Green, Q. Huang, B.M. Corfe, J.P. Bury, S. MacNeil, Int J Exp Pathol  (2011) 92(5):345-356

Tracking nanoparticles in three-dimensional tissue-engineered models using confocal laser scanning microscopy. V. Hearnden, S. MacNeil, G. Battaglia, Methods Mol Biol (2011) 695:41-51

Anatomical site influences the differentiation of adipose-derived stem cells for Schwann-cell phenotype and function. R. Kaewkhaw, R.M. Scutt, J.W. Haycock, Glia (2011) 59(5):734-749

Direct laser writing of 3D scaffolds for neural tissue engineering applications. V. Melissinaki, A.A. Gill, I. Ortega , M. Vamvakaki, A. Ranella A, J.W. Haycock, C. Fotakis, M. Farsari, F. Claeyssens, Biofabrication (2011) 3(4):045005

Three-dimensional alignment of schwann cells using hydrolysable microfiber scaffolds: strategies for peripheral nerve repair. C. Murray-Dunning, S.L. McArthur, T. Sun, R. McKean, A.J. Ryan, J.W. Haycock, Methods Mol Biol (2011) 695:155-166

Degradation, Bioactivity, and Osteogenic Potential of Composites Made of PLGA and Two Different Sol–Gel Bioactive Glasses. E. Pamula, J. Kokoszka,K. Cholewa-Kowalska, M. Laczka, L. Kantor, L. Niedzwiedzki, G.C. Reilly, J. Filipowska, W. Madej, M. Kolodziejczyk, G. Tylko, A.M. Osyczka, Ann Biomed Eng (2011) 39(8):2114-2129

Protein Patterning by UV-Induced Photodegradation of Poly(oligo(ethylene glycol) methacrylate) Brushes. S.A. Alang Ahmad, A. Hucknall, A. Chilkoti, G.J. Leggett, Langmuir (2010) 26(12):9937-9972

Biofilm formation in environmental bacteria is influenced by different macromolecules depending on genus and species. J.S. Andrews, S.A. Rolfe, W.E. Huang, J.D. Scholes, S.A. Banwart, Environ Microbiol (2010) 12(9):2496-2507

Using poly(lactide-co-glycolide) electrospun scaffolds to deliver cultured epithelial cells to the cornea. P. Deshpande, R. McKean, K.A. Blackwood, R.A. Senior, A. Ogunbanjo, A.J. Ryan, S. MacNeil, Regen. Med. (2010) 5(3):395-401

Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes. M. Massignani, I. Canton, T. Sun, V. Hearnden, S. MacNeil, A. Blanazs, S.P. Armes, A. Lewis, G. Battaglia, PLoS One (2010) 5(5):e10459

A micro-incubator for cell and tissue imaging. C. Picard, V. Hearnden, M. Massignani, S. Achouri, G. Battaglia, S. MacNeil, A. Donald, Biotechniques (2010) 48:135-138

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering. H.L. Morris, C.I. Reed, J.W. Haycock, G.C. Reilly, Proc Inst Mech Eng H (2010) 224(12):1509-21

Intrinsic extracellular matrix properties regulate stem cell differentiation. G.C. Reilly, A.J. Engler, J Biomech (2010) 43(1):55-62

Short bouts of mechanical loading are as effective as dexamethasone at inducing matrix production by human bone marrow mesenchymal stem cells. A. Sittichokechaiwut, J.H. Edwards, A.M. Scutt, G.C. Reilly, Eur Cell Mater (2010) 20:45-57

Directed single molecule diffusion triggered by surface energy gradients. P. Burgos, Z. Zhang, R. Golestanian, G.J. Leggett, M. Geoghegan, ACS Nano (2009) 3:3235-43

A ruthenium(II) polypyridyl complex for direct imaging of DNA structure in living cells. M.R. Gill, J Barcia-Lara, S.J. Foster, C. Smythe, G. Battaglia, J.A. Thomas, Nat Chem (2009) 1:662-667

