The Microscopy Imaging Core is located in the Research Core Facility on the sixth floor of the Biomedical Research Institute. This is a shared user facility that provides access to advanced microscopes as well as the expertise of dedicated imaging specialists. The microscopes are open to all members at LSU Health Shreveport and outside users. We are happy to assist you with instrument training, experimental design, image acquisition, and image analysis. We encourage you to contact us prior to beginning a new project.
All users must be trained by the RCF staff before using the microscopes. For training request, please contact Dr. Margo Bienkowska-Haba.
How to use a microscope in the core?
Consult and select a microscope → Schedule a training session when you are almost ready with your samples → Training with on hand practice followed by a quiz → Your first imaging session must be operated with the help of a core staff → Your second imaging session must be scheduled during work hours just in case you need assistance → You are free to image independently!
- Are you new to fluorescent microscopy? Check out this paper to avoid errors and bias! http://jcb.rupress.org/content/218/5/1452
- Check out this new course on Bioimage analysis from iBiology! https://www.ibiology.org/online-biology-courses/bioimage-analysis-course/
The instruments in the Microscopy Imaging Core are very sensitive and extremely expensive, so must be treated with care by ALL users. Incorrect or careless use could render these systems unusable for extended periods of time, hampering multiple research programs. Even under service contract, repairs can take weeks to be completed. In addition, service contracts do not cover repair of damage due to user negligence. Thus, to ensure that equipment is available and functions properly for all users, the following rules apply to all use of the Core microscope facility.
PDF version of policies can be downloaded here.
The Microscopy Imaging Core operates as a Biosafety Level 2 laboratory and the microscope rooms are equipped with class 3b and 4 lasers, so all users must abide by the following safety measures:
- No food or drink is allowed in the microscope rooms at any time.
- When a laser is in operation, access to the laboratory is limited to only those person(s) necessary to perform the experiments.
- The presence in Core labs of individuals other than those performing experiments (e.g., students for an educational purpose) must be approved in advance by Core Staff.
- No alterations to hardware or software may be made by Core users. Any alterations must be approved and carried out by Core Staff. Any approved modifications to microscope configurations must be returned to the previous configuration when the work is complete.
- If any microscope component is broken or malfunctioning, users should notify Core Staff immediately. Prompt notification will lead to prompt repair.
- Damage to equipment during use must be reported immediately to Core Staff. If reported, only the cost of repair will be charged to the associated lab. Failure to report damage to the equipment will result in loss of access of the user to the facility as well as charges to the associated lab to cover repairs.
- All users must be trained by a Core Specialist and be approved to use the equipment. Established users are not permitted to train new users under any circumstances.
- Refresher training may be required for any user who has not used the facility equipment for 6 months or more.
Violation of these rules can result in retraction of facility privileges for the associated lab.
In order to ensure equal access for personnel of all labs, all users must adhere to the following rules for microscope reservation:
- Users may reserve microscope time through the online calendar no more than 2 weeks in advance unless first cleared with Core Staff based on experimental necessity.
- Users must include phone number (and email address) in the online reservation.
- Approval for after-hours/weekends use must be given by Facility staff and is restricted to experienced users who have demonstrated a high degree of proficiency and independence on a given instrument- at least 10 imaging sessions following training are required before a user may apply for after-hours privileges.
- Peak hours are 9am-5pm, daily.
- For use with fixed samples, advance reservations for use during peak hours are subject to a 4-hour/day maximum on all systems. Users wishing to reserve a block of time longer than 4 hours must use off-peak hours.
- The four-hour limit per system does not apply when imaging live samples.
- Users may extend their session on the day of the reservation if that time is still available.
- Users may not advance-reserve more than 12 hours of peak usage time per week (9am-5pm). No laboratory may advance-reserve for more than 24 hours of microscope time per week, not including same-day and after-hours reservations.
- Each laboratory is limited to 2 time slots a day/microscope.
- Reservation for overnight imaging may not extend into peak hours of the next day.
- If your experiment requires uninterrupted imaging exceeding the stated time limits, please contact Core Staff for approval.
- If a user is found to use an instrument during an unscheduled time or significantly beyond the scheduled time (including after hours), the user's PI will be notified, and the user's after-hours access may be revoked at the discretion of Core Staff.
- Sign-up slots may be cancelled at any time for required maintenance or repairs.
Any unapproved reservations in disagreement with the rules will be deleted.
- Please start your session punctually (on regular hours as well as evenings and weekends) and fill out the log sheet.
