Spinning Disc confocal training
Topics that will be covered in training:
Microscope
o Turning the imaging system on and off
o Laser safety
o Objectives: Magnification, NA, protective mechanism, multi-immersion.
o Cleaning objectives ONLY with lens tissue paper
o Loading and focusing specimen
o Imaging modes: brightfield, DIC, epi-fluorescence, confocal scanning
Acquisition
o Lasers and detectors: PMT or Camera
o Excitation / Emission spectra
o Signal – adjustment of laser power/detector gain/confocal aperture (pinhole)
o Saturation and quantitative data analysis
o Set up and save personal imaging settings
o Optical sectioning, scanning, confocal aperture
Software
o Pixel size, image format, averaging, scan rate
o Z stacks, maximum projection, zoom, ROI
o File format, saving and exporting files as tiff images; File naming
Additional
o File saving, transfer to server, local drive cleared monthly
o Positive and Negative controls
o Experimental design: live imaging, time-lapse, quantification
o Other available instruments and capabilities, image processing
Please read the attached local rules for the confocal microscope and the general facility
rules.
All users must sign the list in the facility to confirm that they have read and understood the
local rules.
These files can be found on the server:
\\cri-share\Core\light_microscopy\public_folders\Microscopy Health and Safety 2014\Microscopy
Facility_local_rules_2015
If there is a problem, or for more advice, please contact the facility staff
Email: microscopy@cruk.cam.ac.uk
Please also keep an eye on the FACILITY WEBSITE for information on
seminars, workshops and microscope demo systems, the latest application
notes, news and general facility information.
www.lightmicroscopy.cruk.cam.ac.uk
Spinning Disc_training_sheet_user_copy2015
Local Rules for the Use of the Improvision Spinning Disc confocal
Location: Cancer Research UK Cambridge Institute, Light Microscopy Facility, Room 034F
Laser Safety Officer
Issued under the authority of
Date
Richard Grenfell, richard.grenfell@cruk.cam.ac.uk
Stefanie Reichelt
Light Microscopy Facility Manager
14 Jan 2015
These local rules cover the use of the lasers and fluorescent light sources attached to the Improvision spinning disc
confocal located in room 034F. They cover the normal use only. They implement the University’s laser safety policy at
a practical level and form part of the University’s duties under section 2(3) of the Health and Safety at work etc Act
1974.
Description
1. Class 3B Diode 405nm laser
2. Class 3B Diode 491nm laser
3. Class 3B Diode 561nm laser
4. Fluorescent light source containing mercury
Authorised Users
Only light microscopy facility staff and persons who are adequately trained by the light microscopy facility staff and are
on the authorised users list may use the confocal systems.
Class 3B lasers
 Do not disconnect any of the optical fibres that route the laser beam to the microscope.
 Do not change samples or filter cubes while the laser is scanning.
 Do not introduce fingers or other objects into the beam path at the objective.
 Do not insert reflective material into the beam path at the objective.
 Avoid looking directly into the light beam at the sample.
Fluorescent light source
 Avoid looking directly into the light beam at the sample.
 Ensure that the light source is blocked before changing the sample or objective.
 Once turned off, allow the bulb to cool for at least 20 minutes before switching back on.
 In the rare event that the lamp breaks or bursts leave the room and report to a facility staff member or an
appropriate safety person. No one must enter the room until told it is safe to do so.
General
 Use lens tissue to remove oil from the objectives after use.
 Clear up any spillages or glass breakages immediately and report them to facility staff
 Dispose of all sharps in the sharps bin provided.
Summary of hazards
If the rules are not followed:
 Class 3B laser radiation and prolonged exposure to the fluorescent light source can cause long-term damage
to the eyes and skin.
 Exposure to toxic mercury vapour
See the laser risk assessment for additional rules
Contingency Plans
 In the event of any eye injury or suspected eye injury, proceed straight to Addenbrooke’s Accident and
Emergency and tell staff that it concerns a laser eye injury. Get advice from Moorfields Eye Hospital (020
7253 3411)
 In the event of skin injury or suspected skin injury, proceed straight to Addenbrooke’s Accident and
Emergency (01223 217 118).
Spinning Disc_training_sheet_user_copy2015
Light Microscopy General Rules for Microscope Booking and Usage
New Users
All new users should discuss their imaging requirements and arrange training BEFORE they use any instrument or
computer. We will provide advice, regular lectures and training in imaging techniques, acquisition and analysis
software and new microscope methods.
Training
All users have to be trained before they use any equipment in the imaging facility. This will include a 2-hour training
session before using the instrument for the first time as well as further instructions once the user is familiar with the
basics. Please contact us if you wish to arrange training for any of the instruments.
The Webpage contains further information on the provided equipment as well as protocols and user manuals. Please
read the full details of the protocol for the equipment you are planning to use before your training session. The training
session topics include how to acquire, save, export, and transfer an image as well as the general microscope
operation.