Diffusion Studies of Nanometer Polymersomes Across Tissue Engineered Human Oral Mucosa. V. Hearnden, H. Lomas, S. MacNeil, M. Thornhill, C. Murdoch, A. Lewis, J. Madsen, A. Blanazs, S. Armes, G. Battaglia, Pharmaceutical Research (2009) 26:1718-1728

Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes. S.W. Botchway, M. Charnley, J.W. Haycock, A.W. Parker, D.L. Rochester, J.A. Weinstein, J.A. Williams, Proc Natl Acad Sci USA (2008) 105(42):16071-6

If you have published any data/images acquired using the Kroto Imaging Facility, please send the details to Dr Nicola Green, so it can be included in this list

Registering as a New User

The confocal facility is available for use as a service facility, where samples are brought for imaging by the Experimental Officer. If you wish to use the facility in this way please contact Dr Green to discuss your requirements. However, due to time limitations this option is only available for single experiments with very small sample numbers.
Alternatively, individuals can be trained as independent users, allowing them to book space and access the equipment as required. If an individual wishes to access the confocal and intends to use the equipment without working under the complete supervision of a registered user they must register and be trained as a confocal user. This option is recommended particularly if you have a large number of samples to image or expect that your research will require repeated use of the facility. In all cases support and advice is available from the Experimental Officer.

In-hours (8am - 6pm) access to the Kroto Research Institute no longer requires a swipe card. You only need to request swipe card access for out of hours use of the equipment. Out of hours access can be obtained using your CICS login at https://bars.shef.ac.uk/. Please select the option "Kroto main/link bridge 24/7". All requests require up to date fire training and out of hours training. To obtain the appropriate health and safety training please visit https://hs.sheffieldf.ac.uk/.

Laser Safety

Safety Training
Since the confocal facility has class 3B and class 4 lasers, all confocal users must also be registered laser users. The standard single photon lasers are all part of the Class 3B LSM510 laser module, which has an average power output up to 100 mW at 458 nm. The two-photon laser is a Ti-Sapphire 600-1000 nm tuneable Class 4 laser with an average power output of 2000 mW.

Users must obtain laser safety training and this can be completed online via https://hs.sheffield.ac.uk. Login with your CICS username and password and work through all three laser training modules within the radiation section, study the local rules and then do the assessment.

Undergraduate and taught masters students will not be able to access the online course. Any students who need to use the confocal microscope as part of their final year project will need to contact Nicola Green separately to obtain access to a copy of the laser safety training course.

Laser User Registration
You must also register as a laser user using the online Laser Work and Users database. If you are a new laser user you should first click "apply" to request access via this website. Please then complete the online form by entering your personal details. Note - there is no need to upload training documents unless you are wanting to apply using your training from another institution, however please write "For confocal use" in the "Training" text box.

IMPORTANT: In the dropdown section "Department", please select your home department, and in the section "Show Lasers from" please select  Materials - Kroto/NC.

You must also indicate the laser(s) to be used. The lasers have been named on the system as shown in the table below and these numbers should be used when you apply to register as a laser user. You will first need to know if you are going to be using the upright or inverted microscopes or both, then you must register to use all the class 3B lasers that are indicated for use on the appropriate microscope. However, DO NOT register for two photon class 4 laser use unless you have already received training with this laser or have discussed your requirements to use this laser with me.

Laser ID

Laser details

MNC-16 Argon 458, 477, 488, 514 nm laser - upright confocal (Class 3B)
MNC-17 HeNe 543 nm laser - upright confocal (Class 3B)
MNC-18 HeNe 633 nm - upright confocal (Class 3B)
MNC-19 Titanium-sapphire two photon laser (Class 4)
MNC-20 Argon 458, 477, 488, 514 nm laser - inverted confocal (Class 3B)
MNC-21 HeNe 543 nm laser - inverted confocal (Class 3B)
MNC-22 HeNe 633 nm - inverted confocal (Class 3B)

If you need further information to complete this step please contact Dr Green.

