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Fluorescent microscope guidelines and charges

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Nikon Ti-E Inverted Microscope with Double Port FRET System

Nikon Ti-E MicroscopeThe MCB Microscope Facility houses a Nikon Ti-E inverted microscope with Z-stacking capability, and a motorized stage and CO2 incubator for live imaging of cells and FRET analysis. The Nikon Ti-E microscope is equipped with a top quality colour camera to capture bright field, phase contrast and fluorescent images from 4X to 100X magnification from live cells, as well as fixed samples. The microscope is also equipped with two, low light, cooled EMCCD (Electron Multiplying Charge Coupled Device) monochrome cameras for simultaneous detection of FRET signals. Phase contrast and Normarski DIC optics facilitate the acquisition of high contrast and high resolution images at low magnifications. Narrow band filter blocks allow fluorescent imaging in the UV, FITC and Texas Red range.

The microscope is linked to NIS-Elements AR imaging software which will allow image acquisition up to 6D (X, Y, Z, time, Lambda (wavelength), multipoint), archiving and analysis. This is particularly important for live cell imaging where information from all 6D are organized within one integral platform. Furthermore, the NIS-elements analysis software includes the capabilities for object counting, automated measurements, region of intent measurements and time measurements including image ratio ing of multi-channel images. The NIS Element software can also be used to stitch large images together seamlessly. Most importantly the NIS-Elements software has a built in image-database for the archiving of all image and meta-data captured on the microscope.

All images captured on the microscope will be stored on a dedicated 10 TB server (ROSALIND) managed by ICTS at UCT, from which users can download their images via the internet. All data stored on this dedicated server will be automatically backed up for 6 weeks from date of aquisition. The open source OMERO software will be used for users to view and manage their image data on the ROSALIND Data Store. This solution will be integrated into the University of Cape Town's long term data management strategy.


Microscope booking

All sessions on the MCB Inverted Fluorescent microscope should be reserved using the Bookkit App. Initial training must be undertaken if the user is unfamiliar with the Nikon Ti-E fluorescent microscope.

Restrictions under Level 2

We will be restricting the use of the MCB Inverted Microscope to two users a day, with one user per session. An interval of an hour should be allowed between bookings.

Registering on Bookkit

All existing MCB users of the Inverted Fluorescent Microscope will have already received an email to join the Facility on Bookkit.
Existing (trained) users of the microscope from other Departments (i.e. not MCB) need to contact Keren.Cooper@uct.ac.za and ask Keren to send them an invitation to join Bookkit, and to arrange access to the MCB building.

You will need to have a project registered on the Fluorescent Microscope Bookkit platform in order to book. This information is used for billing, and for reporting on the use of the microscope.
Please note: If your project or fund number changes, you will need to notify us. Any additional project you work on will also need to be registered.

Contact Keren.Cooper@uct.ac.za to arrange a training session. Once you have been trained and have registered on Bookkit, Keren will send you an invitation via Bookkit to join the MCB Fluorescent Microscope Facility.

Signing up to Bookkit

You will have received an email from the MCB Inverted Microscope Facility to join Bookkit.

  1. Login to Bookkit.
     
  2. Accept the invitation; Fluorescent Microscope should be listed in the drop-down list under “My Accounts” on the left sidebar menu.
     
  3. Select the Fluorescent Microscope Facility and then click on “Lab Dashboard” (directly below Fluorescent Microscope Facility on the left sidebar menu.
     
  4. You will see two items listed under Equipment:

    • the “Inverted Fluorescence Microscope – Fluorescence Microscopy” and
    • the “Inverted Microscope - Brightfield only”.

    Both represent the same instrument, but we have included two options for booking to accommodate the differing prices for users.
    Check that the microscope is free (i.e. under both options) and select the “Inverted Fluorescence Microscope – Fluorescence Microscopy” option if you plan to use the Hg lamp and Fluorescence in your imaging. Select “Inverted Microscope – Brightfield Only” if you will not be switching on the Hg lamp). NB: Please check the other instance of the instrument to make sure you are not double booking!
     
  5. An “Equipment-Details” screen will appear, and you will see the calendar of bookings. Click on the day/time you would like to start and drag to block off the time for which you will be using the microscope. If you make a mistake, double click on the booking and select “OK” to delete it.
     
  6. Below the calendar in the “Notes” field – add a short description of the work you will be doing (type of tissue, fluorophore, reason for viewing etc.). Then click the “Book” button directly below the “Notes”.
     
  7. Your booking should now show on the calendar in green. If you double click it, you will be able to edit it or cancel the booking.
     
  8. We will be using the booking calendar to charge for the use of the MCB Inverted Microscope.

Cancelling your booking

Research plans change constantly, and you may not need your booking. But please make sure you cancel your booking at least 24 hrs beforehand. All users will be automatically notified that a slot has become available when you cancel your booking.

You will be charged for your slot if you do not cancel your booking.

To edit or cancel a booking: Click on “My Bookings” on the left side menu. Click the drop-down arrow (to the right of the status column) and select “Details”. You can also double-click on your booking in the equipment calendar to access the booking details. On the Details screen, click “Cancel booking” to cancel or “Edit booking” to alter your booking.

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User charges

Fluorescent MicroscopeNON-COMMERCIAL USERS

Brightfield microscopy only
R40 per hour (unassisted) | R190 per hour (with technical assistance)
Administration fee (per invoice): R50

Fluorescence microscopy
R80 per hour (unassisted) | R230 per hour (with technical assistance)
Administration fee (per invoice): R50

 

COMMERCIAL USERS

R300 per hour (unassisted) | R1000 per hour (with technical support)
Administration fee (per invoice): R200


Heath & safety guidelines

All Covid Health and Safety Measures apply to users of the MCB Inverted Microscope Facility.