- If a user is late to their reserved session by more than 1 hour, another user may take over the time slot after contacting the user with the reservation.
- Extreme attention must be paid to the safety of microscope objectives. Always be aware of the lens position to avoid breakage on the slide or microscope stage and always lower the nosepiece before switching objectives.
- If you finish more than 30 min early, please update your end-time on the booking and let the next user know that the instrument is available to use.
- Arc lamps must be kept on for 30 min minimum and need to cool for 30 min before being reignited.
- Check the online booking calendar at the end of your session; leave the system on if the next user is booked within 2 hr., otherwise turn it off. Keep lasers in standby mode whenever possible to prolong their life.
- Leave the microscope clean and ready for the next user. Use lens paper and cleaning solution to clean any oil/water objectives you used. Permanent damage can be caused by failing to clean objectives properly.
- Users are responsible for cleaning up the microscope tables upon completion of imaging work. Slides, coverslips, specimens, and other tools must be removed from the microscopes and the air table should be cleaned.
- To ensure efficient core operation, overbooking instrument time is strongly discouraged (see limitations in advance-booking).
- If you need to cancel your session, please delete your reserved time on the booking calendar as far ahead of the start time as possible. The latest cancellation time to avoid charges is by 5 pm on the evening preceding the reservation.
- If you cancel your session late (last-minute withdrawal), YOU are responsible for ensuring the microscope is turned off, if appropriate. The person before you will leave the microscope on if someone has reserved after them. This is particularly important if you are to be the last user of the day.
- In case of recurring last-minute cancellations/changes, the charge will be made for the entire time slot missed, including after hours and weekends, by an hourly rate.
- If you fail to cancel your reservation and don't use the system, an hourly rate charge will be made for the entire reserved time, including after hours and weekends.
Please credit the microscopy facility in your publications by mentioning it in the acknowledgements as “The Microscopy Imaging Core at the Research Core Facility at LSU Health Shreveport”. This will enable us to demonstrate our importance to the research programs at the university and help to ultimately obtain additional equipment and funding critical for meeting the needs of our users. Authorship would be appreciated in case of a scientifically significant contribution, but not for routine work.
Acknowledgement may be written as:
“Confocal images were generated using the instruments and services at The Microscopy Imaging Core at the Research Core Facility at LSU Health Shreveport”
- Hourly charges are billed in ½ hour increments, which are rounded to the nearest ½ hour.
- RCF bills quarterly and send internal transaction form with combined fees directly to departmental business manager.
- All bills issued by RCF should be paid within one month.
- Due to institutional accounting system, all contracts are based on fiscal year calendar (July 1st – June 30th).
- Each contract purchased by PI is assigned to the individual user only.
- Transfer of contracts to another user from the same lab need to be well justified (e.g. termination of primary user employment).
- For new PI/users, first contract may be time-adjusted.
- Contract rates are discounted at 30% for each subsequent user in each laboratory.
- Contracts are not refundable under any circumstances (e.g. equipment failure).
- Contract options (available only for on Campus users):
- 12 month contracts begin on July 1st of each year. Fee includes 300 hours (h) of microscope use. Additional hours above the limit will be charged half of the regular hourly fee.
- 6 month contracts begin on either July 1st or January 1st of each year. Fee includes 200 h of microscope use. Additional hours above the limit will be charged half of the regular hourly fee.
- 3 month contracts begin on either the 1st of July, October, January, or April of each year. Fee includes 100 h of microscope use. Additional hours above the limit will be charged half of the regular hourly fee.
A PDF version of the Microscopy Imaging Core Rates & Fees can be downloaded here.
|AxioObserver + Apotome||$100||$15||$300||$600||$900||$7.5/h||$25|
|Leica TCS SP5 Confocal||$200||$40||$800||$1600||$2400||$20/h||$65|
|Olympus iXplore CSU W1 Spinning Disk||$200||$45||$900||$1800||$2700||$22.5/h||$70|
|Nikon A1R Confocal & Super-Resolution||$200||$50||$1000||$2000||$3000||$25/h||$75|
|Zeiss LSM 510 Coherent Multiphoton||$200||$50||$1000||$2000||$3000||$25/h||$75|
|Assisted use with imaging Core Staff||$75/h||$85/h|
|Imaris||$500/year (non-CMTV members)|
|Nis-element, CellSens, Zen||Free|
- Quantitative colocalization
- Analyze Immunohistochemistry images in ImageJ
- Make Binary & intensity measurements on colocalized channels (Nikon-Nis-element)
- How to open a multichannel Z-stack image in Image J/FIJI
- How to convert a image to TIFF format in Image J/FIJI
- Create inset in an image for publication
|RCF offline 1||RCF offline 2||RCF offline 3||RCF offline 4|
|Computer||Dell OptiPlex 3010||HP Z440||Dell Precision 5820||Dell OptiPlex 5040|
LAS AF 2.7.3
|System||Windows 7, 64-bit||Windows 7, 64 bit||Windows 10, 64 bit||Windows 10, 64-bit|
|RAM||4 GB||64 GB||128GB||64 GB|
|Location||F 6-27||F 6-27||F6-13||F 6-13|
RCF Offline 1 and 2 are free to all LSU Health employees and outside users for image analysis purpose, and no reservation is needed. License dongles are required for full access to some of the software. Please contact Dr. Chaowei Shang for the dongles.