Please note  - if you are already registered as a laser user you must modify your details to indicate that you now intend to also use the lasers in S19.

Fire Safety, COSHH and Risk assessments

All users must also have fire training, which should be updated annually. In addition, people requiring access to the equipment out-of-hours must fulfil all the out-of-hours requirements for the Kroto Institute, including possessing out-of-hours training and requesting out-of hours access on the booking site. All work must have the appropriate COSHH and risk assessments related to the work. There is an exisiting risk assessment for confocal work that users can modify for their own needs (ID 10955).

Confocal Training

All new independent users must complete a New User Registration form. Users must also complete an on-line theory course. As part of this you must complete the virtual microscope sections and complete the test at the end of the course and bring the certificate to the practical training session.

Training will not be provided until the registration form has been completed, signed by the project supervisor/grant holder, a grant number provided for the work and the on-line training has been completed.

Training is on a one-to-one basis for about 2 hours. After this training the user is encouraged to work more independently under the guidance of the Experimental Officer.
One-to-one training is charged at the standard price for confocal use - i.e. £32.50 per hour, and trainees are encouraged to supply a sample of the type and with the sorts of stains that they plan to be using in the future.

General training and support is provided by Robert Dickinson and you should contact him to arrange your inital training. If you have more complex needs please contact Dr Nicola Green to discuss your requirements further.

Supported User Request Form

Confocal Facility User Policy

Confocal Booking and Charges

Access to the Kroto Research Institute and Confocal Facility

The Kroto Research Institute is open during office hours (8am - 6pm). Out of hours access is by swipe card using your Sheffield University uCard. If you would like to request your uCard is activated to provide access to the KRI please go through the online Building Access Request System. Please note that if you wish to request out of hours access you will need to have up to date fire and out of hours training.

The entrance to the Confocal Facility itself is via the main door into the second floor lab, S20. Access to S20 requires an activated ibutton. Individuals who already have an ibutton but require it activating to access the S20 area should contact the technical manager of this laboratory, Vanessa Singleton. For users without an ibutton, there are some which can be borrowed for the duration of the booking but must be returned after use. These can be obtained from either Dr Nicola Green (room F07) or the technical staff (room S10).

Charges

Please note that there is a charge for the use of the facility on an hourly rate. This is to ensure that the facility is used in a prudent and well managed manner and bookings are limited to the time required, providing reasonable access for all users. The charge does not fully cover the costs of running, servicing and supporting the facility but is intended to recoup some of these costs. Please ensure that the grant holder/supervisor of the project is aware that you are using the facility and that they will be charged.

On booking the facility, users provide a grant number against which this charge will be made.

All individuals will be charged £33.00 per hour for use of the confocal microscopes within the facility. The same charge applies to both Kroto members and other individuals from within the University.

The widefield fluorescence microscope is free to users within Tissue Engineering, and is charged at £5 per hour for all other users. However all use whether free or charged must be booked using the equipment booking website.

The offline PC and analysis software can be used free of charge and if required at a specific time it is recommended that it is booked via the website. Anyone who has booked a particular time on the PC will have priority over someone using the PC without such a booking.

How to Book the Facility

Independent Confocal Use
All independent users are required to book the facility using the Equipment Booking Website. To do this you will require a username and password, which will be provided after the new user registration and training has been completed (see above). This will allow you to make and edit your own bookings by simply logging in and selecting the times and the specific microscope that you require. If you require the two-photon facility, remember to book this along with the required microscope.

Supported Confocal Use
All other individuals should view the booking site to determine confocal availability and contact Dr Nicola Green to arrange bookings at mutually appropriate times. It is possible to provide samples for imaging by the Experimental Officer. In this case you are expected to be present during the imaging process and you will need to complete the Supported User Request Form prior to any work commencing.

Use of widefield fluorescence microscope
If you wish to book the widefield Olympus or Leica fluorescent microscopes but have not got a username and password for the booking website please contact Dr Nicola Green or Robert Dickinson to arrange a booking.

