The Microscopy Imaging Core at Penn State College of Medicine provides consultation and training in ultra-high-resolution imaging of cells and tissues in fixed or live states. The core also provides expertise in quantitative image analysis and consultations on microscopy-related research projects.
The Confocal, Super-Resolution STED and Deconvolution microscopes are useful in 3D imaging of any biological systems and spatial distribution of multiple fluorescently-labeled sub-cellular objects, including various cellular proteins complexes (multiple colors) simultaneously in live or fixed biological conditions.
The College is also equipped with a Multimodal Multiphoton Microscope, which is capable of performing deep-tissue fluorescence (multiple colors) and harmonic generation imaging in ex vivo, in vivo or intra-vital small animal imaging.
The IMARIS, VOLOCITY and HUYGENS workstations are excellent tools for 3D reconstruction from a series of 2D optical sections as well as complex quantitative microscopy computations.
Cryo- and transmission electron microscopes enable ultra-high-resolution 3D imaging of sub-cellular objects as small as protein complexes and antigen-antibody interactions at the ultra-structural level.
The Auto3dem and EMAN2 workstations are useful for cryo-EM or TEM image processing and 3D reconstruction.
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Instrumentation and Services
The IMARIS, VOLOCITY and HUYGENS (Room C1732A) image processing work stations facilitate 3D and 4D reconstructions to visualize and analyze multiple color images from fluorescence and confocal microscopes.
- Surface renderings
- Fluorescence intensity measurements
- Complex cell tracking measurements
- Voxel counting
- Batch processing
- Drug bio-distribution measurements
- Accurate colocalization analysis
- Many other complex image processing, statistical calculations depending upon researcher’s needs
Leica SP8 Inverted Confocal (Room C1730) is capable of generating submicron level spatially and spectrally resolvable multicolor 3D or 4D fluorescent images in live or fixed cells/ tissues.
- Tunable emission filter technology (AOBS) and tunable pulsed white light laser source
- Fully automated operational procedures
- High numerical aperture immersion objectives
- Differential interference contrast (DIC) imaging
- Line and area scanning capabilities with simultaneous or sequential scanning options
- Ultra-high-speed live cell imaging, complex cell tracking experiments, FRAP measurements, ratio metric experiments, complex FRET measurements,
- Tile scanning (scanning whole slide)
- Hybrid signal detection systems which allow 3D or 4D image generation at much lower light energies
- Time-gated fluorescence signal collection and single photon counting
- Sophisticated live cell stage heater and CO2 chamber for perfusion system (Tokai Hit, Japan) that enables complex live cell experiments continuously for several days
The JEOL 2100 Cryo-EM (Room C1724) collects data that can be processed into high resolution 3-D maps. Samples in solution are vitrified, preserving biologically relevant structures samples include:
- Small proteins
- Large whole cells and sub-cellular environments
- Dynamic complexes
The JEOL 2100’s tilt stage and SerialEM software also enable 3D tomography.
Auto3dem or EMAN2 (Room C1725) work stations enable 3D reconstruction and visualization of Cryo-EM images.
PyMOL, Chimera, Veda, or Situs software program facilitates fitting, and interpretations of Cryo-EM data.
DeltaVision Elite Inverted Microscope (Room 1728) is uniquely designed for generating multicolor 3D or 4D fluorescent images in ultra-high speed live cell imaging mode as well as ultra-fast tile scanning mode.
- Precise optical sectioning capability and deconvolution software
- Multi-wavelength switching excitation light source
- High numerical aperture immersion objectives
- Highly sensitive cooled CCD and EM-CCD cameras
- Differential interference contrast (DIC) imaging
- Tile scanning (scanning whole slide)
Nikon A1 MP+ with Spectra Physics Insight DeepSee Femtosecond Laser (Room 1730) is designed for high-resolution ultra-high speed deep tissue ex vivo, in vivo or intra-vital small animal fluorescence (multiple colors) and harmonic generation imaging in upright microscope platform with spectral detection capability.
- Ultrahigh-speed hybrid scanning capabilities
- Ultrashort pulse IR laser tunable from 680-1300 nm
- Highly sensitive GaAsP detection systems
- Low magnification high resolution water dipping objective lens
- Tile scanning (scanning whole slide)
- Fast video rate imaging enabling deep tissue visualization of rapid reactions in living organisms, including dynamics and cell interactions and various other physiological events in real time
The JEOL 1400 TEM (Room C1727) is capable of generating ultra-structural nanoscale images from fixed cell/tissue samples or multiplexed immune-labeled samples.
- Computer-controlled operations
- Resolution up to 3 Angstroms
- Magnification up to 370,000X
- Capable of collecting data suitable for 3D reconstructions of negative-stained samples
Procedures, Protocols and Forms
The instruments in Microscopy Imaging are very sensitive and enormously expensive. These instruments must be treated with care by all users, as improper or careless use could render these systems unusable for extended periods of time, hampering multiple research programs. Even under service contract, many things will take weeks to get fixed, and service contracts do not cover negligence on the part of the users. Thus, to keep all of the equipment functioning properly for everyone, the following rules apply to all jobs done on the shared microscopes. These rules and regulations are subject to change at any time.
