FV500 Confocal Microscopy
The FV500 Confocal Microscopy Facility at the Masonic Cancer Center is committed to providing a state-of-the-art confocal microscope in a collaborative environment.
Confocal microscopy uses fluorescent probes to identify, characterize, and colocalize cells or cell structures in tissues.
Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens.
The FV500 Confocal Microscopy facility provides training, instrumentation, and software for confocal microscopy. The FV500 is a user-operated facility providing 24-hour access to a state-of-the-art instrument. Trained users prepare their own samples in advance, and can acquire images on a dedicated automated microscope or analyze data on a separate workstation. This facility is available only to Masonic Cancer Center members and their staff.
To see the complete specifications download the Leeds brochure (pdf).
Margaret Ramnaraine, Scientist
Olympus FluoView 500 Confocal Laser Scanning Microscope
In the FV500, Blue diode (405nm), Argon (488nm), Green Helium Neon (543nm), and Red Helium Neon (633nm) lasers are available. The fifth PMT detector, dedicated for transmitted light imaging, is outfitted for DIC images with which the confocal fluorescence images may be overlaid.
Automated software control of the FV500 lasers, filters, and BX61 microscope provides a simplified and user -freindly set-up of the system's hardware. The fully automated BX61 microscope allows complete control of the stage (xy) scanning and sample sectioning (z). In addition, time-lapse is possible on the FV500 with imaging rates of up to 4 frames/sec.
- BX61 upright motorized research microscope
- FV500 laser scan head
- Laser combiner and Argon, Gr HeNe and Red HeNe lasers
- Fiber optic delivery system
- Transmitted light detector
- BX61 handswitch, prior motorized stage control joystick, transmitted light source
- Microscope control unit
- Blue diode, Argon, Green HeNe and Red HeNe laser power supplies
- Prior motorized stage controller
- Mercury burner power supply
- FV500 control unit and power supply
- FV500 computer
- Computer monitor and surge-protected power outlet
The confocal microscope is housed in 506 Masonic Cancer Research Building (MCRB) (formerly named Cancer Center Research Building - CCRB) in a secured room. In order to get in you will need to have ID card access.
- Fill out the access form (pdf) with user information, proof of Hazardous Chemical Waste Training, and ID card number,
- Turn in to the confocal administrator (Margaret, 470 MCRB, 322 CR, or MMC 806),
- Complete advanced training course given by Leeds Microscope or otherwise demonstrate familiarity with the FV500 system.
Sign up using the online reservation schedule. Reservations are on a first-come, first-serve basis. If you must cancel, do so in advance.
If you are more than 15 minutes late for your reserved time, you forfeit that time IF someone else is waiting.
Report any problems in the "comments" column of the sign-in/out log, add a note to the event in the online calendar and inform Steve Ricchio (612-209-1086), Ken Kilby (651-270-9379) (Leeds, imaging and microscope, respectively), and/or Margaret (612-626-3672, email@example.com), so the problem can be addressed and service can be scheduled.
Scheduled maintenance has priority sign-up, but you will have ample notification in the reservation schedule.
From time to time, information will need to be passed on to the PIs and/or user. This will most often be by e-mail. It is therefore important to have your e-mail address and a contact phone number on file.
Users must provide documentation of Hazardous Chemical Waste Training to the confocal administrator. Hazardous Chemical Waste Training is required for all Cancer Center access and needs to be updated annually.
It is recommended that users have Blood Borne Pathogen training. Users must take appropriate precautions when working with human-derived samples or transfected animal tissues. Formalin fixation DOES NOT guarantee decontamination.
Samples containing biohazardous material(s), e.g. human-derived tissue or retrovirus, must be noted in the sign-in/out log.
Users must dispose of their samples in their own laboratory.
Absolutely no eating or drinking is allowed in the confocal microscope room (506 CCRB).
Although safety measures are in place to protect personnel from the potential hazards associated with the use of high energy lasers, under no circumstances are the covers to the laser housings to be removed, except by authorized service personnel.
When you are finished, lower stage and wipe objectives clean of oil with lens paper and clean stage of any oil, glass chips, or mounting media.
Change the objective to 4x when you finish and return the stage to non-escaped (up) position.
If someone is waiting to use the confocal, leave the automated components (BX-UCB and stage controller power supply) on.
If someone is waiting to use the confocal, leave the laser(s) on.
Only turn on the laser(s) you need, not all of them, to save the tube life. (See dye list for laser used.)
There are two computers, one attached to the FV500 confocal system for acquisition and one workstation for analysis. Neither computer is on the network and there is and will be NO Internet access to these computers. If people are waiting to acquire data, users should move to the second station to perform analysis.