Please note the following conditions when booking equipment:-

  1. All bookings must provide the grant number against which the facility charge will be made, failure to do so without prior arrangement may result in bookings being cancelled
  2. Bookings cannot be made more than 4 weeks in advance without permission from the Experimental Officer
  3. Any booking which is cancelled more than 24 hours before the start time will not incur a charge, unless a user does this repeatedly in a manner that prevents fair and reasonable access for other users.
  4. Any booking which is cancelled or reduced within the 24 hours before the start time will be charged for the full period originally booked unless the user also emails the confocal users at the same time to inform them that the equipment has become available.
  5. The system allows users to edit or delete their own bookings up to 3 hours after the start time. This is to allow people to extend or reduce their booking time during use.
    However, if the booking is deleted after the start time users will still be charged in all cases. If the booking time is reduced after the start time users will also be charged for the full period originally booked unless an email is also sent out at the same time informing users that the equipment is now free.
Links and Downloads

How to Use the Microscopes

There are protocols available for download (see links on right hand siide) which cover confocal imaging and the setting up of both the upright and inverted microscopes, together with a walkthrough for imaging using the multiphoton laser. You can also obtain a Powerpoint tutorial Zeiss LSM 510 Tutorial which includes instructions on confocal imaging.

Finally, the Zeiss manual describing the LSM 510 Meta confocal system and software for obtaining and analysing images, and the manual which describes the use of the Confocor system for FCS use, are both also available for download.

LSM Image Browser

In order to view the images that you have obtained from the confocal microscopes you need to download the LSM Image Browser software - please contact Nicola Green to obtain access to the software.

Once downloaded the software will allow you to view your images on your PC, modify your images, add scale bars and labels, process z-stacks and perform some basic analysis of the data. It also allows you to export the images as JPEG or TIFF files for videos for use with other software.

There is also a dongle which will allow acces to LSM Image Examiner software. This can perform the aspects of the image analysis software which is greyed out in the free version. If you require this software please contact Nicola for access to the disc and dongle. The dongle restricts access to one user only at any time, but once access is granted the dongle can be booked out as required.

Online Microscopy Courses

There are some excellent online courses available that cover confocal microscopy and much more.

iBiology have produced an online microscopy course which is freely available, and consist of videos on all major topics in light microscopy given by leaders in the field, labs demonstrating specific techniques at the microscope, and short tips. These are a great resource for all microscopy users in the life sciences and definitely worth a look.

There is also a very good confocal training website which includes light microscopy and confocal theory, a practical element and an assessment. This is now compulsory for all new users of the facility but existing users, particularly those who only use the facility infrequently, might find it helpful too.

Confocal Theory

There is so much information available on the Internet which explains the theory of confocal microscopy in greater detail than has been provided here. A good place to start is with the confocal manufacturers, who all provide well written websites with a large amount of useful information to help you get the most out of your confocal experiments.

Go to Zeiss Microscopy and Imaging for theory, hints and tips for improving your images, specific techniques and applications and some help with fluorophore selection. Zeiss MicroImaging Online Campus also provides more information, articles and tutorials. It is also worth looking at Olympus, Nikon Microscopy and Molecular Expressions, whilst Helsinki University has a confocal tutorial on the principles of confocal microscope and some information on image optimisation.

Multiphoton Imaging

Again there is much information available regarding the theory behind multiphoton imaging. The sites listed in the confocal theory section above also cover multiphoton imaging. Nikon also provides a good explanation of the theory and techniques while Zeiss lists a number of useful references.

Fluorophore selection

When planning multiple label experiments, or if attempting to deal with background autofluorescence, the careful choice of probes is critical to obtaining good quality images.

For some useful tutorials, which allow you to examine the compatibility of particular probes, see the manufacturers websites described above. There is also a helpful tutorial provided by Invitrogen.

Finally there is some good information on the care and cleaning of objectives.

Useful Downloads