- No foods are allowed in the microscope rooms at any time.
- No sitting/standing on the anti-vibration table.
- No installing software onto the computer.
- No using the computers for purposes unrelated to microscopy (for instance, checking email, surfing internet, etc.)
- The microscope room is equipped with Class IIIb and Class IV lasers. Therefore, unauthorized entry into the facility is prohibited at all times.
- Before training, please arrange a 15-minute appointment to determine the applicability of the facility equipment to your research goals. Please send an email to Dr. Thomas Abraham, Director of Microscopy Imaging, at email@example.com for appointments.
- Fill out a User Registration Form.
- Users need to attend three hours of training before being allowed to sign up to use the equipment independently. Additional collaborative opportunities, including image processing, image analysis and data interpretation, etc., are available for those who need it.
- Refresher training may be required for anyone who has not used the facility equipment for six months.
- Users can schedule initial training only with Dr. Abraham and subsequent instrument scheduling with Wade Edris. Users will not train other users under any circumstances; each user has to receive training and be approved to use the instruments. Any violation of this rule will result in retraction of facility privileges.
- Users may sign up for use of the microscopy equipment between 8 a.m. and 6 p.m. weekdays. After-hours and weekend appointments may be made only after consultation with Wade Edris.
- You may schedule equipment time up to six days in advance. See scheduling links.
- Cancellation must be given at least 24 hours in advance of the requested time slot, preferably sooner. Failure to cancel in advance will result in the following penalties:
- First offense: a warning
- Second offense: time slot missed billed in full
Investigators who must cancel who can find someone to fill in the canceled time will receive no penalty.
- Investigators finishing early must contact the next person signed up.
- Signup slots may be cancelled by the facility staff at any time to schedule needed maintenance or repairs.
- Users must include their phone number and email during online signup.
- In the login book, users must also include lasers, PMTs, etc., that will be used.
- Use of the facility is a privilege. Access to confocal computer system will be activated only after successful completion of all training sessions. Investigators will use their Penn State Health ePass ID and password for login, once those have been activated following successful completion of training.
- Users must follow the standard operating procedures (SOP) when operating the instruments. If unsure of any procedures, users should contact Dr. Thomas Abraham or Wade Edris for further information.
- Any misuse of the facility will result in immediate prohibition.
- Investigators should check the schedule to see if someone else is signed up after them. Follow the Shutdown Procedure included in the standard operating procedures. Make sure all equipment is cooled down and turned off properly. Each investigator is responsible for logging off properly to avoid extra time charges, and for turning off all equipment.
- 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 up. Users must wipe oil/water with lens tissue or Q-tips from all oil/water objectives used.
When publishing images generated from the Leica SP8 Confocal or Leica SP8 STED or Nikon Multiphoton Systems, include relevant SIG grant number(s) in the “Acknowledgements” section:
- For Leica SP8 Confocal: 1S10OD010756-01A1 (CB)
- For Leica SP8 STED: PA Tobacco Settlement Fund
- For Nikon Multiphoton: 1S10OD018124-01A1 (TA)
Safety for all users and Core personnel is a top priority. Since the facility is a shared laboratory and is designated as a Biosafety Level II facility, there are certain guidelines that all users are responsible to be aware of and follow. This policy includes guidelines for the safe use of lasers as well as procedures for imaging any biohazards and proper cleanup procedures. Any imaging of cells or materials classified with Biosafety Levels 2 or 2+ (including but not limited to live primary cells, cells containing pathogenic viruses) must be approved by both the Institutional Biological Safety and Recombinant DNA Committee and independently by the Microscopy Imaging operator, Wade Edris, and the Microscopy Imaging Safety Committee before scheduling or using the instruments.
Not all BL2+ work will be approved; no Biosafety classification work above BL2+ is ever accepted.
Rules for the Safe Use of the Confocal Microscopy Facility
The Confocal Microscopy Facility operates as a Biosafety Level 2 laboratory. These rules are reviewed and revised as necessary each year, and each facility user is responsible to review them on a regular basis to insure they are in compliance. Briefly, the rules for safe use of BSL-2 materials as applied specifically to the Confocal Microscopy Facility are as follows:
- Admission to the laboratory is restricted to authorized personnel when work with human samples or known infectious agents is in progress.
- All human, primate, biohazardous or genetically modified cells must be assigned appropriate BSL levels for research use by the Biological Safety and Recombinant DNA Committee (Ralph Keil, PhD, Chair) before they are allowed to be used in the Confocal Microscopy Facility. See the Biohazardous Materials, Use in Research and Instruction policy for details (internal access only; ePass login required).