For most users, the use of RCF Offline 3 and 4 cost a minimal fee to maintain the yearly service contract with Bitplane Imaris. You get free remote support from the Imaris (Bitplane) team.
Biosafety for Live Sample Imaging
1. RCF Basic operating procedure for live sample imaging
All samples must be prepared in your lab following biosafety rules.
All live samples must be carried to the microscope room in a sealed secondary container with an absorbent material at the bottom of the container. Parafilm must be used to wrap the cell culture dish during sample transfer. Remember to take off the parafilm before you start the experiment for CO2 perfusion.
During sample transfer, you should keep one hand clean without gloves, and another hand with a glove on to hold samples.
Wipe the dish or chamber insert, the dish holder, and the microscope stage surface with 70% ethanol after each experiment.
Clean hands, no gloves. Area: Room doorknobs, light switches, flashlight, chairs, CO2 tanks, sliding doors of microscope box, computer, keyboard, mouse, temperature wrapping band, microscope system parts that are outside the microscope box and below the microscope stage.
With gloves. Area: Your samples, dish/chamber inserts, live cell dish holder, microscope stage.
There is a small plastic tray inside the microscope box to hold live cell dish holder. Place your gloves in the secondary container you brought with you. Never put gloves directly on the computer table.
If any medium spills out, wipe down the surface immediately with dry paper towel, then wipe down the dry surface with 70% ethanol 3 times. If medium spills out onto the microscope objectives, wipe the objectives with ethanol pads 3 times. Dispose of contaminated gloves immediately into the biohazard box in room F6-13. Inform RCF staff as soon as possible that a spill has occurred.
2. RCF Advanced operating procedure for live sample imaging
BSL (Biosafety level) Overview
BSL-1: Agents that do not cause disease in healthy human.
BSL-2: Agents of potential moderate biohazard to personnel and environment. (Lentivirus, HIV, HBV, all human cells which may contain pathogens, e.g. Hela cells).
BSL-3&4: Agents and microbes that can cause serious and lethal disease via the inhalation route (airborne pathogens).
BSL-3&4 live sample imaging is NOT ALLOWED in the microscopy core.
General Biosafety Guidelines (resource: Addgene)
Adenovirus: This virus can cause mild to severe respiratory disease in humans. Ethanol cleaning does not inactivate adenovirus; 10% bleach (0.5% sodium hypochlorite) should be used instead. Use of a replication incompetent adenoviral system (BSL-2) reduces risk but requires multiple rounds of plaque purification.
Adeno-associated virus (AAV): This virus is replication-incompetent and not known to cause disease in humans. If it is prepared using a helper plasmid rather than helper virus, it can often be handled at BSL-1. If prepared using a helper virus, it should be handled at BSL-2. Ethanol cleaning does not inactivate AAV; 10% bleach (0.5% sodium hypochlorite) should be used instead.
Lentivirus: Lentiviral systems are derived from HIV, but their organization across multiple plasmids and the deletion of many HIV proteins lowers the probability of generating replication-capable virus. These systems are handled at BSL-2/2+.
Retrovirus: Retroviruses are classified based on the cell types they infect. For retroviruses that do not infect human cells, BSL-1 may be appropriate; if human cells can be infected, BSL-2/2+ is appropriate.
Detailed resource: https://www.addgene.org/biosafety/
Basic Microscopy Information
Confocal microscopy - 39 things that can go wrong
Image processing: what's allowed and what's not
Leica Science Lab Microscopy Information
Extra Microscopy Information
Fluorescent dye exication and emission spectra look up
Scanning techniques and photobleaching
Tag and Turbo fluorescent proteins (Evrogen)
Fluorescent probes for super-resolution imaging: STED, FPALM, STORM