Access to the computer is by secure log-in (by PI and PW). Access to the Fluoview program is by PI UN and will open the last settings used by that PI.
When finished you must:
- exit the Fluoview program
- transfer your data files to removable storage
- log out of your session on the computer and 4) shut down the computer if there is no one signed up after you.
Each PI is assigned 1.5 GB on the FV500 computer and 5 GB on the workstation. You are responsible for maintaining your own section of the hard drive, no more space will be allocated. Data files more than 1 month old will be purged on a regular basis by the administrator, so delete your files as they are backed up.
It is suggested that only instrument setting and user profiles be stored on the FV500 computer hard drive and only the most current data on the workstation.
Data files should be archived on zip disk (100 or 250 MB), CD-R or DVD-R when acquisition and/or analysis is completed.
This instrument was purchased through an NCRR Shared instrumentation grant and should be acknowledged in any publications. The following statement is suggested: "We would like to acknowledge the use of confocal microscope made available through an NCRR Shared Instrumentation Grant (#1 S10 RR16851)."
Please provide a reprint of the paper so we can have it on file for our report.
It is crucial that all users complete the sign-in/out log whenever using the confocal system (blue folder on workstation desk).
The usage log will be used to compile report(s) to the funding agency.
The advisory committee will meet twice a year to make, review, and update policy. It will also evaluate any problems and financial decisions.
- Download Access Request Form (PDF), fill in user information and mail [MMC806], e-mail firstname.lastname@example.org filled in form saved in Acrobat or fax [4-3913] to Margaret Ramnaraine.
- Provide employee ID number [included on access form] so that your training records can be accessed in PeopleSoft.
- Download operating manual (PDF) and handbook (PDF).
- View a training presentation (PDF).
- Read and review all materials. You will be tested on the contents.
General Confocal Training
A general course on confocal microscopy and consultation on confocal microscopes are available through University-wide Imaging Centers. Contact Mark Sanders (612-624-3454, email@example.com).
Training on how to use this specific microscope is provided on an as-needed basis by the confocal administrator. Complete and turn in access form (PDF), then call or e-mail Margaret (6-3672, firstname.lastname@example.org) to arrange scheduling.
Mark Sanders will provided hands-on training at a cost of $20/person. When a time has been scheduled, have your PI (or whoever is paying for the training) fill out a work order on the BIPL web page. In the Comments field, write "Olympus Confocal Training" and a list of the name(s) of people to be trained. Fill out one work order per PI.
Training will be scheduled when 4 requests have been received with a maximum of eight people per session. It will take about 2-3 hours, and in about one week your U-card will access the confocal microscope room, the stairwells, and the elevator for after hours/weekend use.
It is recommended that your first session on the confocal be under the direction of someone who is familiar with the FV500 to ensure correct set-up of your settings.
A copy of the handbook (PDF) will be available next to the instrument to act as a prompt to aid in using the confocal microscope system.
A training presentation (PDF) is available for viewing as a refresher.
Proof of up-to-date Hazardous Chemical Waste Training will be checked in PeopleSoft. Card access will not be granted until documentation is verified. Non-compliance with safety rules will result in revocation of user privileges.
Absolutely no eating or drinking is allowed in the confocal microscope room. (506 MCRB). This includes waste from food or drink (i.e. wrappers or cups) in the trash.
Blood Borne Pathogen Training is recommended, but not required.
Steve Ricchio, Digital Imaging Specialist, Leeds Precision Instruments, Inc., office 763-546-8575, cell 612-209-1086, email@example.com
Ken Kilby, Sales Representative (Microscopes), Leeds Precision Instruments, Inc., office 763-546-8575, cell 651-270-9379, firstname.lastname@example.org
Angela Goodacre, Marketing Manager, Imaging Systems Applications, Olympus America, Inc., voicemail 800-645-8100 ext 6004, email@example.com
Mark A. Sanders, Program Director, University-wide Imaging Centers, 612-624-3454, firstname.lastname@example.org
Margaret Ramnaraine, Scientist, Confocal Administrator, 612-626-3672, email@example.com
- Olympus Fluoview FV500 Training, Presented by Leeds Precision Instruments, Inc. (pdf)
- Olympus Fluoview FV500 Laser Scanning Confocal System Operating Instructions (pdf) and Safety Manual (pdf)
- Olympus Fluoview FV500 Multi Point Time Lapse Software full version of the stage control manual (pdf)
- Shared Confocal Microscope User Handbook: Policies and Practices (pdf)
Cell Biological Applications of Confocal Microscopy. Ed. B. Matsumoto.