- All samples that are intended for imaging and all unfixed biohazardous cells intended for analysis must be clearly identified and approved in advance by the Microscopy Imaging Safety Committee. Generally this is covered by the above process of evaluation by the Biological Safety and Recombinant DNA Committee.
- In general, unfixed primary human and primate cells will not be allowed to be imaged on the Confocal, although some exceptions may be made if the cells have been certified as biohazard/pathogen-free. Any need to image unfixed cells must be justified and pre-approved by the Microscopy Imaging Safety Committee. Some otherwise “forbidden” cells may be permissible on the Confocal microscopes if they are fixed according to an accepted process documented to inactivate HIV and other biohazards. In any case, protocols for safe containment and cleanup must be submitted to and approved by the Microscopy Imaging Safety Committee and the Confocal specialist, Wade Edris, before scheduling or performing any imaging of BSL2 or BSL2+ materials. Discuss requirements for imaging any BSL2 or BSL2+ materials with Wade Edris well in advance of the need to image the materials.
- All procedures must be performed carefully to minimize the creation of aerosols.
- All samples must be logged onto the facility in the logbooks provided. Samples that have been fixed for imaging on the Confocal microscopes may be monitored and recorded to ensure that the fixation is adequate.
- Work surfaces are to be decontaminated with 10 percent household bleach after any spillage of sample or hazardous material and after each user completes their work.
- Instrument decontamination shall be performed according to protocols posted on each instrument, an addition to any special procedures required for particular samples (as decided by the Microscopy Imaging Safety Committee).
- All spills, however small, must be recorded in the accident book and reported to the Confocal microscopy specialist, Wade Edris.
- All liquid and solid waste and disposal is to be removed from the laboratory as biohazardous material by each user.
- Gloves must be worn whenever handling or running biohazardous samples on the microscope.
- Hands must be washed after handling biohazardous materials and before leaving the laboratory.
- Regardless of biosafety level, thorough cleanup of the microscope – including oil-immersion and other lenses – and the sample stage and other surfaces, is required after every imaging session. Failure to clean up properly will first result in a warning; continued failure to leave the microscope in a completely useable, ready state for the next researcher will result in loss of privileges on the microscope.
- Neither syringes nor hypodermic needles should be used in the facility without the express consent and knowledge of the Confocal specialist, Wade Edris. This consent must be obtained each time syringes or hypodermic needles are used; consent for use today does not imply that consent is given for later dates.
- No eating, drinking, smoking or applying cosmetics in the work area.
- No mouth pipetting.
- No animals are permitted within the laboratory.
In addition to the above guidelines and because this facility uses Class III, the following Laser Safety guidelines shall also be observed:
- Access to the laboratory will be limited to only those persons necessary to the running of experiments when any laser is operating.
- Only qualified personnel shall operate or adjust any laser.
Any questions regarding these guidelines may be directed to the Confocal microscopy specialist (Wade Edris, 717-531-0003, ext. 284149 or firstname.lastname@example.org) or to the director of the Microscopy Imaging Facility (Dr. Thomas Abraham, 717-531-0003, ext. 285486 or email@example.com).
In the event of a potential retrovirus exposure
This item refers to exposure of skin or mucous membranes to infectious materials, and is modeled after the NIH program “3 Emergency Steps to Take in the Event of a Potential HIV Exposure.”
Remember that the risk of infection is very dependent upon the titer of virus and the route of exposure. Thus the risk of infection by contact of intact skin with infectious body fluids is probably truly zero, and although the risk of infection by contact of mucous membranes or non-intact skin with infectious body fluids may be extremely small, it is likely to be non-zero.
The emergency steps are:
- Immediately initiate first aid at the work site.
- Contaminated skin should be meticulously cleaned for 10 minutes using a povidone iodine solution (such as Betadine) and copious amounts of water.
- Contaminated eyes and mucous membranes should be irrigated for five minutes using water.
- Notify your supervisor, if they are immediately available.
- Report to the hospital emergency department to activate the invasive accident protocol (needlestick protocol), which will include evaluation, counseling and provision of antiretroviral treatment if deemed appropriate. Do this immediately (within the half-hour).
- In the event of an emergency (e.g., fire, water leaks), investigators should act calmly and ensure their own personal safety first. If possible, shut down all running equipment, cover equipment if possible, and exit the building.
- Notify Wade Edris or other core staff immediately and phone the campus emergency line at 717-531-8888 (on-campus, dial 8888).
Work With Microscopy Imaging
For details on fees, contact Kathy Stauffer at firstname.lastname@example.org.
To reserve time on one of the light microscopes, sign in to iLab.
To use iLab, you must have an account, be assigned to a PI’s lab and have funding identified before you will be able to reserve time and use the microscope. See core staff for questions.
To reserve time for the transmission electron microscope, use the calendar here.
All potential users of light microscopes are required to have completed training prior to using the microscopes. See training requirements and find out how to schedule training.