Please fill in the Microscopy Project Form and e-mail to us for your discussion
Microscope Booking
1. Microscopes and workstations have to be booked using the resource scheduler. Between the hours of 9am and
6pm, on any one microscope, booking is restricted to a maximum of three hours per day (except for bookings for timelapse experiments). Users requiring longer sessions can book in the evening after 6pm or at weekends.
2. Please inform us as soon as possible if you need to cancel a booking, preferably at least the day before so that
others can use the slot. Anyone can come into the facility and use a system which is not used at any time. In this
case, the user should make a booking for the system so that we know the usage of each instrument.
3. Note that due to time wastage where the microscopes are booked and then not used for long periods of time, the
policy is that a booking will be cancelled if the user is more than 30 minutes late for their allotted time. Anybody
wanting to use the scope will be given access and the booking will be amended. The new users should also be
considerate with their demands, for example if the late user has live material which might have taken some time to
prepare or is more precious than fixed slides. If someone else is already on the system they can stay on until
someone else arrives.
4. It is not always necessary to use the confocal microscope. Wherever possible slides should be screened on a widefield system, then the best ones can be imaged on the confocal.
5. If you require operator assistance, you should inform us at least the day before your appointment.
6. The computers in the microscopy facility are for your scientific usage only. Although they are connected to the
network, please do not use the internet, especially while acquiring images on the confocal microscopes.
Cleaning
Objectives are cleaned ONLY with lens tissue and the cleaning solution provided. Immersion Oil is an aggressive
solvent. Avoid contact with your skin. Do not leave oil on the table surface or the keyboard or computer monitor. The
reason for this is that the oil will dissolve plastic knobs etc. Do not allow oil to spill into the mechanism of the
microscope, since it removes the correct lubricant and can damage gears and racks. If you drip oil clean up the
surface using paper tissues and the cleaning solution. Do not use lens-cleaning tissue except on objectives,
eyepieces or other optical surfaces.
If you find encounter a problem with the microscope or computer, report it immediately to the facility manager or staff.
Live Samples
Any live samples that are brought into the facility for imaging should be in sealed containers to prevent any spillage.
Any containers with liquid, including cell culture dishes, should be sealed with parafilm then placed inside a plastic box
or other container with a tightly fitting lid.
Spinning Disc_training_sheet_user_copy2015
Data Storage
Each user should log into the computer with the username and password provided by the CRI IS team. Images should
be stored initially on the local computer. This is particularly critical for time-lapse and fast acquisition experiments.
Data should then be transferred to the server at the end of the imaging session.
User Data Storage Agreement
This Agreement is about the storage of data on local hard drives of computers situated in the Cancer Research UK
Cambridge Institute Light Microscopy Facility. This includes the Workstations 1 – 4, all computers attached to any
microscope and the computers of the Microscopy Facility Staff. It is not about data that is stored on any network drive.
No user should store any data on any local hard drive outside the time the system has been booked for analysis. This
means you can copy data from the network or any other trusted source onto the local hard drive and analyse, modify
or do whatever you like with them. However, at the end of your session you must transfer your data back to any
trusted medium and delete the data temporarily stored on the local drive.
This is to ensure the local hard drives contain enough space for the users to work on their data. Furthermore, the local
hard drives are set up for performance (RAID 0), which means if the local drive crashes all stored data will be
irrecoverably lost. Therefore, the local hard drives are not a safe storage option.
All data stored on local hard drives may be deleted without further notification but at the latest after three months if
they have not been accessed. This will be done automatically.
NEVERTHELESS, WE ASK YOU TO DELETE YOUR DATA AT THE END OF YOUR SESSION. PLEASE DO NOT
RELY ON THE AUTOMATED DELETION.
In essence, it is your responsibility as a user to make sure your data are saved/stored in a secure location and not
locally.
Dedicated Imaging Servers
Image1 and Image2 have been specifically set up for Microscope Users to transfer and store their imaging data.
Please note that access to Image1 and Image 2 is arranged once microscopy training is completed.
\\uk-cri-limg01.cri.camres.org\IMAGE1
\\uk-cri-limg01.cri.camres.org\IMAGE2
CompuCyte iCys Rules and Guidelines
The iCys should be booked according to the following blocks:
Morning
9am – 1pm
Afternoon
1pm – 5pm
Evening/Overnight
5pm – 9am
You do not need to book the whole block, for example if you only need 1 or 2 hours for your scan, but please book
within the block and not over the blocks (please do not book 11am – 2pm for example). Please limit overnight
bookings to a maximum of two per week per person.
Saving and accessing your iCys run files
There is a shortcut to the iCys run files on the server on each workstation desktop.
IMAGE1\light_microscopy\iCys run files\iCys 3.4.12 (from 01_2012)
IMPORTANT: We have had problems when saving files with very long names onto the server. Longer file names are
truncated which makes them unreadable and causes problems when trying to open your file for analysis. To avoid this
please keep your run names to a minimum.
----------------------------------------------------------------------------------------------------------------------Please complete below to confirm that you have read and understood the information in this leaflet, then email a copy
to Microscopy@cruk.cam.ac.uk
NAME
DATE
Spinning Disc_training_sheet_user_copy2015