It is going to be essential that users of the MCB Inverted Fluorescent Microscope take the following precautions when working in Room 432.1.

Following advice from Nikon, the microscope should be cleaned by applying 70% ethanol to paper towel, and wiping surfaces of contact on the microscope carefully.

Users should never spray 70% ethanol directly on the microscope. Apply 70% ethanol to paper towel and use the paper towel to clean surfaces.

It is the responsibility of each user to clean (using 70% ethanol), all the desk top surfaces, the microscope points of contact, the keyboard and the mouse when they start working in the microscope room. They should repeat the procedure before they leave.

The microscope points of contact include

  1. The eyepieces
  2. The focusing wheel
  3. The X-Y navigation joystick
  4. The Fluorescent Shutter control

Do not touch the rest of the microscope. All operation should be via the computer interface.
You can take additional precautions of wearing disposable gloves and placing cling film across the keyboard. A roll of cling film will be provided (but not disposable gloves).

Additional cleaning
One of the scientific officers will be responsible for cleaning all work surfaces (NOT the microscope) with diluted F10 disinfectant once a week.
Users must wash their hands on entering and leaving the Microscope Facility. Hand soap, paper towel and 70% Ethanol will be provided.

Users are required follow the guidelines for working in a BSL-2 Laboratory.
The microscope is located within a bio-safety level 2 (BSL-2) laboratory. If viewing live BSL-2 cells (guidelines on BSL levels can be found here), the following rules MUST be adhered to:

  • Microscope users must bring all necessary H&S consumables with them (gloves, etc). These items as well as all slides, cells and reagents must be removed after use for disposal in their own labs.
  • Cultures must be securely sealed.
  • Cultures must be transported in secondary containment.
  • The imaging process must be transient.
  • Cultures must be returned immediately to a BSL-II lab.
  • Users must have a plan of action in the unlikely event of a spill/breakage.

Further precautions
We will initially limit use of the microscope to two researchers per day, one user per session.

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User guidelines

1. Microscope Use

You will need to pass a competency test before you can be accredited to work on the microscope independently. Arrange a time with Keren Cooper to demonstrate your ability to carry out the following instructions:

1.1. Switching on and off
a) Switch on the microscope and the lamps, and understand the function of each component.
b) Not to switch on the Intensilight (the Hg Lamp) within one hour of it being switched off
c) To switch off at the end of your session.

1.2. Precautions when using oil
a) Clean immersion oil from the lens using lens cleaning tissue without dragging the 40X objective through the oil. If this happens, you will be expected to clean that lens as well.

1.3. Use of Slides and coverslips
a) Load the slide onto the stage correctly.
b) Slides should be clean and free from excess mounting fluid.
c) Only clear varnish should be used. No glitter will be allowed.
d) Varnish must be dry before use.

1.4. Initial scanning and focussing
a) Only the 4X and 10X objectives should be used for this purpose.
b) Focussing and navigating your way around the slide.

1.5. Software
a) Find the Ti pad and the camera control pad
b) Capture an image

1.6. Disposal and removal of samples
Users must remove and dispose of all their samples in their own Labs. No disposal methods (waste bins or bottles etc.) will be provided.


2. Access control

You will be given access to the MCB Fluorescent Microscope Facility (4.32.1) and the Rosalind Server once you have past your competency test and have been accredited as a MCB Fluorescent Microscope User.


3. Computers and Data Storage

3.1. Instructions on saving your microscope images on the Rosalind Server
A specialist Server, called Rosalind, is available for you to save your microscope images remotely. This will save the microscope PC from becoming clogged up with user images and will also allow you to access your images when you get back to your laboratory. Part of your payment for the use of the microscope includes the safe storage of all your images for 6 weeks. Your data will be backed up during this period. It is your responsibility to find a solution for the storage of your images beyond this 6 week period.

3.2. Saving your image files
A folder with the day's date will automatically be created on the Rosalind Server as the A: drive when you activate NIS elements from the microscope computer. All image files should be saved in this folder. Any files saved to the microscope PC will be deleted. The server can then be access by mapping a drive for it on your own computer.

Mapping a drive to the Rosalind Data Share
You have to be logged in to the server in order to map the network drive to MCB Central Data share. If you are off-campus, you will also need to be logged into the UCT VPN.

Windows
1. Open Select "This PC" in the efile explorer (or right click it)
2. Select "Map network drive" from the taskbar (or the right click dropdown list)
3. Next select a drive letter, e.g. O:
4. Type in the path to the folder ie. researchdata.uct.ac.za/MCB
5. Check "Reconnect at logon" to automatically remap your drive when you logon

Linux
Use the following command to mount the CIFS share
Command Line:
Example:
user@host:~$ mount.cifs
–o username=wf 3456,password=password,rw,nounix,iocharset=utf8,file_mode=0777,
dir_mode=0777 //researchdata.uct.ac.za/MCB /mnt/MCB

The network drive will appear in your left hand side screen
Right click on ROSALIND, and you will be able to map the network drive so that you can always see it when you log onto your computer. You can assign a letter for the network drive.


Resources

MicroscopyUConcepts in Fluorescent microscopy

MicroscopyUNikon Matching Fluorescent Probes with Nikon Fluorescence Filter Blocks

MicroscopyUNikon Fundamental principles of FRET with fluorescent proteins

Microscopy Resource CentreOlympus Microscopy Learning Centre

ZeissEducation in micrscopy and digital imaging

ZeissIntroduction to fluorescent microscopy

Contact
For enquiries, please contact Keren Cooper (keren.cooper@uct.ac.za).